At the time the first pancreas transplant was performed by Kelly and Lillehei in 1966,
insulin therapy for diabetes was generally available but administered in a form that
is known today as “conventional therapy.”
1
In this era, as many as half of all juvenile onset diabetics did not reach the age
of 55 years. Early mortality from accelerated cardiovascular disease, renal failure,
and hypoglycemia-related events were commonplace. The early low success rate and mortality
of pancreas transplantation by comparison were also suboptimal. As will be characterized
in the succeeding 8 chapters, the outcome of “best medical therapy” with newer forms
of insulin and insulin delivery systems along with dramatically improved outcomes
of islet and pancreas transplantation and novel β-cell sources hold great promise
for those afflicted.
Among the great strides in diabetes research was the Diabetes Control and Complications
Trial (DCCT).
2
This trial, published in 1991, showed that intensive insulin therapy when compared
with “conventional therapy” dramatically reduced the incidence and progression of
the microvascular complications of diabetes, nephropathy, neuropathy, and retinopathy.
Thus, with intensive insulin therapy, the mean hemoglobin A1C was improved to 7% compared
with 8.3% in the conventional group. The improved microvascular outcomes and measured
hemoglobin A1C came at a substantial price, namely, a greatly increased incidence
of hypoglycemic events requiring third-party intervention. After 2 decades, the 2
groups have also diverged with respect to mortality; recent reanalysis of the original
study groups demonstrates that those individuals who received intensive insulin therapy
groups had lower overall mortality.
3
Current best practice includes the availability of insulin pumps and newer forms of
synthetic insulin as well as pharmaceutical agents that augment insulin action. Unfortunately,
the widespread application of the therapeutic measures taken in the intensive therapy
arm of the DCCT is not the norm. Analysis of data from the 67 centers reporting to
the US type 1 diabetes (T1D) exchange shows that even today, more than 20 years after
the DCCT, the average hemoglobin A1C for treated patients is 8.3%. Thus, outside of
a clinical trial, such as the DCCT, actual practice achieves suboptimal outcomes.
A remarkable report of the current state of diabetes care is published in the Journal
of the American Medical Association in January 2015. This report shows that in a modern
era of diabetes care, mortality remains higher than the general population. For men
and women, the life expectancy for those reaching 20 years of age is 11.1 years and
12.9 years less than the general population, respectively.
4
These sobering findings, which have been thoughtfully summarized in an accompanying
editorial by Katz, provide a meaningful context for an international conference dedicated
to summarize the current state of pancreas and islet transplantation and chart the
way forward with an ambitious research agenda.
5
The need for a cure for diabetes through transplantation, stem cell–based therapy,
regeneration, newer insulin delivery systems, and devices that warn of hypoglycemia
have been brought into sharp focus by these reports which show that the progress in
care for diabetics has hit a plateau. Innovation will be required to improve the quality
of life and lower morbidity and mortality for those with insulin-requiring diabetes.
Against this backdrop, the International Pancreas and Islet Transplant Association
(IPITA), in collaboration with the Transplantation Society (TTS), held a scientific
workshop in Oxford England, May 7 to 9, 2014, to review the current status and needed
research agenda of 8 current or nascent β-cell replacement therapies: whole organ
pancreas transplantation, isolated islet transplantation, artificial pancreas (AP),
immunological tolerance, xenotransplantation, encapsulation technologies, β cell regeneration,
and stem cell derived β cells. Thirty-two scientists and clinicians representing 4
continents, 7 countries and 29 institutions, with dedicated expertise in these areas
were recruited to participate in 8 topical workgroups along with representatives of
the NIH (National Institute of Diabetes and Digestive and Kidney Disease, National
Institute of Allergy and Infectious Disease), Diabetes Research and Wellness Foundation,
the Juvenile Diabetes Research Foundation (JDRF) and industry. In advance of the meeting,
the workgroups prepared summaries of their respective topic highlighting the state
of their field and the research agenda needed to move the therapy forward to optimal
clinical application. Presentation and full group discussion at the meeting generated
revised summaries presented in the 8 sections below. These reports are followed by
the results of a conference attendee survey examining how the participants view the
future of β cell replacement therapies.
ALLOGENEIC PANCREAS TRANSPLANTATION
Current State of the Field
Pancreas transplantation has been available as a cure for diabetes since its first
application in 1966. The most common application is simultaneous pancreas-kidney (SPK)
transplant for the uremic T1D patients who are free of major surgical risks. Solitary
pancreas transplants in the nonuremic diabetic have been largely reserved for patients
with brittle diabetes and hypoglycemic unawareness despite best medical therapy or
after a successful kidney transplant. Unlike in the European Union where the application
of whole-organ pancreas transplantation continues to expand, the number of pancreas
transplant cases in the United States has decreased by 35% since the year 2003. The
reasons for the decline in the United States are that the number of waitlist patients
has decreased and the utilization of donor organ pancreas has declined markedly. The
decline in pancreas utilization in the United States followed publication of the pancreas
donor risk index (PDRI) which has created an environment of selectivity in the United
States.
6
The PDRI has also been validated in the United Kingdom. However, the trend for pancreas
utilization in the United Kingdom and Europe is divergent with the US European policies
strongly favoring the effective use of regional teamwork in pancreas procurement which
include policies regarding cryopreservation solutions and the technical details of
pancreas procurement. Thus, European-wide policies have led to much better pancreas
utilization while achieving excellent results. Thus, although PDRI has been validated
on 2 continents, the addition of uniform procurement and preservation policies can
further enhance utilization beyond what can be achieved by the high selectivity created
by simple reliance on the PDRI alone. Countrywide policies regarding pancreas allocation
to islets and pancreas transplantation in Australia have fostered an environment of
cooperation and better utilization.
Decreasing waitlist could be attributable to better insulin therapies, reduced enthusiasm
for pancreas transplant secondary to data showing limited survival benefit,
7
and better outcomes with islet transplantation.
8
Declining organ utilization followed publication of studies that aggregate risk factors
for early graft failure. As such, transplant surgeons are oriented to donor selection
strategies that avoid early graft failure using a predictive index. Studies on strategies
to increase organ utilization by avoiding early graft failure with active interventions
have been scarce over the past decade. Donor selection and intervention studies in
pancreas transplant have been largely focused on early outcomes, specifically avoidance
of early graft loss from thrombosis and sepsis. Studies of long-term graft survival
and the impact of pancreas transplantation on diabetic morbidity and mortality is
hampered by the lack of a clear definition of graft failure. Without an internationally
accepted endpoint of pancreas transplant function, the literature on the impact of
pancreas transplantation on long-term diabetic morbidity can be viewed as observational.
Need for Systematic, Comprehensive Documentation of Pancreas Transplant Outcomes Worldwide
Graft failure criteria cannot be defined clearly unless the goals of pancreas transplant
are defined. Is insulin independence the goal of all pancreas transplants? Does partial
function of the pancreas benefit the patient in providing stable glycemic control
and abrogation of hypoglycemia unawareness? This may be especially relevant for type
2 diabetics (T2D) with high baseline insulin requirements. Further, does partial function
provide long-term benefit in incidence or improvement of secondary complications?
Should pancreas transplant in total pancreatectomized patients have a dual outcome
endpoint for exocrine and endocrine function?
One option would be to have absolute insulin independence as the goal. This would
clearly be the most accepted and easily auditable endpoint for all patients. However,
when applied across the gamut of patients undergoing transplant, it clearly is restrictive.
Another option would be to have different pathways for pancreas transplant with predefined
endpoints for each pathway: type 1 with/without hypoglycemia unawareness, T2D nonobese
with/without secondary complications, surgical diabetics (postpancreatectomy), and
so on. This would require a well thought out algorithm, with clear endpoints in each
limb and additional data, such as C-peptide, HbA1C, quantification of insulin requirement,
and details on hypoglycemia unawareness and secondary complications to name a few.
Currently, none of the abovementioned variables are collected by the United Network
for Organ Sharing for the Scientific Registry of Transplant Recipients outcome analysis.
Another important attribute of a system defining organ function endpoints should be
verifiability. Currently, pancreas graft failure is self-reported, and because the
definition is vague, available data may be regarded as unreliable. Death in most countries
can be verified using census or other governmental databases. Insulin use and oral
agents can be tracked based on prescription data obtained from third-party payers.
9
Clear auditing guidelines need to be established for any outcome endpoints.
There is opportunity to learn from our islet transplant colleagues in maintaining
a detailed database resulting in the ability to look at outcomes with well-defined
criteria.
10
If the Collaborative Islet Transplant Registry database were to be linked with the
UNOS database for pancreas transplants, both databases would be enhanced with the
ability to compare “apples to apples”. Granted, the total number of patients is smaller
and at least the current funding in the United States is inadequate. Any attempt to
increase the granularity of data in pancreas transplantation is met with resistance
from the centers largely due to the “unfunded mandate” issue when it comes to data
reporting.
Any effort to impact pancreas transplantation positively in the next 10 years will
have to start with crystallization of functional endpoints that are widely accepted
and verifiable. If achieved, this would lead to a better understanding on the relative
impact of pancreas transplants on the burden of diabetes in comparison with other
options.
The Research Agenda
Indications for Pancreas Transplantation
The majority of solid organ pancreas transplants are carried out in patients with
chronic renal failure associated with T1D. In patients not yet requiring dialysis,
the timing of transplantation is based on the anticipated timepoint of needed renal
replacement therapy. Typically, a glomerular filtration rate (GFR) of 20 mL/min is
accepted as the upper limit of renal function at which it is reasonable to list a
patient for a SPK transplant—this is partly driven by the need for equity with respect
to other patients competing for donor kidneys.
Assessment for pancreas transplantation is substantially focused on cardiovascular
risk—the majority of patients with diabetic renal failure have some significant degree
of cardiovascular disease. Units have developed different protocols to assess this,
most including some modality of myocardial functional testing (perfusion scintigraphy,
echocardiography, cardiopulmonary exercise test, and so on). Research into the predictive
ability of these and other factors is needed. Age is a good predictor of cardiovascular
disease in this population of patients, and in most patients older than 60 years,
the risk-benefit of the combined procedure is judged to be unfavorable. In patients
judged to be at a lower risk of perioperative cardiovascular complications, there
is consistent circumstantial evidence of the life expectancy benefit of combined pancreas
and kidney transplantation.
11
The place for SPK transplantation in patients with T2D is incompletely defined, although
this procedure is carried out in increasing numbers by a several units.
12,13
Numerous questions require answers: Does transplantation provide similar long-term
benefit as in T1D? Should this be restricted to noninsulin-resistant patients? Should
this be restricted to nonobese patients? These and other questions should be addressed
by collaborative and carefully designed clinical trials. In patients who do not require
renal replacement therapy, the evidence that pancreas transplantation prolongs life
is less clear. Recurrent episodes of hypoglycemia, often driven by the desire for
tight control and long-duration diabetes, are both life-threatening and highly disruptive
to lifestyle, and this is the most widely accepted indication for pancreas transplantation
alone. Many patients, however, are not unaware of their hypoglycemia but may nonetheless
be very compromised by the complications of diabetes. In particular, patients with
rapidly progressive retinal, renal, or neurological complications are often referred
for consideration of pancreas transplant alone (PTA).
This creates problems for a number of reasons: first, the outcome of PTA is poorer
than that of SPK in nearly all published series, reducing any advantage of normalising
pancreatic function. Second, the effect of successful pancreas transplantation with
respect to halting the progression or reversing the secondary complications of diabetes
has not been convincingly documented in randomized trials. There are many publications
based on limited numbers in uncontrolled, observational studies, and the consensus
from these shows a beneficial effect; however, publication of positive observation
studies may be hampered by publication bias: negative studies are unlikely to be published.
However, the quality of evidence is poor, and the risk of publication and other forms
of bias considerable. It is clear that a scientifically rigorous approach to this
issue is needed to establish the true benefit of PTA beyond reducing hypoglycemia
unawareness. Diabetic patients who undergo PTA may contribute greatly to our knowledge
of pancreas transplantation: such patients often have less-advanced secondary complications
of diabetes, and longitudinal studies in such patients may provide key information
as to the effect of pancreas transplantation on the progression of diabetic retinopathy,
neuropathy, vasculopathy, and nephropathy. Similar studies in SPK transplant recipients
(with established renal failure) are compromised by the very advanced stage of secondary
complications—for example, it is hard to measure the effect of pancreas transplantation
on retinal disease in a patient who has already received extensive laser therapy.
Another problematic group of patients are those with a significant degree of renal
impairment, but not close enough to requiring renal replacement therapy (typically
GFR 35 to 45 mL/min) to warrant kidney transplantation. Such patients are not generally
eligible for SPK transplant listing and are usually denied PTA listing as well, because
of the concern that the effect of calcineurin-inhibitor therapy might accelerate the
decline in renal function and bring forward the need for dialysis. Such patients,
therefore, are often denied transplantation until such time as their renal function
has deteriorated (see above), and often express concern that they are placed at risk
of deterioration of nonrenal complications in the meantime. In fact, the degree to
which renal function deteriorates under these circumstances is not clear, and some
evidence suggests that this might be less than was once thought.
14
This requires further investigation, particularly with the use of non-nephrotoxic
immunosuppressive drugs (sirolimus, belatacept), that have not been formally tested
in the context of pancreas transplantation.
15,16
Pancreas Preservation
The majority of pancreas transplant units rely on static cold preservation using University
of Wisconsin, histidine-tryptophan-ketoglutarate or Celsior solutions. The majority
of published studies suggest no difference between these solutions, although there
are 2 publications, which suggest inferior outcomes in histidine-tryptophan-ketoglutarate-preserved
organs
17,18
especially with donation after circulatory disease (DCD) organs and longer preservation
times.
Cold ischemia time is an important factor in graft outcome, with a hazard ratio of
1.13 in the Donor Risk Analysis of Axelrod et al.
6
The combination of several risk factors (eg, older age, longer cold ischemia time,
DCD status) has a substantial impact of the likely outcome of pancreas transplantation.
There is clearly a strong argument to develop a means of preservation that reduces
the ill effect of longer preservation times.
The use of the “2-layer” method, whereby the organ is suspended at the interface between
University of Wisconsin solution and the oxygen carrier perfluorocarbon, is effective
in small animal models of pancreas preservation, in the context of islet isolation.
In human pancreases, this method also shows a benefit in organs initially preserved
in University of Wisconsin solution and those preserved for a prolonged time,
19,20
but has not been subjected to a randomized trial in solid organ transplantation.
Hypothermic machine preservation (HMP) has become increasingly popular in the preservation
of marginal and DCD kidneys and is of proven outcome benefit in the context of expanded
criteria donor organs. However, there is very little published information in the
pancreas. Leeser et al
21
carried out HMP in 4 human pancreases and demonstrated improved islet function after
isolation, but there are also concerns that HMP may be damaging to the very fragile
endothelium of the pancreas.
Oxygen delivery is increasingly recognized as important in organ preservation, particularly
of marginal organs. Experimental work in the porcine pancreas suggests that venous
oxygen persufflation at ice temperature effectively improves the viability of the
pancreas, and clinical studies are planned.
22
There is much interest in normothermic machine perfusion in the context of the lung,
22
kidney,
23
and liver.
24
Early attempts to perfuse the pancreas in the same way have been problematic, and
perfusions of more than a few hours have not proved successful in experimental models.
Two-layer preservation, persufflation, HMP, and normothermic machine perfusion are
all potential targets for clinical research studies, although the technical challenges
of machine perfusion (especially normothermic) are not yet sufficiently solved to
warrant clinical studies immediately. Key endpoints in such studies would need to
include surrogate markers of ischemia-reperfusion injury and reperfusion pancreatitis.
Immunosuppression
Immunosuppression after pancreas transplantation follows a consistent pattern in almost
all units. Induction therapy is almost universally used, with either nondepleting
antibody treatment (basiliximab) or depleting antibody treatment (thymoglobulin or
alemtuzumab). There is little systematic evidence as to which of these is better in
the context of pancreas transplantation, although there is evidence from kidney transplantation
that alemtuzumab is superior to basiliximab as a means of minimizing rejection in
low immunological risk patients and that alemtuzumab is equivalent to Thymoglobulin
with respect to rejection but may be superior with respect to postoperative infection.
There are several publications with respect to alemtuzumab in pancreas transplantation
25-27
but the only reported randomized trial is unpublished.
28
Although there has been no adequately powered randomized trial of induction therapy
in pancreas transplantation, it is unlikely that this would provide meaningfully different
guidance to that which is emerging from kidney transplant studies.
Steroid avoidance (or sparing) is particularly desirable in pancreas transplantation,
and there is evidence from several quarters that the use of alemtuzumab induction
enables this to be achieved safely.
Within maintenance therapy, the majority of units use tacrolimus-based immunosuppression;
cyclosporine is rarely used as primary therapy although it is used as a secondary
therapy in cases of tacrolimus intolerance. The importance of tacrolimus nephrotoxicity
(and β-cell toxicity) is debated, and there is a view among some clinicians that the
current low tacrolimus level regimens that followed the Symphony study have substantially
altered the risk profile of this drug.
29
However, many clinicians believe that the long-term use of calcineurin-inhibitor medication
does limit the lifespan of the kidney and that a non-calcineurin inhibitor maintenance
regime is desirable. This view is strongly supported by the findings of Budde et al.
30
Patients with an intermediate level of renal dysfunction pose a specific problem.
Such patients may not have a sufficient level of renal function to be able to tolerate
safely the incremental deterioration in renal function that my occur with tacrolimus,
but do not qualify for a combined pancreas and kidney transplant, being still some
years away from the need for dialysis. In this group of patients, trials of novel
immunosuppression would be of interest, although the numbers of patients that fulfil
the description make the design of a phase 3 trial challenging.
Innovative immunosuppressive regimes might include the use of belatacept and/or sirolimus.
Neither drug has the immunosuppressive potency of tacrolimus and would need to be
combined in such a way to provide adequate protection from rejection. The use of alemtuzumab
may achieve this, or possibly, the use of tacrolimus during the early, high-risk period
postoperatively. Kidney transplant trials of sirolimus have generally suggested that
this is a drug which may be best introduced at an interval after transplantation.
Belatacept has been tested in kidney transplant patients and shown to be nontoxic
and well tolerated, albeit with higher rejection rates than cyclosporine. The combination
of belatacept and sirolimus has recently been tested,
31
with and without the addition of donor bone marrow (which appeared to have little
effect on outcome in this small study). The association with posttransplant lymphoproliferative
disorder in Epstein-Barr virus-negative patients is of concern, although this is an
uncommon scenario in adults.
Further opportunities will come with the use of cell therapy. Early trials of T regulatory
(Treg) cell strategies are now in progress in kidney and liver transplantation, and
early-phase studies in the use of mesenchymal stem cells have generated optimism.
32
The pancreas may be a good environment for the phase 2 studies that such strategies
will need.
Immune Monitoring and Clinical Assessment of the Failing Pancreas Transplant
Monitoring the graft postoperatively is probably the greatest challenge in pancreas
transplantation. Particularly in solitary pancreas transplantation (in which there
is no donor-specific kidney to help with graft monitoring), graft surveillance is
very subjective—the move away from bladder drainage, although desirable for many reasons,
has removed a useful biochemical marker of allograft function. It is very likely that
this is an important factor in the higher rate of late graft loss in this group of
patients. Trials of immune monitoring are in progress in both kidney and pancreas
patients to try to identify an “immunological fingerprint” that predicts imminent
rejection.
33
Other approaches to immune surveillance include endoscopic biopsies—facilitated by
placing the graft in a more accessible location with the duodenal anastomosis to the
proximal jejunum or duodenum.
34,35
Percutaneous biopsy is carried out by many units, but generally as a diagnostic (ie,
confirmatory) test rather than for surveillance.
The hypothesis that portal venous drainage is preferable with respect to the alloimmune
response is unproven but may be a viable area for research particularly in the context
of more sophisticated methods of diagnosing an early immune response. A small number
of units routinely drain the pancreas into the portal venous system, although there
is a lack of good evidence for an outcome benefit.
36
The immunologic monitoring for pancreas transplant recipients has largely been directed
at conventional monitoring of the alloimmune response. Unfortunately, this monitoring
has been ineffective at monitoring the reoccurrence of the autoimmune response. Recipients
of pancreas transplants are presumed to be “preimmunized” in terms of having memory
autoreactive cells. To design more selective and effective (and possibly milder) immunosuppressive
approaches, it is important to establish validated techniques capable of accurately
monitoring both alloreactivity and autoreactivity. It is also important to recognize
that insulin resistance in the absence of an immune response can contribute to graft
failure. Along these lines, standardized metabolic testing will be essential to differentiate
between graft failures secondary to insulin resistance versus an autoimmune or alloimmune
response to treat reversible causes of endocrine insufficiency. Finally, an accurate
assessment of graft failure (resistance versus β-cell loss) will facilitate more accurate
definitions of graft function for utilization in registry data.
Pretransplant Screening of the Pancreas Transplant Recipient
The immunologic screening for recipients of SPK transplants, pancreas after kidney
transplants, and PTA is essentially directed at the same immunologic work-up as performed
for kidney transplantation. This work-up is only directed at the detection of alloantibody,
but there is literature that suggests that pretransplant work-up looking at markers
of autoimmunity should also be considered. The monitoring of the autoimmune response
can include the measurement of titers of autoantibodies associated with T1D, including
glutamic acid decarboxylase, IA-2, islet-specific glucose-6-phosphatase catalytic
subunit-related protein, and ZnT8. It is unclear what clinical relevance these autoantibodies
may have as it relates to whole organ transplants. However, there is evidence that
the detection of a cellular response against autoantigens before transplantation is
predictive of outcome (see below). Prescreening for the autoimmune response before
transplantation may have even a higher relevance of the scenario of retransplantation.
The immunologic screening before transplantations is an area which needs further investigation.
The presence of an autoimmune response before transplantation may guide the choice
of immunosuppression. Whether current methods of immunosuppression can block the memory
response associated with autoimmunity is uncertain.
Most transplant centers are evolving to the “virtual” crossmatch for screening compatible
donors. This requires single-antigen luminex beads to identify relevant anti-major
histocompatibility complex antibodies which should be avoided for a given donor. The
use of flow cytometry to detect T-cell and B-cell alloreactivity using flow cytometry
can also be used, but, for cost reasons, may be limited to highly sensitized recipients.
Currently, pretransplant immune monitoring is limited to the standard detection of
preformed alloantibody. The role of an additional screen for markers of autoimmunity
may provide further guidance with respect to immunosuppressive strategies and their
impact on the autoimmune response.
Assessment of the Failing Pancreas Allograft
Pancreatic allograft dysfunction can be a gradual process and is frequently asymptomatic.
It can be detected by a gradual escalation in Hgb A1C, or incidental elevations in
serum amylase and lipase. Unfortunately, it is frequently discovered by the new onset
of hyperglycemia. Early detection is imperative to identify reversible causes of graft
dysfunction. Broad consideration for the etiology of dysfunction include: alloimmune
rejection, recurrent autoimmunity, insulin resistance (T2D), chronic calcineurin inhibitor
toxicity, graft pancreatitis in the absence of rejection, cytomegalovirus, posttransplant
lymphoproliferative disorder, and bacterial or fungal infection. An algorithm to distinguish
between the above factors requires a standardized clinical assessment. The clinical
assessment should be triggered by incidental elevations in serum amylase and lipase,
a gradual increase in fasting blood sugars and/or an increasing HgbA1C or hyperglycemia.
The assessment should include an ultrasound of the pancreas with Doppler, and potentially
magnetic resonance imaging or computed tomography depending on the expertise at the
center. Blood cultures and cytomegalovirus cultures should be performed if there is
suspicion for an infectious etiology.
Although ultrasonography can provide indirect evidence of acute rejection, its most
important application is to guide a percutaneous needle biopsy using an 18 gauge core
biopsy needle. Poor visualization of the pancreas secondary to adjacent bowel or patient
body habitus may preclude transcutaneous biopsy. Occasionally, a suitable window can
be found with CT guidance, and rarely an open/laparoscopic biopsy is required. In
the absence of a biopsy, a suspicion for acute rejection can be made based on ultrasonography
findings and laboratory findings consistent with rejection (elevated lipase/amylase).
Gene signatures associated with rejection have been validated for both kidney and
liver transplantation, but not pancreas transplantation.
37
Of equal significance, the rejection “signature” appears before the clinical onset
of rejection. Serum markers for rejection in the scenario where a pancreas biopsy
is unattainable would be particularly helpful. Unfortunately, these markers need to
be validated for pancreas transplantation, and validating gene signatures for both
alloimmunity and recurrent autoimmunity should be done. The ability to pick up signals
of recurrent autoimmunity as well as the development of a de novo alloimmune response
before clinical deterioration would greatly facilitate the management of the pancreas
transplant recipient. The availability of tissue greatly facilitates the ability to
distinguish between the multiple etiologies leading to pancreatic graft dysfunction.
The Drachenberg/Banff guidelines for the diagnosis of rejection were recently updated
and will not be reviewed herein.
38
Early recurrent autoimmune disease can be identified by islet-centered lymphocytic
inflammation (isletitis), but more frequently late recurrent disease is associated
with the absence of insulin producing β cells using immunohistochemical stains.
Immune Monitoring of the Alloimmune and Autoimmune Response
There is an increasing amount of data demonstrating serologic markers of alloimmunity
and autoimmunity associated with pancreatic graft dysfunction. Again, it is unclear
what the significance of the autoantibodies (GAD, IA-2, IGRP, ZnT8) associated with
diabetes is in terms of the development of dysfunction. In islet transplantation,
the increase in these titers and epitope spreading was associated with graft loss.
39
Other reports could not find an association between autoantibody and islet graft outcome.
40
With regards to the findings of autoantibodies and pancreas transplantation, Sibley
41
found that there was no evidence of autoimmune recurrence in non-HLA identical recipients
and no islet cell autoantibodies. However, Bosi et al
42
found that 9 of 23 pancreas transplant recipients (non-HLA matched) developed ICA
antibodies and 7 of 9 went on to develop graft loss with 2 to 35 months after detection.
There have been more recent reports of increasing autoantibody titers and epitope
spreading in recipients of pancreas transplants that were associated with inferior
outcome.
43
Interestingly, there are an increasing number of reports suggesting that monitoring
the cellular response against autoantigens is strongly associated with graft dysfunction.
Much of this literature relate to islet allograft survival and will not be reviewed,
but is nicely summarized in a review from Abreu and Roep.
44
The assays for monitoring the cellular response to autoantigens have not been standardized,
but effective assays that can be validated and performed at multiple laboratories
will be an important advancement. The most elegant demonstration of recurrent autoimmune
disease after whole-organ pancreas transplantation came from Vendrame and demonstrated
the progression of allograft dysfunction associated with the development of autoantibodies
and a CD4− T-cell response against GAD.
43
These autoreactive cells were isolated from biopsies and detected and sorted using
class II tetramers. When these were cotransplanted with human islets into immunodeficient
mice, they caused diabetes. In the same report, other CD8+ lymphocytes reactive against
IGRP autoantigen were detected using class 1 pentamers. A more recent report from
Japan shows similar evidence for recurrent autoimmune disease after SPK transplantation
from a DCD donor.
45
Clearly, a standardized/validated approach to monitoring autoreactive cells with different
specificities will be important prognostically and will help guide the immunosuppressive
interventions to prevent graft loss after pancreas transplantation. At the same time,
these studies may provide insight into the etiology of late graft loss in the absence
of an alloimmune response. The tetramer-based assays are compromised by the large
volume of blood necessary to detect responsive cells. Other assays using major histocompatibility
complex multimers permit direct ex vivo quantification of autoreactive cells and require
significantly less blood.
46
This assay, the Diab-Q kit, requires low blood volumes, and its results correlated
with clinical outcome in islet recipients. Another monitoring strategy determines
the cellular response against overlying peptides of the autoantigen GAD.
47
Although this assay has not been validated, it is attractive in that the assay is
not HLA restricted.
Insulin Resistance
Finally, pancreatic graft insufficiency/failure may be related to the development
of insulin resistance in the absence of alloimmunity or autoimmunity. In these cases,
recipients may have a gradual increase in fasting glucose, or a gradually escalating
Hgb A1C. This may be related to weight gain, steroid use, or calcineurin inhibitor
toxicity. Because all of these may be reversible, it is important to determine whether
increases in fasting glucose and Hgb A1c are related to insulin resistance. The gold
standard for determining insulin resistance is a clinical research center-based assessment
using the euglycemic-hyperglycemic clamp studies. These studies are labor intensive,
and require a stay in a clinical research center, thus are not adapted to office-based
assessment of pancreas transplant function. However, the homeostasis model assessment
of insulin resistance is gaining widespread use as a result of its simplicity and
validity and is based on fasting blood glucose (FBG) and insulin levels.
48
The homeostasis model assessment score correlates well with the euglycemic-hyperglycemic
clamp in terms of assessing insulin resistance after pancreas transplantation. The
detection of insulin resistance is important, in that it may be reversible by altering
immunosuppression, weight loss, addition of oral hyperglycemic agent, or glucagon-like
peptide (GLP)-1 agonists.
In summary, a complete clinical assessment of the failing pancreas graft should differentiate
between alloimmunity, recurrent autoimmunity, and the development of insulin resistance.
Because treatment for each of these etiologies leading to poorer glycemic control
requires different strategies, appropriate immunomonitoring and metabolic testing
may be able to identify reversible causes for graft dysfunction and loss. Standardization
of these noninvasive assays for monitoring the alloimmune and autoimmune responses,
as well as metabolic testing for insulin resistance, will provide essential data in
terms of early intervention to prevent graft loss. Nonetheless, the validation of
noninvasive markers of recurrent autoimmunity as well as metabolic tests for insulin
resistance represents a major “gap” in pancreas transplantation. At the same time,
validated tests to differentiate between causes for graft insufficiency/failure will
be extremely useful in terms of accurately reporting outcomes of pancreas transplantation.
Prospective Randomized Comparison of Pancreas and Islet Transplant Outcomes With Best
Medical Therapy
Prolonged insulin independence (47% at 3 years) has been demonstrated in the modern
era of islet transplantation.
49
In addition, selected groups have reported 50% or greater 5-year insulin independence
rates in islet transplants,
50
thus comparing it favorably to solitary pancreas transplants. This comparison, despite
its weaknesses, compels us to strongly consider a prospective randomized trial comparing
pancreas and islet transplants.
There are obvious challenges to designing and conducting this trial—when is the right
time (pending Food and Drug Administration [FDA] application for registration of islets
as a therapeutic agent), what is the design—randomization points and patient counseling.
Early randomization could lead to a high patient dropout rate, late randomization
may lead to patient apprehension about not knowing whether they would undergo major
surgery). Several additional questions arise, particularly reimbursement issues.
Inclusion and exclusion criteria could be controversial. The typical islet recipient
(low body mass index, low insulin requirements, early or no secondary complications)
is not the typical pancreas transplant recipient. Whether patient selection as currently
performed would need to change to enable a clinical trial is an area of uncertainty.
Another important consideration is whether the best medical therapy should be compared
with both transplant options. Can we get the patients and endocrinologist investigators
on board?
Despite these challenges, if this trial can be performed credibly and successfully,
it will go a long way in answering key questions that could shape the trajectory of
both pancreas and islet transplants in the future. In addition, it will provide a
unique opportunity for cross-fertilization and collaborative efforts between investigators
that have been previously focused solely in the pancreas or islet transplant fields,
as well as the endocrinology community.
Secondary Complications and Mortality
The impact of pancreas transplantation on secondary complications of diabetes has
been documented by several investigators. Most notably, in patients with T1D mellitus
who did not have uremia and have not received a kidney transplant, pancreas transplantation
did not ameliorate established lesions of diabetic nephropathy within 5 years after
transplantation, but did so at 10 years posttransplant.
51
Improvement in motor, sensory, and autonomic indices in patients with diabetic neuropathy
after pancreas transplantation has been reported.
52
At the Manchester Royal Infirmary, 20 SPK transplant recipients were studied before
and 6 months after SPK transplantation; these were compared with 15 normal volunteers
using retinal confocal microscopy: SPK transplant recipients compared with normal
controls had a marked decrease in nerve fiber morphometry and nerve fiber length and
density dramatically improved.
53
Thus, the beneficial effect of pancreas transplantation on diabetic retinopathy seems
highly logical. However, studies to date do not use strict case-control method comparing
transplanted patients versus untransplanted controls. Also, risk factors, such as
blood pressure, baseline degree of disease, renal function, and type of immunosuppression,
are not controlled.
Does Pancreas Transplantation Protect the Kidney Transplant?
The effect of pancreas after kidney (PAK) on the kidney transplant has been marred
by controversy due to lack of clear controlled data. One United States Renal Data
System study of PAK in diabetic kidney recipients showed PAK was protective of renal
function in all groups, and documented that a GFR between 30 and 39 mL/min was a risk
factor for kidney failure after PAK.
54
In a long-term study (5 years) of the efficacy and safety of pancreas transplantation
alone, it was shown that in 51 patients with sustained pancreas transplant function,
kidney function (serum creatinine and glomerular filtration rate) decreased over time
with a slower decline in recipients with pretransplant filtration rate less than 90
mL/min.
55
Live donor kidney (LDK) transplant alone has been shown to provide a survival advantage
in T1D patients compared with deceased donor transplantation. Moreover, recipients
of an SPK transplantation had statistically significant patient and kidney graft survivals
compared with those T1D patients who received a kidney transplant alone.
56
Interpretation of Young's data is difficult because the donors for those who received
a deceased donor kidney (DDK) alone were significantly older than the donors of SPK
transplantations. Nevertheless, it appears that kidney survival after multivariate
analysis appeared to be superior when a pancreas transplant was performed simultaneously.
It has been shown that early pancreas graft failure in SPK transplant recipients is
associated with an increased risk for subsequent kidney failure and death.
11
Patients with end-stage renal disease and T2D have been shown to benefit from SPK
transplantation in a selected series. A commonality of most of these series is that
the T2D recipients were not obese and did not have excessive insulin requirements.
Thus, lean, insulinopenic T2D have similar outcomes as T1D recipients of an SPK transplant.
Sampaio et al
57
showed that T2D recipients of SPK transplants were not at increased risk for death,
kidney failure, or pancreas failure when compared with recipients with T1D.
Attempts to document clear survival benefit after pancreas transplantation have been
hampered by limitations and controversy. A large study looking at 4-year survival
for transplanted patients versus those on the waitlist suggested a survival benefit
in SPK transplantation but a survival disadvantage in PAK and PTA.
7
However, the same population was reanalyzed by a different group showing survival
benefit for all groups of pancreas transplants—SPK, PAK, and PTA.
58
Thus, studies on the effect of pancreas transplantation on patient survival have been
affected by the lack of control for renal function, lack of comparability of study
groups: SPK versus DDK versus LDK. They have been highly impacted by selection bias
(choosing ideal patients for transplantation) and not controlling for the effect of
renal function over time. Further, patient risk factors allocated to choice between
SPK, DDK, and LDK are not the same.
Proposal for a Randomized Trial in Pancreas Transplantation
Do we need a randomized trial comparing SPK versus kidney transplant alone? There
are significant factors that would support the rationale for such a trial. Animal
studies of benefit are compelling. Nevertheless, the best medical therapy of diabetes
has improved dramatically with progressive reductions in diabetes related mortality
and improvement in quality of life. Studies in the past asserting SPK transplant's
benefits are poorly controlled especially for renal function. Case selection bias
inherent in prior studies can be overcome. Outside the transplant community, pancreas
transplantation is not accepted, particularly among endocrinologists. This is further
complicated by the fact that the endpoint of pancreas transplant function has never
been clearly defined. It is acknowledged that among members of the pancreas transplantation
surgical community that pancreas transplantation is effective at reducing mortality
and arresting secondary diabetic complications. As such, most would suggest that the
time has passed for a randomized trial of SPK transplantation versus kidney transplantation
alone. A strategy for analyzing the impact of transplantation-based β-cell replacement
on mortality and renal function (as well as other secondary complications) would be
for a head-to-head randomized trial of pancreas and islet transplantation. Recent
reports of near equivalence in the 5-year graft survival of PTA and islet transplant
alone (ITA) make a head to head trial appropriate. Based on these considerations,
the following trial is proposed comparing pancreas and islet transplantation. The
SPK transplantation could be compared with simultaneous islet kidney (SIK) transplants.
In this head-to-head trial, the assumption will be made that no control group receiving
a kidney alone is feasible both because the vast majority of the transplant community
consider kidney transplant alone inferior to SPK transplantation. Moreover, patient
acceptance and knowledge of SPK transplantation is very high. Consequently, enrollment
in a trial comparing SPK and SIK transplantations to kidney transplantation alone
is likely infeasible. However, nonuremic diabetics receiving either ITA or PTA or
previously kidney-transplanted diabetics receiving a islet-after-kidney transplant
(IAK) or PAK could be randomized to a third arm of untransplanted controls. In the
case of IAK and particularly PAK, there remains significant scientific disagreement
regarding the potential benefit of pancreas transplantation on mortality, future renal
function, and other secondary complications.
Summary of Research Priorities
(1) Develop carefully designed, well-controlled clinical trials that define the impact
of pancreas transplantation on mortality and secondary complications, particularly
renal function. Developing validated measures of pancreatic organ function will be
required for success.
(2) The greatest obstacle to growth of pancreas transplantation is low organ utilization
rates. Preservation strategies leading to improved early graft survival and function
and increased utilization are needed.
(3) Concern about new onset and recurrent renal dysfunction markedly limits growth
of pancreas transplantation. Clinical trials of non-nephrotoxic immunosuppression
are needed.
(4) Detailed clinical studies verifying the risk of recurrent disease are needed to
exclude other causes, such as β-cell exhaustion and alloimmune causes. Refine immunologic
detection and prevention strategies against recurrent autoimmunity.
Two parallel randomized trials of whole organ pancreas versus isolated islet transplantation
are recommended: (a) SPK versus SIK, and (b) PTA and PAK versus ITA and IAK versus
best medical therapy.
ISLET ALLOTRANSPLANTATION
Current State of the Field
Over the last 10 years, islet allotransplantation has developed into an established
treatment modality for subjects with T1D complicated by hypoglycemia unawareness,
and the procedure is currently reimbursed for this indication in several countries.
At present, the primary goal of islet transplantation should be optimal glycemic control
without severe hypoglycemia, rather than insulin independence. Importantly, this must
be routinely achievable with a single islet infusion. A standardized approach to evaluation
of clinical outcomes will be essential for further developments in β-cell replacement.
A recently completed multicenter prospective phase 3 study
59
demonstrated that:
(1) Islets can be manufactured reproducibly at multiple sites using a common manufacturing
process.
(2) Independence from exogenous insulin can be achieved in about half of islet recipients
at one year from infusion, with 1 or 2 infusions needed.
(3) Glycemic control is excellent even when insulin independence is not achieved.
(4) Hypoglycemia unawareness is treated effectively by islet transplantation, with
associated freedom from severe hypoglycemic events.
Islet allotransplantation is also an acceptable therapy for patients with end-stage
renal failure and T1D, either simultaneously with or after kidney transplantation.
60
A comparison between islet and pancreas transplantation in combination with a kidney
transplant demonstrated achievement of similar HbA1c levels in the 2 groups. Islet
recipients were less likely to achieve insulin independence, whereas pancreas recipients
had substantially greater procedure-related morbidity.
61
Because of the limited overall availability of human organ donors, islet allotransplantation
is unlikely to provide a cure for all those affected with uncomplicated T1D, unless
islet expansion becomes a reality. In addition, “closed-loop-systems” using implantable
glucose sensors to control insulin administration may enable good metabolic control
without the need for systemic immunosuppression in uncomplicated T1D.
62
However, we fully anticipate that improvements in the outcome of islet transplantation
will, within 5 to 10 years, make islet transplantation an appropriate therapy not
only for the current indications of hypoglycemia unawareness and grossly unstable
glycemic control, but also for all people currently considered eligible for pancreas
transplantation. Many of the recent and future advances in islet allotransplantation
will benefit clinical islet transplantation overall in the future. This applies whether
stem cell or xeno sources are used as the alternative cell source and/or immune tolerance
or immunoisolation protocols are used to obviate the need for immunosuppression. To
achieve these goals, however, a number of obstacles need to be overcome.
Pancreas Allocation
In most countries, allocation of donor pancreases for whole pancreas transplantation
still takes priority over pancreases for islets.
63
Clearly, if reimbursement for islet transplantation is implemented, parity of organ
allocation is essential for the islet transplant service to be effectively delivered.
The United Kingdom has pioneered a joint pancreas allocation system which is currently
being evaluated. It is based on a point system and organs allocated to the matched
patient at the top of the joint waiting list, regardless of whether they are listed
for whole organ or islets. Overall, it is important that any decision to allocate
donors with specific characteristics preferentially to pancreas or islet transplantation
should be based on rigorous studies of clinical outcomes.
“Competition” Between Whole Pancreas and Islet Transplant
Whole pancreas and islet transplantation are still often seen as competing therapies.
64
This is not only the case for organ allocation, but also for patient referral and
patient selection. Using analysis of stratified outcome data, we need to work toward
unified, joint programs of β-cell replacement, with treatments tailored to individual
patients, rather than treatments being largely dictated by referral patterns and physician
preference. We anticipate that, within 5 to 10 years, islet transplantation will be
the preferred therapeutic procedure for β-cell replacement, as a result of the metabolic
efficiency and the superior safety profile of the islet in relation to pancreas transplantation.
However, in the meantime, it is essential that these 2 different modalities are considered
complementary, rather than in direct competition.
Funding and Reimbursement
The encouraging results of islet transplantation over the past decade mean that, from
a clinical point of view, islet transplantation can rightly now be considered as a
clinical treatment rather than an experimental tool. However, for this transition
to be fully realized in terms of islet transplantation becoming a standard therapy
worldwide, full reimbursement by health care providers needs to be implemented. Although
this has been achieved in a number of countries around the world (eg, United Kingdom,
Switzerland, Canada, and so on), this remains an ongoing challenge in many countries
including the United States.
Regulation
Over the last decade, islet isolation has become a highly regulated procedure in most
countries. This has added enormously to the cost and complexity of the isolation procedure
and as a result has propagated the importance of islet transplant networks, in which
1 or 2 centers isolate human islets within “state of the art” designated isolation
facilities for a network of implanting centers.
The degree of regulation of pancreatic islets and the pathway to licensure for an
islet product vary in different countries.
65,66
However, all islet isolation and islet transplant teams must work with their regulatory
bodies to ensure that islet personnel are closely involved in the development and
interpretation of the cell processing regulations, rather than them simply having
them imposed from the outside.
The Research Agenda
Pancreas Procurement, Preservation, and Islet Transport
To expand the donor pool, novel strategies of optimized pancreas procurement, pancreas
preservation, and islet transport are essential. First, a unified approach to optimal
pancreas retrieval during organ procurement is essential, regardless of whether the
pancreas is being procured for whole organ or islet transplantation. Indeed, several
studies have demonstrated that a dedicated surgical team currently is the most important
determinant for the clinical outcome of islet allotransplantation.
67
Second, research into the optimal approach to pancreas preservation, whether persufflation,
normothermic perfusion, or perfluorocarbon incubation
68-70
is vital (see previous section on whole organ transplantation). Finally, the stringent
regulation associated with human islet isolation described above, means that islet
isolation/islet transplant networks will increasingly become the norm. Ideally, each
islet isolation facility should support a population of 10 to 20 million. However,
for networks to realize their full potential, optimization of islet transport and
more efficient systems for islet shipping need to be developed.
Optimization and New Strategies for Islet Isolation
Although great progress has been made in standardization of islet isolation, significant
improvements are still needed. The pancreas digestion step, in particular, remains
an empiric undertaking, dependent on the relatively haphazard interaction of administered
bacterial enzymes with the endogenous enzymes of the donor pancreas. In addition,
currently the same techniques are used regardless of the huge variability in human
donors. Research efforts should focus on understanding the detailed molecular ultrastructure
of the pancreatic islet-exocrine matrix in the full range of donors (age, BMI, and
so on)
71
and on developing new, targeted clinical grade enzyme (recombinant) blends that can
be used on all available donor pancreases.
72,73
Research should also continue into the selective inhibition of the activated pancreatic
enzymes. In parallel, alternative, nonenzymatic technologies for cell separation,
for example, photodynamic technologies, and so on, should be investigated.
74,75
Islet Preconditioning and Culture
The introduction of a pretransplant period of islet culture has been hugely beneficial
from a logistical (patient work-up, radiology, and so on), physiological (islet recovery,
and so on), and regulatory (functional and safety release criteria) perspectives.
However, this period of islet culture also presents an opportunity for novel interventions
before islet implantation which could provide exciting opportunities to improve clinical
outcomes. Potential strategies include coating with compounds that promote oxygenation
and islet engraftment,
76
compounds that reduce instant blood-mediated inflammatory reaction (IBMIR),
77,78
compounds that enable “in vivo” islet imaging,
79,80
compounds that secrete local immunosuppression,
81,82
and compounds inducing protection against hypoxic stress.
83
These approaches are collectively termed “islet preconditioning” and clearly require
close collaborations with those in the fields of nanotechnology and tissue engineering.
The role of peri-islet “scaffolds” is another area needing extensive research, with
exploration of the associated issues of islet-exocrine interactions, for example,
paracrine influences, signaling, and so on.
84-86
Moving such developments through regulatory agencies is likely to be complex, because
they involve cellular products, devices, and drugs. The issue of commercialization
of human cells is also problematic. Scientific international organizations, such as
IPITA and TTS, together with patient advocate groups should engage in facilitating
discussions with regulatory bodies to make these developments possible.
Defining the Islet Product
The current approach to establishing identity, potency, and purity of manufactured
islet remains crude and variable between centers. Indeed, there is currently no basis
for predicting clinical outcomes based on product characterization. Improved measures
would facilitate more stringent release criteria as well as enabling meaningful comparisons
between centers and for the purposes of rigorous scientific studies.
87
Strategies to Stratify Recipient in Terms of High Risk Versus Low Risk for Islet Survival/Function
The development and implementation of reliable and accurate methods to stratify recipients
according to pretransplant predictors of high risk versus low risk for islet survival/islet
function (eg, immunologic and metabolic signatures)
88
should enable a personalized approach to the optimization of clinical outcomes after
islet transplantation.
Novel Sites for Implantation
Although the liver is easily accessible and has a number of advantages compared with
some other anatomical sites, it is probably not the “ideal site” for islet implantation.
Research should continue to focus on identifying alternative implantation sites
89
which will ensure that islet transplantation remains a straightforward, well-tolerated,
minimally invasive procedure, but that ensures improved islet graft survival and optimal
glycemic control.
“In Vivo” Islet Imaging and Biomarkers for Islet Survival and Rejection
The development and implementation of reliable and accurate methods for “in vivo”
islet imaging
90-92
is essential to obtain a better understanding of clinical outcomes, that is, islet
engraftment, the gradual loss of function, evaluation of the optimal site, and technique
for implantation. In addition, identification of a panel of validated biomarkers within
the blood would greatly enhance our understanding of islet graft function.
Strategies to Minimize the Need for Systemic Nonspecific Immunosuppression and Eventually
Induce Tolerance
Several approaches are supported by strong preclinical results and are close to being
introduced in clinical studies. Approaches currently being introduced in clinical
studies include
93
: cotransplantation of mesenchymal stem cells (MSCs) or Treg cell; islet pretreatment
(anti-HMGB1, islet transduction to facilitate engraftment, and inflammation); islet
encapsulation (both macroencapsulation and microencapsulation—see below); recipient
treatment using nondepleting monoclonal antibodies (mAbs). Efficient modalities found
in experimental studies should be appropriately applied to preclinical study. The
obviation of immunosuppression would be the quantum step required to enable islet
allotransplantation to be implemented in younger patients, including children. It
must be remembered that this remains the ultimate aim of islet transplantation.
Summary of Research Priorities
(1) Optimization of pancreas procurement, pancreas transport, and development of targeted
methods for islet isolation to improve functional islet yield to permit routine single-donor
insulin independence
(2) Standardization of definition of released islet product to enable accurate comparisons
between centers and enable accurate prediction of islet graft outcome.
(3) Development of novel strategies for islet preconditioning to improve islet engraftment
and islet graft longevity.
(4) Definition of suitable alternative anatomical sites for islet implantation.
(5) Strategies to minimize or eliminate the need for immunosuppression, enabling the
ultimate goal of islet allotransplantation to be reached, that is, the transplantation
of children
ISLET XENOTRANSPLANTATION
Current State of the Field
Progress made in human islet transplantation in the past 15 years has made commonplace
the restoration of near-normoglycemia, insulin independence, and protection from severe
hypoglycemia with a low risk of procedural complications in immunosuppressed T1D recipients.
49
These favorable outcomes can now be achieved in single-donor islet transplant recipients,
both with refined induction therapy
94
and calcineurin inhibitor-sparing maintenance immunosuppression.
95,96
Increasing evidence indicates that insulin independence can be maintained in more
than 50% of recipients for 5 years
50
and that islet transplants have slow progression of microvascular complications.
97
Improving human islet allotransplant efficacy and safety outcomes have inspired investigators
to develop more widely available cell-based diabetes therapies. Preclinical safety
and efficacy data obtained in the last 10 years in the stringent pig-to-nonhuman primate
(NHP) islet transplant setting,
98-109
and preliminary safety data obtained in recent pilot clinical trials,
110
suggest that xenogeneic pig islets can possibly be developed into an islet β-cell
replacement therapy with broad applicability. However, several hurdles remain to be
overcome.
In the preclinical pig-to-NHP model (Table 1), several groups have achieved insulin
independence for longer than 180 days with near-physiologic control of fasted and
postprandial glucose in a small number of NHP after porcine islet xenotransplantation.
98-103,106-109
Except for 1 report
101
that demonstrated long-term survival of embryonic porcine pancreatic tissue transplanted
to the omentum of 2 monkeys immunosuppressed with anti-thymocyte globulin (ATG), anti-IL-2R,
anti-CD20, belatacept, everolimus, and FTY720, in all other studies that achieved
long-term diabetes reversal, adult islets or neonatal islet cell clusters (NICC) were
transplanted intraportally into monkeys in whom the rejection prophylaxis involved
induction or both induction and maintenance immunosuppression with CD40-CD154 costimulation
pathway blocking antibodies. Although several studies show the efficacy of anti-CD154
antibodies in preventing islet xenograft rejection,
98-100,103,106,107
1 report indicates that prolonged islet xenograft survival can also be achieved with
antagonistic anti-CD40 antibodies.
102
TABLE 1
Published Studies Demonstrating Prolonged Insulin Independence After Pig Islet Transplantation
in NHP With Surgical or Streptozotocin-Induced Diabetes
Genetic engineering of donor pigs mitigates the IBMIR to intraportally transplanted
pig islets.
103,111
The IBMIR is a major obstacle to engraftment of intraportal porcine islet xenografts
in primates
112
; it is triggered by the contact of isolated islets with blood and causes islet destruction
by complement and coagulation activation products and other inflammatory mediators
released by recruited neutrophils and monocytes.
113-116
Intraportal transplantation of galactose-α 1,3-galactose (αGal)-deficient NICC from
galactosyl transferase knockout (GT-KO) donor pigs
117
increased achievement of insulin independence in rhesus macaques when directly compared
with wild-type (WT) NICC.
103
The improved engraftment of αGal-deficient NICC was likely due to reduced antibody
and complement binding as well as complement-dependent destruction. Profound IBMIR
was also triggered by WT NICC in baboons, with intravascular clotting and graft destruction
occurring within hours.
111
In contrast, and without directly targeting coagulation, IBMIR was minimal, and intravascular
clotting was not observed in baboons after transplantation of NICC from αGal-deficient
porcine donors transgenic for the human complement regulators CD55 and CD59. The extent
that the transgenes CD55 and CD59 contributed to the protection of αGal-deficient
NICC from IBMIR was not directly addressed. The transgenic expression on adult porcine
islets of human CD46, another complement regulatory factor, had little impact on IBMIR
but was effective in limiting antibody-mediated rejection.
100
There is no additional evidence, as of now, of prolonged survival of GM porcine islets
in NHP.
118
Xenografts of αGal-deficient NICC in NHP were not protected from eventual cellular
rejection.
103
Also, αGal-deficient NICC transgenic for hCD55 and hCD59 underwent cell-mediated rejection
within 1 month after transplantation into baboons immunosuppressed with a costimulation
blockade-sparing protocol, including ATG, tacrolimus, and MMF.
111
The NICC with islet β cell–specific expression of LEA29Y, a high-affinity variant
of the T-cell costimulation inhibitor, CTLA4-Ig, were protected from cell-mediated
rejection in humanized mice.
119
Adult porcine islets with expression of CTLA4-Ig, under the control of the porcine
insulin gene promoter, have recently been transplanted into NHP; however, the study
was not designed to test the immune-protective characteristics of the CTLA4-Ig transgene.
107
Encapsulation facilitated restoration of insulin independence longer than 180 days
in 3 porcine-to-NHP islet transplant studies. Intraperitoneal transplantation of adult
pig islets in alginate-polylysine-alginate microcapsules rendered 7 of 9 spontaneously
diabetic monkeys insulin-independent for periods ranging from 120 to 804 days with
FBG levels in the near-normoglycemic range.
120
Macroencapsulation of adult porcine islets in alginate and transplantation into abdominal
subcutaneous tissue as an islet monolayer on an acellular collagen matrix in a macrodevice
maintained FBG levels less than 150 mg/dL for 20 to 28 weeks in 5 streptozotocin-diabetic,
nonimmunosuppressed cynomolgus monkeys; 2 of the 4 control monkeys that received microencapsulated
adult porcine islets under the kidney capsule showed FBG levels less than 150 mg/dL
for up to 2 weeks.
108
A subsequent report by the same investigators showed that coencapsulation of islets
with MSCs slightly improved oxygenation and neoangiogenesis of subcutaneously placed
implants and maintained FBG levels in the near-normal range for up to 32 weeks in
nonimmunosuppressed monkeys; however, the cotransplanted MSC did not substantially
improve or prolong islet xenograft function.
109
The first clinical trial of porcine islet xenotransplantation under a comprehensive
regulatory framework was performed in New Zealand after the authorization by the Minister
of Health under a specific section of the New Zealand Medicines Act, and also after
thorough review performed by the New Zealand Medicines and Medical Devices Safety
Authority, Medsafe, in consultation with the National Health Research Council and
international referees.
110,121,122
This open label, safety, and dose finding phase I/IIa study of microencapsulated neonatal
porcine islets, prepared under GMP from designated pathogen-free donor animals and
transplanted intraperitoneally in 14 nonimmunosuppressed subjects with unstable T1D,
demonstrated the microbiological safety of the tested encapsulated porcine islet product.
123
There was no apparent dose effect of porcine islets, and porcine C-peptide was not
measurable in the serum of any of the transplanted subjects.
110
Nevertheless, the reduced frequency of unaware hypoglycemic events, the lower HbA1c
levels, and the up to 30% lower daily insulin requirements observed in some of the
subjects posttransplant provided indirect and preliminary evidence of islet xenograft
function.
110,122
The same encapsulated porcine islet product was subsequently tested at doses of 5000
IE/kg and 10 000 IE/kg in a phase IIa efficacy trial in 8 subjects with T1D and hypoglycemia
unawareness in Argentina with authorization by the Minister of Health and approval
by the local bioethical committee.
122
This trial confirmed the microbiological safety of the porcine islet product and demonstrated
lower insulin requirements, frequency of unaware hypoglycemic events, and HbA1c levels
in most subjects compared with pretransplant.
Obstacles to Application of This Therapy
Although substantial progress has been made in islet xenotransplantation, 2 significant
obstacles to clinical application remain.
First, a clinically applicable immunosuppressive protocol for preventing rejection
of porcine islet xenografts is currently not available.
118
Regimens that protect islet allografts in NHP and humans from rejection, including
basiliximab combined with FTY720 and everolimus
124
and antithymocyte globulin combined with tacrolimus and mycophenolate mofetil,
125
fail to facilitate long-term islet xenograft survival in NHPs,
98,111
indicating that either the immunity to islet xenografts is stronger than islet allografts
or that additional immune recognition and effector pathways are operative in xenoislet
compared with alloislet transplantation. As summarized above, prolonged porcine islet
xenograft survival has been achieved with protocols based on CD154-CD40 costimulatory
blockade by several groups. However, long-term functional survival exceeding 180 days,
the efficacy milestone to be met in 5 or more of 8 NHP before initiating clinical
trials according to the consensus statement of the International Xenotransplantation
Association,
126,127
has been demonstrated in only a small proportion of transplanted NHPs (1 to 2 of 3
to 7 in several studied cohorts; Table 1).
Lack of more consistent success could be ascribed to failure of the protocol to prevent
rejection or to failure of the preclinical model to accommodate the protocol.
128
Therefore, continued understanding and improvement of the preclinical animal model
is a critical requirement for documenting long-term success on a consistent basis.
Long-term success has also been precluded by a high proportion of engraftment failure
after porcine islet transplantation due to IBMIR and complement-mediated lysis of
islets triggered by binding of preformed xenoreactive antibodies. Engraftment of neonatal
porcine islets has recently improved considerably with the use of GT-KO donors.
103,111
Early posttransplant loss of adult islets from GT-KO can be very significant,
107
possibly due to the activation of complement by transplanted islets via the alternative
pathway.
106
As shown in human islet allotransplantation,
50,129
increasing the proportion of transplanted porcine islets that stably engraft will
substantially improve long-term islet graft function.
Several immunotherapeutics with demonstrated efficacy in inhibiting immune responses
to porcine islet xenografts are no longer available for clinical investigation in
transplantation due to their safety profile. These include in particular anti-CD154
antibodies and also FTY720, anti-LFA-1 antibodies, and LFA-3-Ig. Alternative strategies,
such as antagonistic anti-CD40 antibodies, are being investigated; however, none of
these investigational antibodies will be available for clinical research in islet
xenotransplantation in the very foreseeable future.
The risks of immunotherapeutic protocols being developed for initial pilot clinical
trials must not be higher than the risks associated with immunosuppression currently
used clinically in organ and islet allotransplantation. For islet xenotransplantation
to be applied very broadly in the future, a rejection prophylaxis with a very favorable
safety profile will be required. It is reasonable to assume that immunotherapeutic
protocols will become increasingly more selective and safe as our understanding of
the immunobiology of islet xenotransplantation improves.
The second major obstacle to broader clinical application of islet xenotransplantation
is the high number of designated pathogen-free donor pancreases required for manufacturing
a therapeutic patient dose of neonatal porcine and adult pig islets. The minimum number
of nonencapsulated pig islets transplanted to achieve normoglycemia in NHP has been
50 000 IEQ/kg or greater for neonatal islets
99,102,103
and 25 000 IEQ/kg or greater for adult islets,
98
though much greater numbers have been transplanted in some studies.
100,106,107
Fewer porcine IEQ are expected to be required per kg body weight of human recipients,
whose insulin requirements per kg are 2- to 3-fold lower compared with streptozotocin-diabetic
monkeys.
128,130
The exact number of porcine islets required to restore insulin independence in humans
with T1D remains uncertain, but assuming a yield of 400 000 IEQ from a good adult
porcine pancreas,
131
3 or more suitable adult pancreases would be required for 1 patient dose. Manufacturing
of NICC is much less challenging and costly and islets from neonatal pigs maintain
a proliferative capacity after transplantation; however, the number of donor pancreases
expected to be required per patient dose with commonly used techniques
132
is considerable. Further improvements in the selection of suitable source pigs and
in porcine islet manufacturing will need to be achieved to develop commercially viable
adult and of neonatal porcine islet therapy products.
Housing of designated pathogen-free pigs under strict barrier conditions will require
significant resources. As the islets will be isolated and cultured, likely for several
days, testing of the islets alone (ie, the ‘product’) for the presence of microorganisms
should be sufficient to ensure that no bacteria and fungi will be transmitted, although
monitoring of the herd will be required to ensure no viruses are present.
133-135
The requirement of designated pathogen-free porcine donors may therefore not be as
stringent for islets compared with organ xenotransplantation. Inevitably, porcine
endogenous retroviruses (PERV) will be transplanted with the islets.
136
However, monitoring of humans exposed to various pig tissues and cells has never identified
active PERV replication.
137-140
Although national regulatory authorities, for example, the Food and Drug Administration
in the United States, will insist on monitoring for PERV, clinical xenotransplantation
is unlikely to be precluded on the basis of the presence of PERV alone.
141
Furthermore, if essential, techniques of small interfering RNA could successfully
prevent PERV activation after transplantation.
142
The Research Agenda
The research agenda in nonencapsulated islet xenotransplantation focuses on meeting
the key requirements for initiating pilot clinical trials, that is, prevention of
IBMIR and prevention of cell-mediated rejection with anti-CD154 sparing and clinically
applicable immunosuppression.
The IBMIR is a multifaceted process involving coagulation, complement activation,
cytokine and chemokine release, and granulocytes/monocyte infiltration.
115,116
Many different strategies have been investigated. Targeting coagulation using low-molecular
weight dextran sulfate alone
114
and in combination with tissue factor pathway inhibitor-transgenic islets,
107
and thrombomodulin
111
has at best mitigated but not prevented IBMIR in preclinical islet xenotransplantation.
Strikingly, the use of neonatal islets from GT-KO porcine donors has been more successful,
103,111
pointing to the importance of complement activation via the classical pathway caused
by binding of preformed xenoreactive antibodies to islets. It is probable that preformed
antibody binding to other (ie, nonGal) antigens will also be an initiating factor
in complement activation after porcine islet transplantation in humans. To date, the
identity of nonGal antigens on porcine islets has not been fully determined. N-glycolylneuraminic
acid is likely to be a target in clinical islet xenotransplantation, though this oligosaccharide
is not important in pig-to-NHP islet transplantation because NHP also express it and
therefore do not produce natural antibodies against it.
143
Pigs that express neither Gal nor N-glycolylneuraminic acid are now available for
preclinical research.
144
Recent evidence demonstrated the contribution of the alternative complement pathway
to IBMIR and the mitigation of early loss of adult porcine islets in NHP treated with
human factor H.
106
In view of increasing evidence indicating the activation of classical and alternative
complement pathways, complement-specific biologics, such as compstatin that inhibit
both pathways appear to be promising interventions.
116,145
For the control of inflammatory cytokines/chemokine axis, biological drugs, such as
anti-TNF-α mAb (humira and infliximab), TNF receptor-Ig fusion protein (sTNFR, etanercept),
and IL-1β receptor antagonist (anakinra) are available for off-label use.
146
Finally, the CXCR1/2 allosteric inhibitor, reparixin, has been shown to inhibit the
infiltration of neutrophils into islet grafts in experimental and clinical islet allotransplantation.
147,148
An alternative approach to preventing IBMIR would be to place the islets in a site
where they are not immediately exposed to blood. The gastric submucosal space has
the advantage of being accessible by endoscopy,
149,150
and endoscopic biopsy of the graft may be possible.
150
Other sites are also being explored.
151-153
Taken together, because the IBMIR is a multifaceted phenomenon, the combination of
interventions targeting coagulation, complement activation, and recruitment of neutrophils
and monocytes will be required to ameliorate early graft loss and prolong the graft
survival in porcine-to-human islet xenotransplantation.
Anti-CD154 antibody-based immunosuppressive protocols achieved long-term survival
of WT and genetically modified porcine islets in several NHPs. For the clinical translation
of this immunosuppressive protocol, the replacement of anti-CD154 mAb is required
due to its associated risk of thromboembolic events.
154
Preventing cell mediated rejection of porcine islets using anti-CD154 antibody-sparing
protocols will likely require detailed immune mechanistic studies to determine the
precise immune recognition and effector pathways inhibited by anti-CD154 immunotherapy
in the above-referenced studies.
Induction therapy with anti-IL2R mAb and maintenance immunosuppression with tacrolimus,
sirolimus, and CTLA4-Ig) prevented adult porcine islet xenograft survival on a very
consistent basis in a large cohort of monkeys; however, diabetogenic and other side
effects associated with this protocol prompted the investigators not to consider clinical
development of this protocol (Graham ML and Hering BJ; unpublished).
Whether antagonistic anti-CD40 mAb can substitute for anti-CD154 mAb in preventing
islet xenograft rejection is currently unknown, it is conceivable that anti-CD40 mAbs
are less effective than anti-CD154 mAbs because they fail to mediate Fc-dependent
depletion of activated T cells,
155
and they fail to block the interaction of CD154+ T cells with monocytes, macrophages,
and neutrophils expressing the integrin Mac-1 as an alternative pathway for CD154-mediated
inflammation.
156
To date, the efficacy of only 1 antagonistic anti-CD40 mAb in preventing porcine islet
xenograft rejection in NHP has been reported.
102
Currently available antagonistic anti-CD40mAbs proven to be effective in islet allotransplant
models include chi220,
157
3A8,
158
2C10R4,
159
and ASKP1240 (4D11).
160
The replacement of anti-CD154 mAb with anti-CD40 in a protocol which achieved long-term
islet xenograft graft survival in NHP using ATG induction combined with anti-CD154
mAb and sirolimus maintenance failed to show similar efficacy (Park CG et al, unpublished).
Additional studies exploring the efficacy and mechanisms of action of presumably more
potent antagonistic anti-CD40 mAb, such as 2C10R4 and ASKP1240, used for induction
and maintenance therapy alone and in combination with other immunotherapeutics remain
to be performed. We suggest, therefore, that an anti-CD40mAb–based regimen will prove
effective when neonatal islets from genetically engineered pigs are transplanted.
Anti-CD40mAb-based regimens have shown efficacy in xenotransplantation of hearts and
kidneys from genetically engineered (eg, GT-KO/CD46) pigs in baboons.
161,162
Accordingly, future studies of anti-CD40 mAb should also address the survival of islets
from genetically modified donors.
Additional immune intervention can be harnessed, such as negative vaccination using
ethylenecarbodiimide-fixed donor apoptotic cells
163
and thymus cotransplantation
164
to reduce the immune response or induce tolerance against xenoantigens. Pig islet
transplantation may be enhanced by the cotransplantation of mesenchymal stem cells
(of either recipient or donor origin)
165,166
or donor Sertoli cells.
167,168
Both cell types may facilitate revascularization of the islets, reduce the inflammatory
response, and provide immunoprotection.
What Is the Potential for the Treatment of Diabetes?
A number of key requirements for performing additional clinical trials of porcine
islet products have already been met. First, the regulatory framework established
by national health authorities (including but not limited to the US FDA and European
Medicines Evaluation Agency) and the recommendations made by International Xenotransplantation
Association and the World Health Organization provide a safe and suitable framework
for conducting clinical trials of investigational porcine islet products in T1D. Second,
a surveillance and safety program has been developed to detect, measure, manage, report,
and respond to infectious diseases caused by known infectious agents and, possibly,
previously unknown or unexpected pathogens in individual recipients of pig tissues.
Finally, suitable, designated pathogen-free, WT source pigs have been generated for
planned pilot clinical trials. However, other key requirements remain to be met, including
first and foremost the development of a safe and consistently effective rejection
prophylaxis and the development of a commercially viable porcine islet product. Accordingly,
the significance of islet xenotransplantation in the care of patients with diabetes
in the next 10 years will be determined by progress made in these 2 areas. Equally
important in the determination of the future significance of islet xenotransplantation
will be the progress made in the development of the AP and stem cell-derived sources
of islet cells.
Summary of Research Priorities
(1) Prevention of IBMIR. This will likely require a multifaceted approach including
targeting coagulation, compliment, inflammatory cytokines/chemokines and granulocytes-monocytes.
2. Development of effective and clinically acceptable antirejection regimens. An important
current focus is on the targeting CD40-CD40L interactions with anti-CD40 antibodies.
ISLET ENCAPSULATION—AN ONGOING DEVELOPMENT CHALLENGE
Current State of the Field
Over 40 years of islet encapsulation research has failed to provide an approved clinical
product despite many encapsulation approaches and efforts, including several clinical
trials. This IPITA effort is critical to focus on future research goals and objectives
that have the promise to achieve a successful clinical encapsulated islet product
in as short a time as possible. A major review of encapsulated islet efforts has recently
been published which describes the history and accomplishments of research and development
of islet encapsulation as part of an Advanced Drug Delivery Reviews issue entitled
“Cell Encapsulation and Drug Delivery.”
169,170
There have been 2 major types of encapsulating devices: macrodevices and microencapsulation.
Macrodevices
Macrodevices seek to confine the total transplanted cell volume within a single, confined
device. The appeal of this approach is that the implant is easily transplanted and
retrieved. The primary challenge of this approach, however, is that, when avascular,
it is plagued with inefficient nutrient and product delivery.
Early Extravascular Diffusion Devices
The origin of these extravascular diffusion devices began with the work of Algire,
Prehn, and Weaver (1948-1959) who originated a planar diffusion device for the purpose
of studying the mechanisms of immune rejection of cells and tissues. In the process
of their successful research, they defined membrane biocompatibility, host cell membrane
overgrowth, delays in immune rejection of encapsulated tissues, and prevention of
allograft rejection, but not xenograft rejection. After the development of hollow
fiber technology for renal dialysis, Amicon hollow fibers became the target of inserting
islets inside to use as diffusion devices. The majority of these studies were performed
by Wm. Chick who reported that their long-term results were limited by host membrane
overgrowth. However, this problem for hollow fibers was overcome by the use by the
cytotherapeutics team of tubular devices with altered membrane materials in 1985 to
1995 that enabled encapsulated islet implant success in rodent models. A noncurative
clinical trial was published with subcutaneous implants of this device in 3 types
of human recipients: nondiabetic, T1D, and T2D. This study demonstrated the recovery
of viable and functional human islets after several weeks of implanted islet allografts.
However, the low packing density reduced the clinical interest in this device type
due to the large volume of encapsulated islet hollow fibers that would have been required
for a curative clinical trial. Because this trial was performed without the use of
immunosuppression, it is important to note that 2 of the 9 recipients developed donor
antigen sensitization. This is a potential risk for diabetic patients receiving encapsulated
islets without immunosuppression because this could increase their risk of a positive
crossmatch if they ever required a kidney transplant.
Current Extravascular Diffusion Devices
The next device approach returned to a planar device design by Baxter Healthcare in
the early 1990s to develop a device for their future gene therapy products. It was
a well-designed flat sheet device with a central islet chamber and a tubular loading
port. Although it worked well in rodents, once an alginate matrix was used to prevent
cell clumping and necrosis, results in large animals showed less robust capillary
ingrowth in the outer walls of the polyester outer coat. It became known as the Theracyte
device after it was sold by Baxter to Theracyte. Currently, this device type is the
first choice for 2 companies looking for a diffusion device to encapsulate human embryonic
stem cell (hESC)–derived islets that will assure these cells cannot escape from the
device. Both companies, Viacyte and Betalogics, are making separate modifications
to this device type to meet their needs for this newly developed insulin-producing
cell (IPC) source.
Current Direct Oxygenation of Extravascular Diffusion Device
Reviews of many results of encapsulated islet implants reveal that the major acute
cause of encapsulated islet death is hypoxia. It takes too long for new capillary
development and ingrowth to keep the freshly implanted, encapsulated oxygen-requiring
islets from dying. Since 2005, β O2 has been developing many ways to provide direct
oxygen delivery to the encapsulated islets through peripheral connections to their
implanted device. These β-O2 studies have been successful in rodents and more recently
in large animals. The first individual patient trial for this device showed persistent
islet graft function in the chamber for 10 months with regulated insulin secretion
and preserved islet morphology without immunosuppression.
171
Ongoing clinical trials are planned for the near future.
Intravascular Diffusion Device
In the 1980s and 1990s, the WR Grace company joined in a research venture with Biohybrid
to develop an intravascular device to eliminate the acute loss of islet mass from
hypoxia as well as continuously supply oxygen to the functional encapsulated islets.
After many designs, they developed the “Hockey Puck” device that perfused arterial
blood flow through tubing around which the islets were implanted within the device.
This device demonstrated the longest duration of efficacy for a macrodevice both with
islet allografts and xenografts in diabetic dog recipients to date. The FDA was reviewing
the potential to initiate clinical trials with this device when disaster struck this
model by the unexpected disconnection of the carotid artery cannulae to the device
resulting in exsanguination of the diabetes-cured canine recipients. This complication
closed the program, and except for a few repeats with different approaches, this type
of device has not continued forward for any clinical applications.
Intravascular Ultrafiltration Device
Although a limited number of investigators have tried this approach, the concept is
excellent for the islets because it not only provides continuous oxygenation to the
islets but also eliminates the problems of diffusion of insulin from the device. Even
though the in vitro results were excellent and the early rodent in vivo results were
promising, this lead has not been followed since the 1980s.
Microdevices
Alginate Microcapsules as Islet Diffusion Devices
Alginate and similar hydrogels that formed into microcapsules have produced hundreds
of publications with multiple successes in rodent models of diabetes, but remain with
limitations for achieving significance for large animals and human clinical trials.
The standard islet encapsulating alginate microcapsules are produced by droplets of
sodium alginate mixed with islets into a bath of CaCl2 or BaCl2 that rapidly crosslinks
to form the capsule containing the islet. Standard alginate microcapsules are 500
to 1000 microns in diameter with a significant percentage of the capsules not containing
islets. Because gravity pulls on the islet with its higher density than the alginate
during the drop formation and falling, many of the islets tend to sink within the
droplet so when it is cross-linked, a portion of the islets is on the edge of the
capsule, not adequately protected from immune attack. These large capsules have a
very small percentage of their volume as islet so a potential clinical dose of alginate-encapsulated
islets is very large, even for the peritoneal cavity. Multiple methods have been developed
to make the alginate capsules smaller by reducing the surface tension of the droplet,
such as vibrating the droplet needle or using an air knife. Connecting the needle
with direct current to the calcium bath provides an electrostatic condition that also
results in smaller capsules. However, all of these methods to make smaller alginate
capsules by reducing the surface tension should be replaced with the more recent methods
of making small capsules by the use of microfluidics. Another basic problem is that
the pore size is very open so that a second layer of polyamine or similar substances
are coated on the surface. However, because this second coat makes the biocompatibility
worse, a third coat of alginate is required. The vast majority of published encapsulated
islet results use alginate-encapsulated islets. They have been implanted successfully
in rodents, to a lesser degree in large animals as well as humans. Currently, Living
Cell Technologies has been conducting clinical trials of alginate-encapsulated porcine
islet transplantation in different countries with several collaborators. Ongoing results
have not yet achieved the degree of success desired.
110
Alginate Alternatives
To address these problems, investigators have been working on making Minimal Volume
Capsules of alginate as demonstrated most successfully by the Calafiore group. Other
modifications are to replace alginate with agarose as was done by Iwata's group. Due
to slower crosslinking, they moved to an emulsion approach that results in centered
islets with smaller capsule volumes. Although these results appeared promising in
rodents, this approach has not been picked up by other groups. Taylor Wang has developed
multicomponent islet coatings that also center the islet and have shown good results
in rodents. Large animal studies appear promising for this unique microcapsule.
PEG Conformal Coatings
Jeff Hubbell working with Novocell developed an interfacial polymerization formed
by radicals that causes a thin coating of poly(ethylene) glycol (PEG) to form on the
surface of each islet outward to a desired thickness. Rodent implants were excellent,
but failed in translation to large animals due to bioincompatible reactions and islet
losses that required development of new forms of PEG that were biocompatible. The
PEG-coated baboon islets were successfully implanted subcutaneously into diabetic
baboons with normoglycemia maintained up to 2 years without immunosuppression or insulin.
A GLP study in diabetic baboons was performed, successfully leading to the approval
of a ;phase I/II clinical trial in T1D recipients. However, when only partial function
was achieved in the first 2 recipients, the trials were closed. This PEG technology
has been taken to a second generation by Hubbell who is now collaborating with Converge,
a biotech start up in collaboration with the Diabetes Research Institute with early
rodent and large animal results that are promising.
Nanoscale or Layer-by-Layer Islet Coatings
The Iwata group has been developing even thinner islet coatings with different materials,
such as PEG-lipid coatings with poly(vinyl alcohol) alginate up to 10 layers thick
on each islet with early promising rodent results. More recently, they are trying
to place layers of living cells, such as chondrocytes on the surface of islets, to
protect them from immune destruction.
Novel Thermoreversible Islet Coatings From a Glucose Polymer
Alex Gorkovenko has produced a novel polymer of glucose that is a thermal-responsive
gel that is both biocompatible and has programmable biodegradability. In collaboration
with Prodo Labs, a new islet encapsulation technology is being developed with encapsulated
islet size peaking at 250 microns and centralization of the islets with few empties.
In vitro studies show excellent glucose-stimulated insulin release results with initial
implants into diabetic mice showing graft function.
Corporate Involvement in the Development of Islet Encapsulation
From 1980 to current, there have been over 40 corporations involved in developing
encapsulated islets into a clinical product. The companies range in size from small
startups to large corporations, such as Baxter, Amicon, WR Grace, Metabolex, Gore
Medical, and Johnson&Johnson. The specifics of these efforts are documented in the
Advanced Drug Delivery Reviews article.
170
There are currently 11 corporations actively involved developing islet encapsulation
technology.
The Research Agenda
With a vision to translating islet encapsulation to the clinic within a short timeline
(5 years), the goals are modest, with the convergence of a short-course immunosuppressive
therapy and a focus on allogeneic islet sources. Once the potential of microencapsulation
as a strong tool for mitigating immunological attack is demonstrated, broader goals
seeking to expand the cell source and/or eliminate immunosuppression completely can
be explored.
Alginate Encapsulated Human Islets With Modest Immunosuppression
Because of the ongoing problems with alginate encapsulation, a minimum volume capsule
with modest immunosuppression should be targeted as a likely candidate to demonstrate
clinical efficacy within this time frame. Previous approaches sought to gain it all
without the use of immunosuppression. A first step to the clinic would be to define
a minimal immunosuppression approach that permits islet graft function while reducing
the burden of totally relying on the coating to protect the encapsulated islets. Given
what limited information is known about the capacity of encapsulation to mitigate
immune rejection in a patient that will express both alloimmunological and autoimmunological
responses, the first step to the clinic would be to combine either low-dose or short-course
immunotherapy with encapsulation to define if this combination could have a synergistic
effect. Yet, this minimal approach may create new problems if the alginate coating
is too large to implant into the portal vein. Even if it is sufficiently small for
the implants, it brings the additional burden of alginate bulk in the portal vein
with repeated implants. This research would require minimal volume capsules because
allogeneic islets are initiated in diabetic large animals to determine the risks and
benefits before implanting into diabetic humans. Although previous clinical trials
used the intraperitoneal site, the optimal encapsulated islet implant site needs to
continue as a research target.
Screening New Biomaterials With Low Innate Immune Activation Propensity
The current alginate/agarose polymers suffer from the lack of specific control properties
and purification. Several new and unique biomaterials are becoming available which
can better meet these requirements and should be moved rapidly to large animal studies
once biocompatibility is demonstrated for each candidate.
Nonalginate New Polymers
Several of these new polymers under development need to be tested for biocompatibility
and toxicity with islets followed by islet functional testing. If these are minimal,
they need to be fast tracked into islet functional testing and implanted. Some of
these include Alginate-N3 hydrogel, extracellular matrix microspheres, and extracellular
motifs.
Microfabrication Approaches to Decrease Capsule Sizes: Many of these choices can result
in smaller capsule sizes increasing nutrient/waste diffusion and functional responsiveness
as well as be more amenable to intrahepatic sites.
Bioactive Polymers to Enhance Vascularization and Engraftment
These include known items such as vascular endothelial growth factor or other methods
of encouraging rapid new vessel formation at the time of implant. It also could include
methods of prevascularization of devices to establish a vascular response before implanting
the islets within the device, if it can be loaded after implant. It also could include
the incorporation of VEGF or other stimulants of new vessel growth within the device
that are designed to elute from the device for a period.
Combination of Encapsulation and Local Immunosuppression
The use of systemic immunosuppression is to be avoided if possible. However, localized
immunosuppression adjacent to or from within the encapsulating device may be sufficient
to protect the islets. Polymers could be used to provide controlled release for local
immunosuppression. This approach could also include the addition of human MSCs within
the device. Alternatively, the use of a genetically modified islet source, such as
transgenic pigs expressing LEA294, a high affinity variant of T-cell costimulating
inhibitor CTLA4-Ig, may provide local immunosuppression for encapsulated islet transplants.
Alginate Encapsulated Porcine Islets With Full immunosuppression Either Simultaneous
or After Kidney Transplant
Alginate is insufficient to protect xenograft islets from immune attack so these devices
will always require immunosuppression. The allograft immunosuppression is unable to
protect unencapsulated xenografts but the combination encapsulation and allograft
immunosuppression may be effective in xenografts. The focus of this approach is to
increase the potential donor islet pool using xenografts without excessively increasing
immunosuppression over that needed for allografts.
PEG Encapsulated Human Islets With Immunosuppression
Although the first-generation PEG patent protection was abandoned by Novocell, the
second-generation PEG technology should be tested in humans initially with immunosuppression.
Although there are different surface modifications that can reduce inflammation, the
safety profile for PEG is better than alginates. The combination of PEG and immunosuppression
has been shown to increase early engraftment.
Oxygen Delivering Macrocapsules Without Immunosuppression
Delivery of oxygen gas into the device is under development by β O2 which may shift
the primary cause of encapsulated islet graft failure from hypoxia to an immune cause.
The clinical proof of principle for macro-oxygenation needs to be completed. In addition,
microencapsulation of perfluorocarbon or oxygen-generating materials has been accomplished
and need to be considered for these applications either inside of the islet device
or in close proximity.
Key Methodologies Not Being Used
Intravascular Macrodevice Approach
Although this approach may seem too risky as evidenced by the previous attempts in
diabetic dogs, there have been extensive advances from product development in vascular
grafts and shunts because those studies were completed. A new approach with modern
techniques and shunt materials needs to be considered. The risk of blood interactions
should be reduced by new polymers and intravascular device designs.
Combination of Immunosuppression and Encapsulation
(a) Systemic low-dose or altered dose or additional drug—In the past, encapsulation
approaches have typically attempted an “all or nothing” approach to the use of immunosuppressive
drugs. As such, limited preclinical, and no clinical, studies have explored the potential
synergistic effects of immunosuppressive reagents and encapsulation. Multiple approaches
could be evaluated, such as systemic low dose/altered dose/alternative drugs. Further,
when encapsulated islets are placed within alternative sites, avenues for local delivery
of drugs are highly feasible. Drugs could be eluted from the encapsulation material
itself or from eluting materials placed around the implants.
(b) Local delivery of drugs from encapsulating materials—The addition of different
types of drugs that can be eluted or released from the encapsulating materials need
to be defined and tested to avoid the systemic complications of immunosuppression.
This is especially true for treating patients with diabetes because they need islet
replacement to prevent the complications from reaching an end stage rather than waiting
to perform a transplant after the complications have already done significant damage.
(c) Macrodevice approaches other than theracyte extravascular device—The design of
this device with polyester as its outside supporting structures makes it very hard
to use without ingrowth of the device into the recipient. Either coating the polyester
or replacing it will increase the value of this type of device.
(d) Convergence of vascularized implant site with encapsulated islets—New ways of
combining these 2 requirements need to be focused on to develop novel ways of achieving
this goal. Examples include modification of the local environment of the device with
bioactive surfaces, localized drug delivery, and in situ oxygen generation. Alternatively,
the site could be prepared before implanting the islets so oxygenation no longer is
a restriction after implant.
(e) Reduced capsule volumes and thickness—The days of injecting large volume alginate
or other types of capsules need to be over because these large volumes are not clinically
relevant. The focus should only be on minimal volume capsules that can be achieved
with different approaches already defined.
Novel Encapsulation Materials
New Hydrogels
(1) Glucose polymer—This new polymer has many opportunities to be further modified
with different types of additions to the basic structure of the polymer.
(2) Synthetic materials—Examine PEGs or other novel hydrogels or responsive and bioactive
materials for islet coating technologies.
Define the Optimal Biomarkers and Assays for Predicting Islet Survival and Function
and Understanding Causes of Failure—The current approaches are often missing mechanistic
evaluations that are critically needed to understand the progress and predict the
next studies to be completed.
Areas of Controversy and Impediments
Optimal Implant Site
(a) Unique considerations for encapsulated islets—There is a need to compare the unencapsulated
islet implant requirements with the unique requirements for encapsulated islets for
each implant site under consideration. The specific items below are left in question
form for those areas that have not been firmly established.
(b) Intrahepatic site—There certainly is clinical evidence that intraportal vein encapsulated
islet implants can cause portal hypertension, liver impairment, hemorrhage, and death,
although on a very limited basis. Portal pressures should be routinely measured during
the infusion as is done for unencapsulated islets. Yet, there are several critical
questions that have not been answered. What is the volume of encapsulated islets that
the human liver can handle for a single encapsulated islet implant yet avoid portal
hypertension? Can the encapsulation material degrade sufficiently fast enough to complete
the next implant and still be protective of the encapsulated islets? What is the time
course over which repeated intraportal encapsulated islet implants can safely be performed?
(c) Intraperitoneal Site—Because the human pelvis is shaped as a centrifuge tube when
sitting or standing, injected islets that do not stick to the abdominal tissues fall
to the bottom of the pelvis, compact there, and undergo hypoxic death rather quickly.
Can one make the capsules more sticky to prevent their dropping to the pelvis while
at the same time not have this increased stickiness lead to capsule fibrosis?
(d) Omental site—The omental site has advantages in that a variety of pouches can
be created that can entrap the islets that can also lead to vascularization. Yet,
will the capsules stay within the omentum itself and gain vascularization or does
a pouch have to be created to hold them and does vascularization have to be induced?
(e) Intestinal or gastric submucosa site—Encapsulated animal islets have been injected
in both the intestine and gastric submucosa. Can the encapsulated human islets be
injected into these sites and gain sufficient vascularization without compromising
the integrity of the intestine or stomach while maintaining long-term functional survival?
(f) Subcutaneous site—The subcutaneous site has shown promise in animal models. One
human study has suggested that long term insulin injections before encapsulated implants
lead to sufficient subcutaneous scarring that insulin transport away from the implant
site is delayed and reduced.
170
How normal functioning will the encapsulated islets be in the face of extraportal
circulation? How fast will the insulin delivery out of this site be in the face of
chronic scarring from years of insulin injections? How severe will the local fibrosis
and fat formation become in these subcutaneous implant sites done in the face of insulin
injection scarring?
Type of Device in Terms of Islet Retrieval and Replacement
(a) All-in and all-out device type—This device is designed to be implanted with an
islet load and then easily removed completely when the islets need to be replaced.
Thus, it cannot cause the surrounding tissues to grow into the device. Can such a
device be readily removed with the expended islets and readily replaced without damage
to the patient at that site? Such a curative device may require 200 cm2 surface area
as a minimum and perhaps even 2 to 3 times for a curative response with the required
numbers of encapsulated islets. What sites are amenable to these requirements for
this type of device? How will this approach deal with the immediately postimplant
islet hypoxic losses?
(b) Flush and reload device type—This type of device is designed to be permanently
implanted into the recipient and as such requires the ability to flush out the old
islets and reload fresh islets whenever required. When the islets need to be replaced,
what design characteristics are required to maintain the implanted device within the
host yet still permit effective removal of the expended islets by simply flushing
them out and then loading the new islets into the implanted device? How does one assure
the prevention of encapsulated islets becoming stuck to each other preventing their
flushing?
(c) Combination of all-in/all-out and flush/reload devices—Experimental designs may
require some combination of these very basic requirements to be successful in readily
achieving removal and replacement of islet loads in these recipients.
Enhanced Oxygenation Requirements
There are 2 problems with lack of oxygen for encapsulated islets: (a) immediately
after implantation a significant number of islets die of hypoxia as it takes several
days for new blood vessels to grow and (b) unless islets have sufficient oxygenation,
they cannot maximally produce their required insulin. So, by definition, any encapsulation
system that chronically limits oxygenation levels of the β cells will have a marginal
outcome.
(a) Acute Postimplant Requirement × 2 to 4 Weeks
Direct Oxygen Delivery—β O2 is directly supplying free oxygen on a regular basis to
the encapsulated, implanted islets that will presumably be required for the life of
the islets within this type of device.
VEGF before or acutely with islet—While both approaches need to be tested, it is clear
that VEGF added without any hypoxic tissue targets will not lead to effective vascularization
because there is nothing for the new vessels to attach to, and thus they will disappear.
So timing of treating the device without the islets has to be closely determined.
In addition, adding the VEGF at the time of implant will not by itself help much because
the islets will die off before sufficient quantities of new vessels can grow out.
Vascularization of device before islets—There are other ways than adding VEGF to the
device to grow new vessels before implanting the islets, such as maintained factor
release over time and others. These need to be explored to see if they can benefit
islets survival.
Devices Containing Microencapsulated Oxygen—Microencapsulated oxygen is currently
available. Yet, it is not being used because some believe that it will only stay oxygenated
for a few hours after implant that is not long enough to protect the islets beyond
acutely. However, with the oxygen carrier encapsulated, it cannot escape to the lungs
and will remain in the implant site still binding the oxygen. When it has released
all of its oxygen after implant, one needs to test whether simply breathing 100% oxygen
for several minutes a day will replenish the oxygen carrier with sufficient fresh
oxygen binding to the carrier located with the islets to maintain their function.
(b) Chronic Oxygen Requirement
Adequate oxygen availability is required for normal islet function. With restricted
oxygen, the encapsulated islets may remain viable but cannot function. Easily replenished
chronic oxygen supply with oxygen carriers with islets is required as long as islet
function is required.
Islet Functional Capabilities
Encapsulated versus nonencapsulated islets—For each specific type of islet encapsulation
approach developed, what limitations on islet function are the result of the encapsulation
process, considering both acute and chronic changes?
Duration of effective treatment for a long-term product—There certainly is a difference
in postimplant success for islet autografts and allografts or xenografts. How efficiently
do the encapsulated islets continue to function at what percent of their normal capacity
both acutely and long term? What is the functional duration of the encapsulated islets
functioning at what level of normal responsiveness?
Strategy for islet replacement—To avoid being a simple demonstration using a fraction
of the needed islet mass, a strategy for islet full replacement by encapsulated islets
is required with current technology in which normoglycemia can be fully attributed
to the encapsulated graft. What are the criteria for replacement of encapsulated islets?
What is the priority of encapsulated islet recipients to receive additional doses
of islets over those who have not received an islet implant? What is the role of donor
sensitization in determining which patients get the next doses of encapsulated islets
in cases where low or no immunosuppression is required?
Sticky islet coatings—Sticky islet coatings increase the host response for vascularization
but also increase the fibroblast and immune attack. Is there a method to get just
the right kind of islet coating stickiness? Does host reactivity to the device reduce
over time?
Insulin delivery to portal vein or systemic vein—Encapsulated islet implants into
the portal vein have been limited by capsule size and breakdown rates. What are the
requirements for islets in terms of long term function if they deliver insulin through
the portal vein or do not? Does this site of delivery have any effect on delayed function
leading to reactive hypoglycemia?
Speculation of Potential Therapeutic Utility in 5 to 10 Years
(1) There will be multiple types of encapsulated islet approaches under clinical trials
permitting direct comparisons of different types.
(2) Novel methods of required oxygenation of encapsulated islets will be obtained.
(3) Combination of islet encapsulation with local immunosuppression delivery will
greatly reduce risk of systemic immunosuppression expanding procedure from limited
kidney transplant recipients to widespread application.
(4) Successful human islet expansion will reduce the need for more immune challenging
porcine islet xenografts.
(5) Due to these improvements, encapsulated islet therapy will be expanded to people
with significant T2D as well as meeting demands of people with T1D.
Summary of Research Priorities
(1) Conduct a preliminary trial of alginate encapsulated islet allotransplantation
with a short-course or low-dose immunosuppression. Type 1 diabetics who have already
received a renal transplant would be candidates because they are already obligated
to chronic immunosuppression.
(2) Development of improved biocompatible encapsulation materials and capsule designs.
(3) Define new approaches to gain oxygen delivery to encapsulated islets to improve
both early engraftment and long-term survival.
(4) Define optimal transplant sites that have adequate capacity/surface area and that
circumvent the differences of the intraportal and intraperitoneal sites.
AP AND INSULIN DELIVERY SYSTEMS
Current State of the Field
New treatments for T1D may be suited to different clinical situations and stages of
disease—it is unlikely that 1 form of treatment will be best for all patients in the
foreseeable future. Diabetes evolves over years from diagnosis when significant endogenous
insulin secretion still exists to established disease with barely any endogenous insulin
(C-peptide negative). This progression is associated with increasing lability and
difficulty in achieving glycemic control and increasing prevalence of hypoglycemia
unawareness. Hypoglycemia unawareness in most cases results from physiological adjustment
to recurrent hypoglycemia with reduced counter-regulatory hormone production and reduced
autonomic symptom response. It does not result from structural autonomic neuropathy.
The frequency of severe hypoglycemic episodes is strongly correlated with unawareness.
This is a major clinical challenge in people with established diabetes and has become
the usual indication for pancreas or islet transplantation alone (in the absence of
a kidney transplant). Overnight hypoglycemia is also a significant problem and is
the major cause of the “dead-in-bed” syndrome.
One possible treatment—the AP—can be traced back decades to studies demonstrating
the possibility of external blood glucose (BG) regulation using intravenous BG measurement
and infusion of insulin and glucose.
172,173
With the establishment of subcutaneous insulin delivery
174,175
and minimally invasive continuous glucose monitoring (CGM)
176
as viable diabetes technologies, increasing academic and industrial effort was focused
on the development of closed-loop systems using CGM coupled with a subcutaneous insulin
pump. In 2004, the Advanced Insulin Infusion using a Control Loop project based in
Cambridge reported the first promising results
177
; in 2006, the JDRF Artificial Pancreas Project was initiated; in 2008, the US National
Institutes of Health launched an AP initiative; and in 2010, the European AP@Home
consortium was established. A roadmap toward a viable AP was accepted, which included
sequential steps beginning with automated mitigation of hypoglycemia and progressing
through control-to-range and control-to-target toward fully automated, possibly multihormonal,
AP.
178
By 2010, the AP became a global research topic engaging physicians and engineers in
an unprecedented collaboration. Key milestones of this development are described in
recent reviews.
179-182
In those with early T1D (with preserved insulin secretion; C-peptide positive), it
is possible that the combination of residual β cell function and closed loop insulin
delivery might be safe and deliver very tight metabolic control. This may have both
short- and long-term benefits. In established diabetes, closed loop systems may reduce
or prevent overnight hypoglycemia. It remains to be tested whether in longstanding
diabetes closed loop delivery can restore hypoglycemia awareness to the same extent
that can be achieved by pancreas or islet transplantation. It is also possible that
if used widely and early enough, the reduction in major hypoglycemia, especially prolonged
nocturnal hypoglycemia that occurs frequently with conventional treatment, may be
enough to reduce over time the number of people suffering from problematic hypoglycemia
unawareness.
Current technology has shown that AP is able to improve average glycemic control and
simultaneously reduce moderate hypoglycemia; thus, it is likely but untested that
the AP would be able to prevent severe hypoglycemia known to cause seizures or loss
of consciousness. Ongoing AP trials aim to reduce mild hypoglycemia enough to allow
consistent restoration of hypoglycemia awareness similarly to current studies of islet
transplantation designed to include patients with hypoglycemia unawareness experiencing
frequent disruptive severe hypoglycemic events. There are still technological problems,
such as accuracy of the glucose sensor, network connectivity between the devices comprising
the AP system, and reliability of the insulin pump. Most of these problems are currently
being mitigated by algorithm development, and technology improvements that are likely
to bring the AP to mainstream use in the not too distant future.
181,182
The table below summarizes certain advantages of, and challenges to, AP systems:
Subcutaneous and implantable insulin pumps (CSII, CIPII)—Continuous subcutaneous insulin
infusion (CSII) relies on providing basal and bolus insulin using insulin analogs.
189
Structured education programs teach patients to adjust insulin doses based on premeal
BG levels and carbohydrate intake and can achieve target glucose control with lower
rates of hypoglycemia. Forty percent of the patients referred to the UK Dose Adjustment
for Normal Eating program have impaired awareness of hypoglycemia, which is restored
in 40% of those 1 year after UK Dose Adjustment for Normal Eating.
190
Meta-analysis shows that insulin pump therapy can provide a 4-fold reduction in the
rate of severe hypoglycemia events.
191
Continuous intraperitoneal insulin infusion (CIPII) with an implantable pump is available
in a few countries. In a randomized, cross-over study with CSII in a group of patients
with a baseline HbA1c approximately 8.2%, the use of CIPII was associated with less
severe hypoglycemia, although baseline rates were low, with no difference in improvement
of HbA1c approximately 0.5%.
192
In a second study of similar design in a group of patients with a baseline HbA1c approximately
8.6%, CIPII was associated with a 0.76% reduction in HbA1c compared with CSII with
no difference in time spent in the hypoglycemia range or rate of hypoglycemia events.
193
However, the need for operative placement and reoperation on average every few years
to address complications, such as battery replacement, catheter occlusion, pump dysfunction,
pain, and infection, have limited broader CIPII application.
194
CGM is available in some countries. Since the advent of CGM technology,
195-197
significant progress has been made toward versatile and reliable CGM devices that
not only monitor BG day and night but also provide feedback to the patient, such as
alarms when BG reaches preset low or high levels. A number of studies have documented
the benefits of CGM
176,198-201
and charted guidelines for its clinical use
202,203
and its future as a precursor to closed-loop control.
204,205
Although many studies have excluded patients with problematic hypoglycemia, the largest
trial of CGM use to date showed a 0.5% improvement in HbA1c from a baseline of 7%
or greater with no increase in hypoglycemia for patients 25 years or older who most
often used their glucose sensor during at least 6 or 7 days each week. Another study
demonstrated that CGM in children and adults with a HbA1c less than 7.5% was associated
with a 0.27% improvement in HbA1c and less time in hypoglycemia.
201
In a retrospective clinic-based analysis, use of CGM in a cohort of patients with
problematic hypoglycemia at baseline was associated with a reduction in severe hypoglycemia
episodes from 8 to 1 per year, an improvement in HbA1c from 8.1% to 7.6%, but no change
in hypoglycemia awareness.
206
Low glucose suspend (LGS) is considered a precursor to closed-loop because of the
information exchange between CGM and the insulin pump. Two randomized control trials,
which were enriched to different levels with hypoglycemia, showed significant reductions
in hypoglycemia: automation to simulate pancreatic insulin response showed a 38% reduction
in nocturnal hypoglycemia compared to CGM alone without increasing HbA1c.
207
In the second randomized control trial, LGS versus CSII showed a significant reduction
in severe hypoglycemia in patients with impaired awareness of hypoglycemia that was
associated with less time spent in the hypoglycemia range, and again no change in
HbA1c; however, the number of severe hypoglycemia events was rather low at baseline
and the patients are rather young with short disease duration.
208
Closed Loop Control:
Algorithms
The central part of any AP system is a control algorithm receiving CGM and CSII information
and computing insulin microdoses which are sent for delivery to the insulin pump at
short time intervals, typically every 5 to 15 minutes. Various types of control algorithms
were introduced ranging from relatively straightforward proportional-integral-derivative
controllers
209
to complex model-predictive algorithms,
184
and empirical logic approaches.
210
Essential for the design of a closed-loop algorithm is a modular structure that defines
the control action
211
:
(1) Safety and prevention of hypoglycemia ranging from straightforward LGS to sophisticated
model-based hypoglycemia safety systems
212
;
(2) Control-to-range (known also as treat-to-range) which mitigates both hypoglycemia
and hyperglycemia and aims the maintenance of BG levels within a certain range (eg,
4-10 mmol/l)
213
;
(3) Control-to-target which aims near-normalization of glycemic control mimicking
the pancreas action in health, which always tends to bring glucose homeostasis to
a set point (eg, 5-6 mmol/L).
AP Technology
Forty years ago, the AP was a refrigerator-size device (eg, the Biostator
172
). The first inpatient closed-loop control studies that began 8 years ago used AP
systems based on laptop computers wired to CGM and insulin pump.
209,214-217
More recent studies took laptop-based systems to the bedside of patients in overnight
summer camp
218
and home trials.
219
However, such systems were unsuited for everyday free-living use, and a different
design approach was needed to bring the AP truly home.
220
In 2011, the first portable AP system was developed at the University of Virginia
using an Android smartphone as a computational platform and was tested in 2 pilot
trials of outpatient AP done simultaneously in Padova, Italy and Montpellier, France.
221
These first studies demonstrated that inexpensive consumer electronics devices are
capable of running closed-loop control, and therefore smartphone-based systems may
be 1 possible approach to an affordable AP in the near future.
181,182
The system can be used as individual modules as well as a fully integrated device.
For example, the system can operate in a sensor-only mode providing extension of the
CGM receiver functionality, predictive alarms and capabilities for remote monitoring
of the patient over the internet; or the system can work as a pump “companion” providing
wireless capabilities and basal/bolus advice to the patient.
Clinical Trials
Extensive inpatient clinical studies were conducted in 2008 to 2012 using increasingly
more sophisticated approaches: manual adjustment of insulin delivery according to
control algorithm recommendations,
214,215
overnight-only control,
216
automated day-and-night closed-loop,
217
dual-hormone control using glucagon as an additional control mechanism,
222
personalized control algorithms,
223
and monitoring of additional input signals to the control algorithm (eg, heart rate)
during exercise.
224
These studies demonstrated the superiority of closed-loop control over standard insulin
pump therapy in terms of improved average glycemia, improved time within target range,
and reduced hypoglycemia. Similar results were achieved by different control algorithms
as demonstrated by a comparative study.
225
As noted above, the transition of the AP to ambulatory use began in 2011 and since
then studies have been conducted in controlled outpatient settings (eg, hotel, guest
house),
226,227
summer camps for children with diabetes,
218,228
and patients' homes.
229
Typically, studies that used laptop-based wired systems were restricted to overnight
bedside AP use,
218,219,229
whereas studies using wireless portable systems were full-time, day and night.
226,227
In particular, the smartphone-based system mentioned above logged over 70 days and
200 nights of closed-loop control so far.
226-228
A number of clinical protocols are under way with progressively longer duration and
relaxed setting transitioning to long-term AP trials at home. Preliminary data were
reported at the 2014 session of Advanced Technologies and Treatment for Diabetes in
Vienna from studies at Cambridge, United Kingdom, and the University of Virginia,
showing significant improvement of glucose control overnight.
Pancreas transplantation represents the gold standard β-cell replacement therapy.
Islet transplantation is emerging as a minimally invasive alternative to pancreas
transplantation that can provide near-normal glycemic control while ameliorating problematic
hypoglycemia. As a benchmark, successful pancreas transplantation fully normalizes
glucose homeostasis, normalizing HbA1c while abolishing hypoglycemia, as evidenced
by a CGM study in recipients of SPK transplantation documenting a mean glucose 5.6
± 0.4 mmol/L and 96% of time spent between 3.3 and 7.8 mmol/L not different from 5.5
± 0.4 mmol/L and 99% in a control group of healthy volunteers. In a study of T1D patients
selected for successful SPK transplant (HbA1c ∼5.2%), IAK (HbA1c ∼5.5%) or CIPII (HbA1c
∼7.1%), glycemic control assessed by CGM demonstrated mean glucose of 5.4 ± 1.1, 5.8
± 0.8, and 7.8 ± 1.7 mg/dL, respectively, with significantly less glucose variability
in both transplant groups and no (SPK) to minimal (IAK) time spent in the hypoglycemia
range compared with significantly greater number and duration of events with CIPII.
230
Another nonrandomized study compared patients with a baseline HbA1c approximately
8.3% consecutively treated with either islet transplantation or CIPII and followed
up or 3 years, and similarly demonstrated superior glycemic control (HbA1c ∼6.6 vs.
8.1% at 3 years) and reduction in hypoglycemia with the cellular replacement therapy;
however, adverse events occurred 4 times more often with islet cell compared with
CIPII therapy.
231
In contrast, current use of AP components, including insulin pumps, CGM, and LGS,
can improve, but not normalize, glycemia. Although it is tempting to apply such technology
to those experiencing problems with hypoglycemia, information from this population
is limited. In addition, inaccuracy of glucose sensors and variability in subcutaneously
delivered insulin absorption and action still limit the application of AP technology.
However, closed-loop control, albeit imperfect when compared to cellular replacement,
has the potential to become widespread by virtue of its accessibility, plentiful supplies
(as opposed to limited supply of islets), ease of initiation without surgical intervention,
and no need for immunosuppression.
The Research Agenda
Metrics
The field of islet transplantation has developed quantitative measures of glycemic
lability and hypoglycemia severity that ensure proper identification of those patients
with longstanding C-peptide–negative disease at greatest risk for severe hypoglycemia
231,232
; these tools have been used to assess CGM
233
and should be further used to assess LGS and the emerging components of the AP system.
Sophisticated analysis of glycemic control is possible with large amounts of sensor
data uploaded to the Cloud for remote analysis.
232
Comparison of different technologies will be improved if the same metrics are applied
to all, whereas at this stage, different measures of glycemic control are used.
AP Technology
The transition of the AP to everyday diabetes therapy use is contingent upon seamless
concerted work of a device network encompassing the patient. Achieving reliable system
operation is possible through 2 distinct routes:
(1) CGM-insulin pump communication and all control algorithms are being implemented
in the insulin pump. This “traditional” approach is currently adopted by industry,
for example, Medtronic, Animas, and Roche. Advantages include straightforward system
integration and simultaneous testing of all system components. Disadvantages are increased
system cost, limited flexibility to use devices interchangeably and select the best
components from different manufacturers, slow life cycle of the technology (typically
4-5 years), and potentially slower adoption of new control approaches.
(2) Use of consumer electronics whenever possible. Advantages include:
Contemporary smartphones are: readily available and inexpensive, computationally capable
of running closed-loop control, wirelessly connectable to CGM and insulin pumps and
capable of broadband communication with a central location for remote monitoring and
safety supervision, and no current insulin pump offers all of these capabilities.
The technological life cycle of a smartphone is months, as opposed to years for insulin
pumps; thus use of consumer electronics allows easier hardware updates and keeping
up with contemporary user-interface appearance and device form factor.
Psychological studies show that many patients (particularly children and teenagers)
are reluctant to use their insulin pump in public, missing boluses and slipping into
poor glycemic control when privacy is limited (eg, during school days). However, no
one is embarrassed to use a smartphone, and that may be a key to better patient engagement
and better glucose control.
Disadvantages include difficulties with system integration and regulatory approval.
Bioartificial pancreas
A few years ago, we speculated that it would become possible to combine cellular and
mechanical insulin replacement in a single unified treatment strategy.
234
Specifically, limited-volume islet infusion can be used to initiate, but not complete
the process of β-cell replacement, partially mitigating hyperglycemia and restoring
the counter-regulatory responses to hypoglycemia. Additional insulin can be then delivered
by a closed-loop controller. Such a combination therapy could alleviate the problem
of limited islet supply and at the same time facilitate the work of the mechanical
AP by aiding the control algorithm with partially biologically restored glucose control.
Although such combination therapies have not been attempted, primarily because the
AP is still not ready for long-term use, randomized controlled clinical trials could
be planned to compare cellular versus artificial insulin replacement therapies, as
well as combination “bioartificial” approaches in terms of effectiveness, cost, and
availability of the treatment. It could be hypothesized that: (i) limited islet transplantation
and/or regeneration would partially restore β-cell function resulting in reduced glucose
variability; (ii) artificial closed-loop would then deliver additional insulin, further
reducing glucose variability; (iii) in turn, reduced glucose variability would exert
less stress on the transplanted β cells, thereby increasing their longevity. However,
the addition of the costs and risks of 2 new technologies may reduce the feasibility
of this approach.
Whether a bioartificial pancreas is feasible or not, it emphasizes that AP and cell-based
technologies for glycemic control have the potential to be used together, not only
separately. Treatment for people with T1D should include consideration of these new
technologies as well as conventional therapy. It is reasonable to consider testing
AP in patients early in the course of uncomplicated T1D when glucose counter-regulatory
defenses remain intact, thus minimizing the risk for severe hypoglycemia. For patients
with established (C-peptide negative) T1D experiencing frequent severe hypoglycemia
despite best medical management, islet transplantation appears the most promising
alternative to a whole pancreas transplant.
Summary of Research Priorities
(1) Assessment of state of the art AP technology with standardized measures of glycemic
lability and hypoglycemic severity developed by the islet transplant field.
(2) Full incorporation of consumer electronics (smartphone technology) to allow remote
monitoring/supervision, opportunity for frequent hardware and software updates, and
to negate the psychological stigma of in public pump use.
(3) Consider assessment of combine AP-islet transplant therapy to address the limited
islet supply and need for multiple islet doses and perhaps limit β cell stress, thereby
improving islet performance and longevity.
IMMUNE TOLERANCE FOR ISLET AUTOIMMUNITY AND ALLOIMMUNITY
Current State of the Field
There are no currently widely available, safe, and extensively validated approaches
for establishing tolerance for islet transplantation in humans. Although a large number
of approaches seem to be efficacious in rodent models, most have failed to translate
into success in primate or porcine large animal models. Some success has been achieved
in humans in renal transplant protocols which require low-risk donors and recipients,
extensive immunosuppression, and components of hematopoietic chimerism. Moving forward,
there are several principles to be taken into account. First, when assessing tolerance
for islets and because of the unique characteristics of different immune responses
(eg, memory, cross reactivity, number of reactive clones, and cells), it is very important
to separately measure and assess responses to autoantigens and to alloantigens. Depending
on future potential, responses to xenoantigens will also have to be separately addressed.
Second, it is important to have measures not only of islet function and injury but
also measures of immune reactivity and immune regulation to prospectively monitor
recipients. Third, it is clear from murine studies that there are many components
to immunity, and that tolerance is achieved only by targeting the distinct arms of
the immune response. Broadly speaking, innate B-cell and T-cell responses must be
controlled to induce and maintain tolerance. It is also clear that tolerance is achieved
not only by preventing these distinct responses but also by generating regulatory
phenomena. Fourth, it is clear from murine studies that the most robust tolerance
for both autoimmunity and alloimmunity is achieved in protocols that incorporate some
form of hematopoietic stem cell chimerism along with immune regulation.
235,236
Chimerism successfully prevents the B- and T-cell components of adaptive immunity
while simultaneously generating a variety of suppressor and regulatory mechanisms
(eg, anergy, deletion, suppressor cells).
237
Current clinical studies define various approaches that hold some promise for tolerance
induction or provide a guide for approaches that do not work (Tables 2 and 3). The
ONE Study (www.onestudy.org) is currently validating procedures for generating suppressive,
Treg and mesenchymal stem cells that can be grown and manipulated. Although there
is currently no information yet as to efficacy of these regulatory cells in transplantation,
safety studies have been performed in patients with T1D. Low dose IL-2 has been shown
in human clinical trials to increase peripheral Treg cell and ameliorate autoimmune
HCV-induced vasculitis.
238,239
TABLE 2
ITN Studies
TABLE 3
TrialNet Studies
Current T1D clinical prevention and intervention trials may define lead assays, drugs,
and procedures for overcoming autoimmunity at early or late stages (Tables 2 and 3).
A number of observations, failed trials, and/or validation studies have yielded important
and surprising results that provide extremely important guidelines for translation
of information to tolerance trials and for interpreting putatively interesting signals
in the preclinical literature.
Grant et al
240
reported on the results of an independent laboratory's tests of novel agents to prevent
or reverse T1D in the nonobese diabetic mouse, diabetes prone BB rat, and multiple
autoimmune disease–prone rat models. Methods were developed to mimic human clinical
trials, including prescreening, randomization, blinding, and improved glycemic care
of the animals. Agents were selected by an NIDDK appointed independent review panel.
Agents selected to prevent diabetes at later stages of progression were: a STAT4 antagonist
(DT22669), α 1 anti-trypsin, celastrol, and a macrophage inflammatory factor inhibitor
(ISO-092). Agents tested for reversal of established T1D were: α 1 anti-trypsin, tolerogenic
peptides (Tregitopes), and a long-acting formulation of GLP-1 (PGC-GLP-1). None of
these agents prevented or reversed T1D, whereas the positive control interventions
were effective: anti-CD3 reversed diabetes in the NOD mouse, dexamethasone prevented
diabetes induction in the multiple autoimmune disease–prone rat, and cyclosporine
prevented diabetes in the BBDP rat. This important study highlights the limitations
of much of the primary rodent literature and strongly demonstrates that stringent
confirmatory testing will be required in rodent models before translation to large
animal or human experimentation.
Sarikonda et al
241
reported that combination therapy with anti-CD20 and either oral insulin or proinsulin
does not protect hyperglycemic NOD mice, but the combination with proinsulin offers
limited efficacy in T1D prevention. The ITN, TrialNet, and NHP studies listed in Tables
2 and 3 demonstrate many outright failures or very limited signals for OKT3 (anti-CD3ɛ),
thymoglobulin (polyclonal antithymocyte globulin), α 1 antitrypsin, oral insulin,
rituximab (anti-CD20), daclizumab (anti-IL-2Rα), abatacept (CTLA4-Ig), alefacept (LFA3-Ig),
and canakinumab (anti-IL-1β). Although these results are obviously disappointing,
they also show what does not work so that nonproductive avenues of research are no
longer pursued.
The current state of the art does not define how best to plan the sequence of preclinical
or clinical trials for interventions and markers. For example, should NOD or humanized
mice be used to evaluated treatments for the autoimmune component? What is the best
model to provide supporting data before initiating clinical trials? Should we first
validate biomarkers and then go to clinical trials, versus validate an approach that
achieves tolerance in primates first? If clinical trials are contemplated, who is
best suited to which therapy? How is safety defined in clinical tolerance trials,
especially for treatment of autoimmunity and alloimmunity in T1D, where alternative
treatments (ie, semisynthetic insulins, closed loop insulin pumps) are rapidly improving?
Should trials first be performed in transplant populations with larger numbers of
patients (ie, renal allografting)?
It is important to realize that immunologic interventions can likely be enhanced by
other nonimmunologic approaches in islet transplantation. Thus, there are multiple
opportunities for cross-fertilization with technologies and approaches from the other
workgroups. Encapsulation techniques may obviate the need either to measure immunity
or to develop novel immune or tolerance techniques. It is conceivable that a perfect
encapsulation system will allow long-term graft survival with conventional immunosuppression
accompanied by immunosuppression minimization and weaning. Although xenografts present
a large series of novel antigens to which it is currently very difficult to provide
adequate immunosuppression, encapsulation may overcome these problems by masking antigens
and/or protecting from effector mechanisms of inflammation and rejection. The β-cell
regeneration and stem cell technologies may overcome some toxic effects of conventional
or novel immunosuppression. Likewise, enlarging and continually replacing the β cell
mass may also overcome immune reactivity and help to generate exhaustion in antigen-reactive
clones of T cells and B cells. Conceivably, the combination of encapsulation, regeneration,
stem cells, and xenotransplant along with conventional immunosuppression and monitoring
may achieve the goal of long-term islet function with ease of monitoring plus minimal
toxicity.
Obstacles to Application of This Therapy
There are many unsolved immune barriers imposed by the lack of markers for diagnosing
recurrence, rejection, tolerance; infections which stimulate innate immunity and tissue
inflammation; cross-reactive or antigen-specific memory T cells and memory B cells
for which there is no effective immunosuppression; and hematopoietic stem cell engraftment
without graft vs host disease using nontoxic conditioning. Overcoming these barriers
will require effective and validated biomarkers for immune monitoring, effective anti-inflammatory
immunosuppression which targets innate and adaptive immunity without toxicity, effective
immunosuppression for memory responses, and far more reliable and less toxic procedures
for achieving bone marrow chimerism.
There are many unsolved nonimmunologic barriers which include limitations to islet
mass and quality; lack of specific, sensitive, and reliable islet imaging or functional
monitoring; and islet toxicity imposed by many immunosuppressive and immunomodulatory
drugs and procedures. Overcoming these barriers will require progress in islet isolation,
regeneration and stem cells; validated measures and markers of islet mass and function;
and novel immunosuppressive regimens that obviate the need for islet toxic agents.
The Research Agenda
There are several approaches which could achieve maturity within the next few years
and be ready for large scale clinical trials. As noted above, hematopoietic stem cell
chimerism is validated for proof of concept in rodent, large animal, and human studies.
Additional approaches to enhance this modality may include vascularized bone marrow
and/or intrabone marrow injection of hematopoietic stem cells to improve the establishment
and durability of chimerism. Rodent studies
242
achieving durable chimerism by adding expanded recipient Treg cells and human studies
safely achieving transient chimerism
243,244
provide proof of concept that the combination of Treg cells plus nonmyeloablative
bone marrow transplantation has great potential to achieve durable and safe chimerism.
The T1D early-onset and preventive studies outlined above may soon point the way to
therapies that are easily adapted to islet transplantation. We can expect results
over the next few years, although there are no currently validated therapies or prime
candidates.
Biomarkers are critically required to move the field forward. A technology that will
dramatically advance the field is a validated assay or biomarker that effectively
yields highly predictive data for immunosuppression and tolerance on the one hand,
and antigen reactivity on the other hand. Assays based on T cell ELISpot, serum DSA,
circulating plasma β-cell DNA, or peripheral blood mononuclear cell profiling by FACS
or RT-PCR have been shown at times to be strongly associated with important immune
events and outcomes.
245,246
There are interesting hints that B cells or NK cells are markers or mechanistically
related to tolerance. However, the associations are not so strong that that these
tests are highly predictive or actionable, and none of these tests or combinations
of these tests have areas under the curve greater than 0.95, which is required for
a reliable clinical test. Array platforms that measure DNA simple nucleotide polymorphisms,
messenger RNA, microRNA, proteins, metabolites, or microbiota are all available and
have all been proposed as possible hypothesis generating approaches in small clinical
studies. None are validated in clinical transplantation. For autoimmunity, biomarkers
may have to be validated in the T1D studies and extended to islets. For alloimmunity,
biomarkers may have to be validated in larger studies in bone marrow transplant and
kidney or liver transplants before attempting to translate to islets.
Specific targeting of T and B memory cells to prevent recurrent autoimmune disease
and chronic ongoing rejection is required. No current agents have been shown to be
clinical efficacious in this regard. Although Alefacept did have some effects on memory
by peripheral lymphocyte subset analysis, it was unable to have a clinical impact
on memory responses in kidney clinical trials and it is clear that all other currently
approved drugs are not effective in safely controlling memory responses. Success and
validation may first have to come from bone marrow transplant, kidney, or liver transplants
and then translated to islets.
Encapsulation technologies and novel sites of transplantation may mitigate issues
related to islet quality, islet quantity, islet regeneration, tolerance, innate immunity,
and memory responses. The experimental approaches to site and to encapsulation are
relatively advanced and may provide the ability to move forward rapidly.
It is clear that a combination of several approaches outlined above must be considered.
247
Combinatorial approaches will undoubtedly create significant contractual, regulatory,
and safety hurdles.
What Is the Potential for the Treatment of Diabetes
It is noteworthy that minimal or minimized immunosuppression is widely used and potentially
ready to implement in islet transplantation. A regimen consisting of transient steroids,
transient anti-inflammatory (eg, TNF-α blocker), CNI-free, mTORi-free or transient,
and costimulatory blockade (Belatacept) has some proof of concept. An S1P1 modulator
(Fingolimod) could be added to this regimen. A major barrier to implementing this
approach is lack of validated biomarkers for islet function and immune monitoring.
Additionally, it may be argued that any protocol relying on long-term immunosuppression,
even at low levels, could only be applied in patients with life-threatening complications
of diabetes, such as hypoglycemic unawareness. Given the excellent standard of care
for diabetes that is currently available without transplantation, the application
of transplantation to broader diabetic populations will only be ethically feasible
when approaches that do not require long-term immunosuppression, such as tolerance
or encapsulation, are successful and safe.
The Treg cell therapy will likely be part of a combination therapy approach. In particular,
mixed chimerism induced and sustained by Treg cells plus nonmyeloablative conditioning
may be one of the most likely approaches to be tried in islet transplantation. As
progress is made in Treg cell therapy and nonmyeloablative conditioning in other areas
(eg, autoimmunity, cancer), those results may be translated to islets.
If substantial progress is made in stem cells, encapsulation, β-cell regeneration,
isolation, imaging, biomarkers, or xenotransplantation, each of these areas will synergize
for the induction and maintenance of tolerance.
Summary of Research Priorities
(1) The recent success of chimeric tolerance in renal transplantation potentially
sets the stage for application to islets. The use of protocols with high-level donor
chimerism may simultaneously achieve allotolerance and rid the host of their native
autoimmune prone T-cell repertoire.
(2) Costimulation blockade with simultaneous targeting of CD28-B7 and CD40-CD40L remains
a scientifically attractive approach. New CD40 targeting agents in conjunction with
the recently approved anti-B7 belatacept may permit such testing in the near future.
(3) Trials of innovative regulatory cell based approaches (Treg cells) are also attractive
in that it may be possible to interrupt in parallel auto and alloimmunity with precise
antigen specificity.
STEM CELLS AS A SOURCE FOR β CELLS
Current State of the Field
Diabetes is a debilitating disease characterized by a chronic inability to normalize
BG levels. Transplanting cadaveric pancreata or isolated pancreatic islets can restore
glucose homeostasis, but organ demand outstrips supply and donor quality contributes
to short-term complications. Consequently, there is significant interest in alternative
sources of IPCs. To overcome the limitations of currently available therapies, research
efforts have focused intensively on generating functional β cells or endocrine cell
clusters from stem cells. A variety of stem cell types have been considered as potential
future sources of transplantable β cells which include human pluripotent stem cells
(hPSCs), such as hESCs and human-induced pluripotent stem cells (hiPSCs), mesenchymal
stem cells generally isolated from bone marrow or cord blood, stem cells isolated
from adult tissues, or directly reprogrammed somatic cells. This review will focus
on pluripotent stem cell (PSC) and reprogrammed somatic cell sources. Although the
concept of reprogramming somatic cells directly into β-like cells is in its infancy,
recent exciting progress in the PSC field has led to refined protocols yielding highly
enriched populations of monohormonal insulin-secreting cells and the initiation of
pilot clinical trials.
Pluripotent Stem Cells
The PSCs are characterized by 2 features: their ability to differentiate to any of
3 somatic cell lineages (ectoderm, endoderm, or mesoderm), and their ability to replicate
indefinitely in a stable pluripotent state.
248-250
Because PSCs can evade senescence in culture, they provide an unlimited supply of
cells suitable to meet the demand for a replacement cell therapy.
249
The PSCs can be directed to differentiate to a variety of specific lineages with relatively
high efficiency.
251,252
The hESCs were first isolated and cultured by Thomson using inherently variable mouse
fibroblast feeder cells and serum supplemented media.
253
Significant improvements now allow derivations and stable extended culture under defined
xeno-free conditions yielding cells with a normal genetic karyotype suitable for cell
banking and clinical applications.
254-256
A second pluripotent cell source originates from somatic cells which can be reprogrammed
to a pluripotent state through a process first identified by Shinya Yamanaka and Sir
John Gurdon.
257-259
Reprogramming can be accomplished by forced expression of a combination of transcription
factors (eg, OCT4, SOX2, KLF4, and cMYC or OCT4, SOX2, NANOG, and LIN28), a process
initially reported using retroviral transfection techniques.
258,259
Subsequent studies showed that reprogramming could be achieved using nonintegrating
episomal vectors based on Epstein-Barr virus or Sendai virus delivery systems
260-263
and, more recently, with RNA- or protein-based methods.
264,265
Many human cell types have now been reprogrammed to iPSCs, for example, adult and
embryonic fibroblasts, mononuclear peripheral blood cells, T cells, islet cells, pancreatic
acinar cells, and hair follicle cells, among others. Human iPSCs have been derived
from patients with a variety of different diseases including diabetes.
266-268
Direct Reprogramming
Rather than generating stem cells from somatic cells and subsequently differentiating
them to the desired lineage, attempts have been made to circumvent this process and
induce direct reprogramming of somatic cells to β cells, either in vivo or in vitro.
In this method, enforced activation of key pancreatic transcription factors, usually
in combinations, is used to drastically alter the program of expressed genes thereby
leading to a dramatic change in phenotype, often across typical lineage boundaries.
269
Such an approach has been attempted in several different cell types, such as pancreatic
acinar cells,
270-272
hepatocytes,
273
and fibroblasts.
274
Although somatic cell reprogramming to pancreatic lineages involves epigenetic conversion,
it is not known whether cells transform directly into β cells, or whether they first
revert to a multipotent state and then redifferentiate toward β cells (ie, 2-step
process) or whether certain cell types are more efficiently reprogrammed. Also, whether
such conversions result in stable, robustly functional β cells remains to be determined.
Conversion of hPSCs to β Cells
The overall preclinical research goal of the field is to achieve efficient derivation
of a functional β cell mass in vitro that is able to rapidly and reliably cure diabetes
in mice, both longstanding gold standard preclinical diabetes assays. To achieve in
vitro conversion of hPSCs to β cells, a highly productive approach has proven to be
the application of knowledge gleaned from developmental biology studies. However,
most information about pancreas development has been obtained from organisms, such
as frogs, chickens, zebrafish, and mice, and it is well known that aspects of pancreas
development and islet biology differ between humans and lower organisms.
275-277
Because of this, refinement of differentiation protocols has progressed through a
combination of strategies that include both rational design and empiric testing of
developmentally important effector molecules and monitoring expression of key transcription
factors. These endeavors have also been complemented by an increasing understanding
of the epigenetic landscape and transcriptional profile of human pancreas development
and fully differentiated β cells.
In vivo pancreatic development is a complex process involving sequential lineage restriction
steps to form a composite, well-vascularized endodermally derived organ consisting
of acinar, ductal, and endocrine tissues.
278-280
Based on principles of vertebrate development, a multistep model of β-cell formation
from stem cells in vitro has been proposed,
281
attempting to recapitulate sequential stages of in vivo development including gastrulation,
endoderm specification, gut-tube morphogenesis and organ budding from the gut tube,
and finally organ-specific cellular differentiation within the organ bud. This multistep
model has since provided a foundation for methods and protocols applicable to pluripotent
stem cell differentiation. A full description of pancreas development and the evolution
of β cell in vitro differentiation protocols over the last decade is beyond the scope
of this review, and the reader is referred to the following references: Hosoya et
al, Alexander and Stainier, Clements et al, and Shen.
282-285
Rapid progress is being made to improve and refine in vitro differentiation protocols
to achieve monohormonal β-like cells that express key maturity markers and exhibit
robust glucose-stimulated insulin secretory responses. Many believe that a more mature
differentiated cell will be more advantageous therapeutically as well as provide a
tool to facilitate studying β-cell pathophysiology and testing of novel pharmaceuticals.
Until recently, in vitro–derived IPCs largely exhibited a polyhormonal phenotype
282,283,286-289
and most likely corresponded to cells of the primary endocrine transition observed
in murine and human development,
290,291
which ultimately do not give rise to adult β cells. Instead, adult β cells are thought
to arise from a distinct developmental event, the secondary transition, marked by
transient expression of neurogenin-3 (Ngn-3). Although the precise reasons for incomplete
differentiation under some conditions are still unclear, it is worth noting that immature
phenotypes are also observed when other lineages, such as blood, cardiac, and neural
cells, are derived from PSCs. Polyhormonal endocrine cells appear to have reduced
levels of, or lack, important β-cell transcription factors, such as PDX1, NKX6.1,
and MAFA,
286,287
and in some cases, key β-cell transcription factors, such as PAX4 and ARX, are misexpressed
compared with adult human β cells in vivo.
286,287
From a functional viewpoint, immature hPSC-derived insulin-positive cells appear to
express reduced levels of other important genes including potassium channels, proconvertases,
Zinc transporters, islet-associated polypeptide (IAPP), and urocortin3 relative to
adult β cells.
40
Thus, the expression of these markers was essential for screening conditions that
yielded more mature functional β cells in vitro.
292
Several groups have recently reported improved differentiation protocols, which achieve
monohormonal β cells and better in vitro functionality, promising to finally remove
this longstanding roadblock.
293-295
Interestingly, different protocols were used by each of these groups yet there were
some commonalities. By extending the culture period, including 3-dimensional suspension
culture and exposure to ALK5iII, Shh inhibitors (SANT1 or KAAD cyclopamine), γ secretase
inhibitors to inhibit Notch signaling, and thyroid hormone (T3), these three groups
were able to enhance glucose-stimulated insulin secretion (GSIS) in vitro. However,
Rezania et al
292
showed that their cells exhibited dampened secretory characteristics in perifusion
assays compared with human islets. Furthermore, although similar results in GSIS assays
were achieved with hPSC-derived β cells and human islets, the degree of variability
from preparation to preparation of hPSC-derived β cells was surprisingly high.
292,294
Importantly, the insulin-positive cells displayed a monohormonal phenotype with improved
expression of key β-cell signature genes. Although there were many differences in
the culture conditions among these recent reports, it is still not entirely clear
whether all of the components in each protocol are necessary and whether the makeup
of the cell populations derived by the different protocols is the same. Nevertheless,
these current reports demonstrate rapid progress in the field toward achieving a more
physiologically functional β-like cell in vitro from hPSCs.
Critical progress in efficiently reversing diabetes in mice was also reported this
past year.
292-294
Until recently, when hPSC-derived pancreatic tissues were transplanted into immunodeficient
mice, either under the kidney capsule or into the epididymal fat pad or in an immunoisolation
device in the subcutaneous space, the graft did not regulate glucose or produce secreted
C-peptide immediately.
296-384
Instead, the graft appeared to mature over many
6,13-20
weeks to form functional pancreatic tissue in a time frame similar to the in vivo
maturation of human fetal pancreas.
300
The mature graft ultimately did contain islet-like structures comprised of α (glucagon),
β (insulin), δ (somatostatin), ghrelin, and pancreatic polypeptide hormone-producing
cells and is competent to maintain glucose homeostasis in mice made diabetic with
alloxan or streptozotocin despite imperfect insulin secretory kinetics of hPSC-derived
endocrine grafts.
301
Efficient in vivo endocrine maturation appeared to be dependent on a sufficient number
of Nkx6.1 + PDX1+ pancreatic progenitor cells (PPCs) in the transplanted population,
298
and if/how the cells were encapsulated.
301
Although the delayed correction of chemically induced diabetes by hPSC derivatives
represented an important preclinical milestone, there remained great interest in accelerating
maturation in vivo. Indeed, 2 studies have now reported more rapid (2 weeks and 6
weeks) reversal of murine diabetes after transplanting more mature cells under the
kidney capsule in Akita and streptozotocin models, respectively.
292,294
Now that the derivation of hPSC-β-like cells in vitro exhibiting improved physiological
and phenotypic characteristics of adult, mature β cells resulting in more rapid cure
of diabetes in mice has been achieved, the next questions likely to arise are: what
accounts for the GSIS variability and dampened insulin secretory kinetics in perifusion
assays and can this be improved? Will a macroencapsulation device provide a suitable
environment for mature cells as it does for maturing progenitors? Other questions
concern the delivery of these cells to patients: What cell population is the best
to transplant? Are progenitors sufficient to transplant or is a terminally differentiated
functional population better? Perhaps a mixed or hybrid population is a better choice?
Is a mixture of endocrine cell types necessary or beneficial and to what degree, or
is it sufficient to transplant a graft solely composed of β cells? Finally, and perhaps
ideally, can one derive/engineer a renewable, yet fully differentiated β cell from
stem cells? Many of these questions should be addressed in preclinical animal models.
Reports of pancreatic lineage differentiation and β-like cell formation in vitro have
come from many laboratories with many different cell lines, using a variety of culture
protocols. However, few protocols have been compared head to head; even fewer have
directly compared multiple different cell lines in parallel. Thus, which line and
which protocol provides optimal pancreatic β cell differentiation have not been determined
prospectively. Given genetic and epigenetic differences of different PSC lines, it
is not surprising that different cell lines can behave dissimilarly under the same
conditions.
Analyses of nonpancreatic cell types within differentiation cultures, regardless of
the protocol, have been limited. These nonpancreatic cell types, including but not
limited to undifferentiated cells, could potentially inhibit or enhance ongoing differentiation.
The retention of undifferentiated cells through later culture stages raises the specter
of teratoma formation after transplantation. Most culture protocols published to date
generate heterogeneous populations with some unwanted cells, which brings up several
questions: how pure does the population need to be? How precisely defined does the
cellular product need to be before it is deemed suitable for therapeutic use? Are
certain unwanted cell types acceptable while others are not?
Regardless of whether transplantation of fully functional β cells or PPCs is the therapeutic
platform, both strategies would benefit from technology that modifies the immunogenicity
of the graft, or induces host immunological tolerance, or protects the graft from
host alloimmune and autoimmune responses. Ongoing efforts are addressing this need
through use of immunoisolation devices, modified stem cells,
302
advanced immunosuppression protocols,
303
tolerogenic strategies, or using syngeneic hiPSCs. Macroencapsulation approaches are
particularly attractive because they have the benefit of graft cell containment, reducing
the risk of excessive growth or the spread of cells with teratoma-forming potential
as well as limiting possible alloimmune and autoimmune damages.
Obstacles to Application
The achievement this past year of improved in vitro GSIS and more rapid reversal of
diabetes in mice was a major milestone. Yet, many clinicians may still view the variable
GSIS results and subnormal insulin secretory kinetics in perifusion assays that have
been reported as a substantial obstacle to widespread application of this technology.
Why has this milestone been so hard to achieve and how will we know when we have achieved
it? The field would benefit from a consensus agreement as to what are benchmark phenotypic
and functional characteristics of an in vitro hPSC-derived β cell. Most would agree
that at the very minimum the following should be achieved: (i) a consistent, reproducible
stimulation index without secretagogues of at least 2 to 3, and (ii) immediate or
near immediate reversal of diabetes in mice with a normal glucose tolerance curve
and stimulated C-peptide release in response to IV or PO glucose, which is eliminated
if the graft is removed. A more stringent definition might include results of a panel
of phenotypic markers, GSIS including secretagogues, estimation of proinsulin-insulin
ratios, and insulin secretion kinetics derived from perifusion assays that mirror
human islets. Additionally, determining physiological responses at the single-cell
level may be valuable, given the possible heterogeneity of the insulin + cell populations.
A comprehensive analysis of the resulting β-cell population will benchmark the degree
of functionality achieved and facilitate comparisons between cells produced using
different protocols. If not transplanting a functional β cell, how long will patients
be willing to wait before they are able to eliminate insulin therapy—or how long will
a transplant be allowed to persist before it is deemed a failure? Moreover, many clinical
events could thwart in vivo maturation and the development of functionality, such
as rejection, recurrent autoimmunity, or toxic immunosuppressant medications.
An important part of preclinical evaluation of a possible therapy is to understand
the immune responses to hESC/iPSC-derived PPCs or β cells in immunocompetent hosts
and to devise ways to protect cells from alloimmunity and autoimmunity. To date, these
clinically relevant questions have not been addressed in depth. Although encapsulation
as a transplant delivery system may demonstrate efficacy, it is also possible that
graft damage will still occur because many current devices do not effectively exclude
cytokines. More studies evaluating host alloimmune and autoimmune responses to encapsulated
hESC-derived β cells are needed. In an ideal scenario, customized patient-specific
iPSC lines may obviate the need for immunosuppression. However, existing data are
unclear about whether syngeneic iPSC progeny would be destroyed after transplantation
304,305
and studies to date do not address this question specifically for pancreatic lineages.
Furthermore, it is currently unknown whether syngeneic grafts derived from Type I
diabetes mellitus patients will indeed elicit immune responses or be susceptible to
recurrence of autoimmunity. Thus, more work needs to be done to clarify and better
characterize the anticipated immune responses to syngeneic and allogeneic hPSC-PPC
or β cell grafts. Moreover, based on the absence of autoimmunity in Type 2 diabetes
mellitus patients, it may be reasonable to first test syngeneic iPSC-derived β cell
transplants in this population.
Teratoma formation, or malignant transformation of a teratoma into a teratocarcinoma,
is a concern of any proposed hPSC-based therapy. With teratomas reported in the context
of a number of current in vitro pancreatic differentiation methods, effective, safe,
simple, and inexpensive methods to prevent or limit teratoma formation are needed.
Furthermore, it will be necessary to quantify the risk for a given cellular graft
in order to assess its risk-versus-benefit ratio. Therefore, developing a predictive,
quantitative assay in which to test a cell product would benefit the field. Such an
assay could be an in vivo assay, such as the injection into the hind limb of an immunodeficient
mouse similar to what is currently used to determine pluripotency of cell lines. However,
this method is neither quantitative nor rapid. A rapid, relatively high throughput
in vitro assay would represent a more ideal method, if available. The PluriTest assay
may prove to be valuable for this purpose.
306,307
Ultimately, a fully terminally differentiated purified cell population may have an
extremely low teratoma risk profile, but determining this preclinically for a given
cell population would have merit. Macroencapsulation may very well be the least expensive
and most effective “teratoma prevention” method currently available, but improved,
less fibrogenic, more proangiogenic, and cytokine-excluding encapsulation methods
may be needed. Other methods could require genetic manipulation of the cells before
differentiation and transplantation; however, such methods may carry their own attendant
risks.
Current differentiation protocols use large quantities of expensive growth factors.
Can these be replaced with less expensive small molecules that signal through the
same receptors and produce the same biological results? An example of this is the
use of LDN193189, a bone morphogenic protein antagonist instead of Noggin in recent
studies.
292,294
High-throughput screens for small molecules which can replace expensive growth factors
may not only allow the differentiation process to be less expensive but also more
efficient. Additional optimization will be needed to scale up the differentiation
protocols to generate large numbers of β cells to meet the demands of millions of
diabetes patients.
Finally, regulatory considerations will present significant challenges in this area.
The use of cadaveric islets in transplantation is already regulated in the United
States as a manufactured cell product. Stem cell-derived β-cell products will likely
be regulated similarly by most regulatory agencies. Additional use of encapsulation
devices, scaffolds, other supporting cells, angiogenic agents, or immune modulatory
factors will render the therapy as a combination product. There will be complicated
preclinical data packages for these combination products depending on the perceived
risks of the individual components and their combinations. Genetically modified lines
may be subject to additional regulatory requirements. Academic researchers and industry
stakeholders will have to work closely with regulatory reviewers to manage reasonable
amounts of preclinical work to justify clinical trials. In addition, to use a cell
line clinically, it is critical that cell lines be generated under current Good Manufacturing
Processes (cGMP). Durruthy-Durruthy et al
308
recently published on a rapid and efficient conversion of integration-free human iPSCs
and suggested strategies to convert to cGMP culture conditions. Currently, there are
numerous groups developing cGMP iPSC lines (Cellular Dynamics International, National
Institutes of Health, Riken Institute, Roslin Institute).
Direct Reprogramming of Somatic Cells to β Cells
Another strategy for generation of IPCs in vivo or in vitro involves reprogramming
adult cells directly to a pancreatic cell lineage. A number of groups have demonstrated
that β cells may be generated from non–β cells through ectopic or artificially induced
gene expression of a single transcription factor (Pdx1) alone or with other pancreatic
genes. Ectopic expression of PDX1 is sufficient to convert fetal α-cells to β-cells
in vivo and can promote functional insulin expression in mouse liver.
309,310
Combinatorial transfections of the transcription factor genes Pdx1, MafA, and Ngn3
can reprogram murine pancreatic exocrine tissue or liver to form functional β-cells
in vivo, and the resulting cells appear competent to rescue animals from hyperglycemia.
270,311,312
Ectopic Pdx1 expression can also induce fate conversion in vitro, as demonstrated
in the case of cultured keratinocytes converted to a pancreatic β-cell fate.
313
These aggregate results suggest that ectopic expression of Pdx1, alone or with other
genes, may provide an alternative means of generating functional β-cells. Recently,
Zhu et al
274
reported on the direct reprogramming of murine fibroblasts to definitive endoderm
by a transient expression of pluripotency reprogramming factors in conjunction with
a unique combination of small molecules and growth factors. They then followed a differentiation
protocol to develop pancreatic progenitors that reversed diabetes in a rodent model.
On the other hand, human pancreatic duct reprogramming may be improved by Ngn-3 overexpression,
inhibiting Delta-notch signaling and coexpressing Myt1.
314
These data demonstrate how the plasticity of adult cells, in particular those of related
lineage, might be harnessed to produce β cell phenotypes.
Many challenges on the road to clinical application remain as this strategy is relatively
young. The reproducibility and functionality of resulting cells still need to be rigorously
tested. Can the efficiency of an in vivo reprogramming approach be increased to achieve
a suitable β-cell mass for replenishment of lost β cells in large animals or humans?
Moreover, the long-term safety of reprogrammed cells is not known. An advantage of
direct reprogramming is that cells would be syngeneic to the prospective recipient;
yet, a potential disadvantage is that autoimmunity may limit the emergence of new
β cells in T1DM patients. Nonetheless, recent data provide significant encouragement
that a direct reprogramming approach could 1 day generate new β-like cells ex vivo,
or in a patient.
The Research Agenda
Refined and improved differentiation protocols are needed to achieve consistently
efficient yields of β cells in vitro which exhibit normal insulin secretion kinetics.
A better understanding of the signaling molecules regulating the later stages of endocrine
cell specification, delamination from the epithelium, formation of islet cell clusters,
and achievement of functional GSIS during in vivo development would greatly aid the
field in achieving the milestone of robust functionality in vitro. The field would
undoubtedly benefit from having a central core laboratory for comparisons of cells
generated by different protocols. Putting different cell populations from a variety
of laboratories and companies through a variety of functional assays such as static
incubation assays, perifusion assays, gene and protein expression assays, and mouse
transplant assays, and so on to directly compare cells would be a valuable endeavor
for the stem cell community.
Identifying and isolating derived β-like cells for investigation would benefit from
availability of advanced reporter lines. A human ESC Insulin-eGFP reporter line generated
by Micallef et al
315
has been used in several studies to characterize insulin-positive cell expression
profiles and physiology.
316,317
However, most in vitro differentiation conditions generate insulin + glucagon + polyhormonal
cells. Thus, it would be beneficial if additional reporters such as for Glucagon,
PDX1, or Nkx6.1 could be engineered into the same cell lines. Cell lines such as these
would aid in isolating various cell populations for further characterization and transplantation.
Additionally, engineering antibiotic resistance genes into the insulin, PDX1, and
other relevant gene loci could facilitate reducing the heterogeneity and improving
the purity of the resulting populations. Efficient genome editing methods are now
available to accomplish this; however, they remain costly and labor intensive.
318-320
The current assay for pluripotency and estimating teratoma risk is time consuming
and expensive. An improved predictive assay would support the field by providing a
reliable, rapid, sensitive, and quantitative measure of teratoma risk. Such an assay
would benefit preclinical studies anticipating investigational new drug submission.
Another “safety net” approach might involve developing hPSC lines that contain suicide
genes tagged to pluripotency or progenitor gene promoters which may be valuable for
ensuring removal of undifferentiated or partially differentiated cells thereby reducing
the risk of teratoma formation.
To date in preclinical studies, stem cell β-like derivatives have primarily been functionally
tested in mouse models of chemically induced diabetes, as a proof of principle. Few
other models have been tested because of the paucity of these diabetes models existing
on a suitable immunodeficient background. Therefore, having readily available additional
diabetes murine immunodeficient models would benefit the field. Additionally, more
robust humanized murine models as well as large animal models would also be advantageous
to studying the immunogenicity and dosing strategies of the stem cell-derived β-cell
populations.
Human iPSCs provide a valuable tool for disease modeling
268,321-325
and several hiPSC lines have been generated from patients with T1D and T2D.
266,267,326
From a scientific and disease modeling point of view, it would be useful to have hiPSC
lines from a variety of types of diabetes including monogenic diabetes to take advantage
of these possible in vitro disease models. Repositories could be established and then
these models would need to be characterized and validated. The combination of data
from genome wide association studies, genome editing and hPSC-derived β cells, provide
a powerful set of tools to study the genetics of various forms of diabetes and mechanisms
of pancreas development.
Safety is a potential issue for virally derived hiPSC lines intended to be used clinically
that relate to genomic perturbations and disease transmission. It is hoped that nonintegrating
methods of reprogramming would provide a satisfactory safety profile, but whether
this is the case after transplantation of iPSC progeny needs to be rigorously tested.
Safety concerns regarding oncogene expression, viral immunogenicity, and genetic instability
of hiPSCs may be averted with further improvements for derivation and expansion, making
this a feasible cell source for future therapeutic purposes.
327
Human PSC lines that possess characteristics that thwart or downregulate adaptive
and/or innate immune responses may be valuable cell lines for clinical application.
For example, Rong et al
302
expressed molecules which blocked costimulatory signals and downregulated immune responses
to hESC progeny. Other strategies might involve modifying HLA antigen expression on
hPSCs or expressing proteins that promote regulatory T cells.
Direct reprogramming usually involves adding pancreatic transcription factor genes
analogous to the addition of pluripotency genes for stem cell reprogramming. However,
the process of reprogramming with integrating lentiviral vectors can alter the genome
leading to malignant transformation or reduced differentiation. Although nonintegrating
vectors exist, those containing pancreatic transcription factors are not widely available
for use.
There is a need for better cell delivery devices taking advantage of material science,
3-dimensional culture, nanotechnology, matrix biology, improved oxygen delivery platforms,
and better encapsulation technology. Many encapsulation methods currently induce fibrosis
after implantation in large animals despite promising results in rodents. Materials
and devices that reduce the fibrogenic foreign body host response while still allowing
macromolecular nutrients to bathe cells and excluding immune cells, antibodies and
ideally cytokines would be a valuable transplant vehicle. Combining platforms could
provide a more physiological environment for growth, differentiation, and survival
of the cells long term.
Although islets can function in vivo after transplant as long as they are well vascularized,
challenges still exist with identifying an optimal site for islets and β-cell grafts
derived from hPSCs. Ideally, one would prefer to transplant allogeneic islets (or
human stem cell-derived endocrine cells) into patients into a site that is safe, accessible
with minimal risk, and optimal from the point of view of allowing adequate volume,
is not highly immunogenic, is well vascularized, and is free from instant blood-mediated
inflammation. Considering these requirements, the venous sac may prove a suitable
transplant site for allogeneic islets in human, but only preclinical studies have
been conducted to date.
328
Lymph nodes and the omentum have also been shown to be potential sites for islet transplantation.
329,330
Ultimately, additional bioengineering approaches may be needed to incorporate scaffolds
and ECM molecules to construct well-vascularized tissue from single cells. Several
studies have made headway in making devices or scaffolds for appropriate islet cell
engraftment.
331-333
A clinical study is currently underway to study whether a subcutaneously implanted,
prevascularized scaffold may be suitable for islet transplantation (http://clinicaltrials.gov/ct2/show/NCT01652911),
and this has clear relevance for stem cell-derived cells. Further studies in this
arena and approvals through the FDA will undoubtedly inform future related studies
with stem cell-derived β cells.
Summary and Speculation on Therapeutic Impact
Rapid progress and expansion of knowledge continues to characterize the field of deriving
β cells from stem cells which began approximately 15 years ago, at which time the
transcriptional network of pancreas development and nature of human definitive endoderm
was entirely unknown. We have come a long way since then. Yet, the majority of research
remains in the preclinical realm focused on optimizing methods for the in vitro conversion
of stem cells or somatic cells to high yield, functional cells that resemble adult
human islets/β cells. Given the complexity of the developmental processes scientists
are trying to mimic, it is not surprising that this has proven such a difficult task.
Nonetheless, real progress is occurring, and there is tremendous anticipation that
physiologically normal adult β-like cells will be achieved in the very near future.
Even without achieving this in vitro milestone, companies, such as ViaCyte, Inc (http://viacyte.com),
are moving ahead with pilot clinical trials. ViaCyte, Inc has proposed a phase I safety
and dosage trial combining an hESC-derived pancreatic progenitor cell product delivered
in a macroencapsulation device and transplanted into T1DM recipients. How this trial
and others like it unfold will significantly affect the public's and investor's impressions
of the field's potential. The results will either embolden others to propose additional
clinical pilot trials or send the field back to the drawing board. Still, many unanswered
questions need to be addressed and new technologies devised to support the responsible
development of the field. Given the steady progress and new innovations the field
has witnessed over the last several years, many are confident that the existing challenges
will ultimately be overcome and that an effective and safe stem cell-based β cell
replacement therapy will emerge in the coming decade.
Summary of Research Priorities
(1) Recent reports highlight progress in achieving refined differentiation protocols
for driving human pluripotent stem cells to β-like cells with improved physiological
function and greater capacity for more rapid correction diabetes in mice. However,
further work is needed to understand the reason for why these cells still do not exhibit
normal stimulus-secretion coupling or dynamic insulin release in perfusion assays.
(2) Additional studies evaluating host alloimmune and autoimmune responses to encapsulated
and unencapsulated human pluripotent stem cell-derived β cells, in both the syngeneic
and allogeneic settings, are needed.
(3) The stem cell-derived β cell therapy field would benefit by testing strategies
incorporating new encapsulation technologies, novel cellular deliver methods and sites,
and innovative tissue engineering approaches.
(4) Further experimental work is needed to study the ability to directly reprogram
somatic cells into β-like cells and assess their function in vitro and in animal models.
(5) Teratoma formation is a key safety issue with the potential therapeutic application
of human pluripotent stem cell-derived β cells. Studies which define this risk and
assays that better predict this risk would advance the field.
β CELL REGENERATION FROM PROLIFERATION AND NEOGENESIS
Current State of the Field
There is a great need to find sources of β cells that can be used to replenish those
that have been lost in diabetes. This commentary focuses on the potential of the pancreas
to regenerate β cells that can reverse the diabetic state. There are reasons to be
optimistic that new β cells can be generated by proliferation of existing β cells
and by neogenesis, the production of new islet cells from non-islet cells in the pancreas
or other organs.
The purpose of this short commentary to discuss the potential for β cell regeneration
in the human pancreas that could be exploited to replenish the β-cell deficit of people
with both T1D or T2D. The major questions being addressed are whether β`-cell replication
can be significantly enhanced and whether there are cells in the endocrine pancreas
or other organs that can serve as precursors for the formation of new β cells.
Is There Significant β Cell Turnover in the Adult Human Pancreas?
Some have argued that virtually all of one's β cells develop by the end of young adulthood
and that no new β cells appear during later adult life in humans.
334-336
One argument against significant human β-cell turnover comes from negative studies
using in vivo thymidine analog incorporation and radiocarbon dating.
334
Despite these conclusions, other data support the presence of some level of β-cell
turnover. For example, we know there is a constant loss of β cells as evidenced by
staining for terminal deoxynucleotidyl transferase dUTP nick end labeling
337
and other makers of death, yet β cell mass is well maintained for decades.
338,339
For this reason, there must be some generation of new β cells to keep up with cell
loss. Moreover, evidence suggests some expansion of β-cell mass in obesity and pregnancy.
337,340
The assumption that the process is slow is supported by studies quantifying the accumulation
of lipofucsin, a marker of aging, in a very high percentage of β cells.
341
Evidence for β Cell Replication in Adult Human Pancreas
Much of the evidence that β cells do not replicate comes from studies done on pancreases
obtained at autopsy or from cadaver donors.
335,342
The most commonly used tool for assessing β-cell replication is Ki67, but it is well
known in studies of cancer pathology that the numbers of mitotic figures and Ki67
positivity fall with warm and cold ischemia.
343,344
This has also been recently demonstrated with mouse and pig pancreases subjected to
autopsy conditions and evaluated with Ki67.
345
These findings support the likelihood that the negligible replication rates found
with Ki67 have led to erroneous conclusions. They also fit with the finding that Ki67
positivity in β cells can be found in fresh surgical specimens of pancreas and in
human islets transplanted under the kidney capsule of immunodeficient mice.
346
The actual rate of β-cell birth from replication is very difficult to estimate because
Ki67 positivity is not necessarily equated with the generation of new cells. However,
if β cells in adult humans have 0.4% Ki67 positivity, and Ki67 positivity lasts 12
hours, and if there were no neogenesis or apoptosis, β-cell mass could more than double
in less than a year. However, Ki67 positivity can be found in cells that do not divide
but are arrested in cycle. It can also be associated with DNA damage and apoptosis.
347
Although many unknowns remain, there is now good evidence that there is some capacity
for regeneration of β cells from replication in adult human pancreases.
Evidence for β Cell Neogenesis in Adult Human Pancreas
Islet neogenesis in rodents and humans remains a controversial topic.
348,349
Lineage tracing experiments in mice have provided mixed results, and it is unlikely
that similar studies can be done with human tissue. The evidence in support of neogenesis
remains circumstantial, some of it stronger than others. For example, the presence
of insulin-stained cells in the duct epithelium and the finding of increased numbers
of single and small clumps of β cells in human pregnancy and in other situations may
be suggestive but are hardly definitive.
340,350,351
One cannot be certain that these small clumps of β cells did not arise from replication
of a few existing cells.
However, finding cells in the duct epithelium that costain for insulin and cytokeratin
19 carry more weight
350
because they suggest a dynamic process. In addition, pancreatic intraductal neoplasms
are rarely seen before age 35 years, but are found in 60% of non-neoplastic pancreases
by age 45 years and in 75% by age 55 years.
352
Pancreatic intraductal neoplasms can frequently contain significant numbers of islet
hormone-positive cells.
Is There Evidence That β Cell Growth can be Stimulated in the Adult Pancreas?
The β cell mass is modestly increased by 30% to 50% in insulin-resistant obese human
subjects.
337,339
Another example of increased β-cell mass is the normal human pregnancy.
340
Increased β-cell mass and high circulating GLP-1 levels have been seen in some subjects
after bariatric surgery
353
; however, the cause-and-effect relationship between the two has not been established.
On the negative side, subjects with T2D have been treated for years with GLP-1 agonists
and dipeptidyl peptidase-4 inhibitors, yet no evidence for increased β cell functional
mass has emerged after drug treatment was stopped.
354
Patients with longstanding (over 50 years) T1D can routinely be found to have some
β cells in their autopsied pancreases.
355
The question of whether these cells are resistant to immune killing has not been answered.
These could be a subset of uniquely strong β cells that survived from childhood, but
a more attractive hypothesis is that new β cells are generated continuously from neogenesis
and then killed by autoimmunity. This possibility is supported by the presence of
islet cell antibodies in these subjects, by the finding of many single and small clusters
of β cells in the pancreases, which suggest neogenesis, and by the observation of
some lymphocytes in the few remaining islets of these pancreases.
What Causes β Cell Death?
At least some β cells have a limited life span with an apoptotic death. Perhaps some
cells live for decades, although other die early for unknown reasons. One possibility
is that islets and most other organs have a natural remodeling process, such that
cell birth and apoptosis serve as mechanisms to facilitate structural change.
In the development of both T1D and T2D, there has been much speculation about what
might cause an increased rate of cell death other than immune destruction.
356
Although usually discussed as being important for T2D, the same problems must be faced
by the residual β cells of T1D. The reality is that we have little idea about which
processes are the most important pathways to death, but attention has focused on the
following candidates:
(a) Glucose toxicity (glucotoxicity): Clearly, hyperglycemia, even in the range of
just impaired glucose tolerance, has a deleterious influence upon β cell function,
most notably acute GSIS. There is currently considerable interest in the dedifferentiation
of β cells that occurs in a hyperglycemic environment.
356,357
Some of these phenotypic changes are presumably responsible for dysfunctional insulin
secretion. Importantly, insulin secretion rapidly returns to normal in T2D shortly
after normoglycemia is restored by bariatric surgery.
358
Little is known about the molecular mechanisms through which glucotoxicity might cause
β cell death. The descriptive term “overwork” is often used and it would be helpful
to have this concept better defined.
(b) Lipotoxicity and glucolipotoxicity: Almost all of the evidence supporting the
presence of lipotoxicity or glucolipotoxicity comes from in vitro experiments in which
free fatty acid such as palmitate are added to isolated islets or β cell lines. Palmitate
and other free fatty acids certainly have toxic effects, and as such can be useful
to examine stress and death pathways. However, do FFAs exert adverse effects on β
cells in real life? As of yet, the evidence that this occurs in vivo in human or animal
diabetes is sparse and unconvincing.
359,360
Moreover, it is difficult to find correlations between β cell dysfunction and FFA
levels as subjects develop diabetes, whereas the correlation between rising glucose
levels is very strong.
361
(c) ER stress: There are good reasons to think that ER stress from the secretory demands
of insulin resistance and hyperglycemia leads to the demise of some β cells
362
but we do not have good markers that can follow the process.
(d) Oxidative stress (ROS): Again, there are many reasons to think that oxygen radicals
could cause cell death resulting from β cell “overwork” or the challenges of the diabetic
environment,
363
but this process also lacks markers.
(e) Amyloid: Amyloid deposits are found in many of the islets of people with T2D but
there are also few in normoglycemic obese subjects with insulin resistance. The amyloid
deposits are formed by fibrils of IAPP and a strong case has been made the initially
formed IAPP oligomers can damage membranes and cause cell death.
364,365
The Research Agenda
Can We Significantly Increase β-Cell Mass by Stimulating β-Cell Replication Either
In Vivo or In Vitro?
Much has been learned about the cell cycle mechanisms in murine and human β cells.
366-368
In addition, various compounds that can stimulate β-cell replication have been identified
by high-throughput screening.
369,370
There has been a recent identification of betatrophin, which is thought to be a factor
secreted by insulin-resistant livers that can stimulate β cell replication.
371
Its mechanism of action has not yet been defined. With all of these works, the differences
between mice and humans must be carefully defined because β-cell turnover in mice
is far higher than that in humans. Research on this topic remains active and promising.
It would be ideal if an intervention could be identified that could stimulate β-cell
replication with no side effects just enough to restore that β-cell mass to normal.
However, it is hard to imagine that such a safe-specific drug with definable activity
will be developed in the near future. It should be more feasible to find a way to
enhance replication of β cells from isolated islets in vitro, whereupon the cell product
could be carefully characterized and then could be transplanted.
What Are The prospects for Significantly Increasing β Cell Mass by Stimulating Neogenesis
Either In Vivo or In Vitro?
In Vivo Possibilities
Similar to the situation with replication, an effective in vivo treatment to effectively
induce neogenesis will probably be very difficult to develop. However, a striking
recent result found that in alloxan diabetic mice significant recovery of functional
β-cell mass from terminally differentiated acinar cells could be induced by short-term
growth factor therapy (epidermal growth factor and ciliary neurotrophic factor), thus
providing potential avenues for drug development.
372
Another notable finding is that pancreatic exocrine cells can be reprogrammed to become
β-like cells by injections into mouse pancreas of adenoviruses carrying 3 transcription
factors, Ngn-3, Pdx-1, and MafA, which are important for β cell development and maintenance.
373
In Vitro Possibilities
An increasing number of investigators are focusing on ways to generate β cells from
exocrine cells in vitro. The first encouraging result was a demonstration in 2000
that cultured human duct cells covered with Matrigel could produce new islet cells,
374
a result that was confirmed shortly thereafter.
375
There continues to be a lot of work exploring the potential of converting pancreatic
duct cells,
374,376
centroacinar cells,
377
or acinar cells
372
to new islet cells and even fully formed islets.
To obtain sufficient numbers of islet cells from exocrine cells, it will be necessary
to expand the exocrine cells in vitro. One approach is to exploit natural branching
morphogenesis to create organoids in tissue culture (481). Another is to expand exocrine
or even islet cells in tissue culture through the process of epithelial-mesenchymal
transition and then use various differentiation factors to produce islet cells that
might be used for transplantation.
379
Among recent examples of progress, the Clevers group found a way to activate duct
cells to express Lgr5, a marker for adult stem cells.
380
These have a propensity to develop into organoids, which allows expansion, and then
when transplanted with fetal pancreas into recipient mouse kidneys can form islet
cells. The group of Kim
381
was able to expand purified CD133+ human duct cells into epithelial spheres and then
reprogram them with adenoviruses carrying Ngn-3, Pdx-1, MafA, and Pax6 into human
IPCs. A similar approach has been used by the group of Docherty.
376
Islet Cell Plasticity
There has been considerable interest in the possibility that 1 type of islet cell
can be converted to another. Leading the way, the Herrera group used lineage tracing
to show that after severe β-cell ablation with diphtheria toxin, some of the residual
α cells could assume a β-cell phenotype.
382
With genetic engineering, the group of Collombat showed that expression of Pax4 in
α cells led to the conversion of these cells to β cells.
383
Another example comes from lineage tracing studies with diabetic Foxo1 knockout mice,
in which conversion for α cells to β cells was demonstrated.
357
A major question is whether such conversion happens naturally to any meaningful extent.
A study using extreme ablation with streptozotocin found no evidence for such regeneration.
384
The more important question is whether this is a pathway that can be exploited for
therapeutic purposes. Although challenging, there are enough α cells to hope that
conversion and expansion might provide enough β cells to provide therapeutic benefits.
CONCLUSIONS
Much work is now being done to find a new source of β cells that can be used to replenish
the β-cell deficit of diabetes. There is well-justified excitement about progress
in converting hESCs and hiPSCs to β cells. However, there are also impressive advances
in finding ways to exploit the regenerative potential of cells in the adult human
pancreas. It is essential that these and other promising avenues be intensively evaluated.
Summary of Research Priorities
(1) Evidence is presented that there is a slow rate of β cell turnover in the human
adult pancreas, occurring from replication of existing β cells and the birth of new
β cells through neogenesis. The potential of exploiting this for clinical application
is being explored by many laboratories.
(2) There is also a slow rate of β-cell death in the adult human pancreas occurring
through the processes of apoptosis and necrosis.
(3) There is evidence that the rate of β cell death is increased in T2D, and the contributing
mechanisms are thought to include endoplasmic reticulum stress, toxic amyloid oligomers,
oxidative injury, and the ill-defined processes of overwork and glucose toxicity.
Report of Meeting Survey
The quest for a safe and efficacious form of β-cell replacement is at a unique juncture
in its history with multiple therapies with either existing or potential application
that will in the coming years compete for a place in the care of patients with T1D
and potentially T2D. To gain a sense from the expert group, we assembled in Oxford
as to their opinion of what the field of β-cell replacement would look like in the
future, we conducted a survey on the final day of the meeting. The survey asked each
participant to assign their predicted market share at 5, 10, and 20 years in the future
of clinical β cell replacement activities to each of the therapies we discussed: allogeneic
islets, xenogeneic islets (encapsulated or not), tolerance for allogeneic islets,
tolerance for xenogeneic islets, AP, ES-derived β cells, encapsulated ES-derived β
cells, IP-derived β cells, and β-cell regeneration from proliferation or neogenesis.
For each timepoint, each survey participant assigned 100% of market share among these
therapies.
The results in Figure 1 reveal that at 5 years hence, our panel believes that the
market will be dominated by isolated allogeneic islets and the AP with anticipated
minor contributions from IPs and ES-derived β cells, xeno islets, and tolerance. About
50% of respondents predicted no or negligible contributions from tolerance to xeno
islets, endogenous islet regeneration/proliferation and IPs derived β cells. At 10
years, the most significant increase was in the predicted role of encapsulated ES-derived
β cells and further expansion of reliance on AP. There were also minor increases in
xeno islets, tolerance to allo islets, IP-derived β cells, and continued significant
predicted activity in allo islets. By 20 years, hence, our experts predicted market
domination by IP-derived β cells and AP with contraction of allo-islet activity and
modest expansion of β cell proliferation/regeneration. The survey has obvious limitations
including a small sample size and pool of respondents selected for meeting participation
based on expertise in a given areas that likely carries with it an associated bias.
FIGURE 1
Expected changes in β cell replacement over time. before the conclusion of the conference,
a web based survey was completed by the participants seeking their individual opinion
as to what portion of the β-cell replacement market would be captured by the existing
and potential therapies discussed at the meeting. For each of the time horizons of
5, 10, and 20 years, respondents allocated predicted market share between 9 potential
therapeutic options. The graph shows the average market share awarded to specific
therapies at 5, 10, and 20 years.
Summary
Biologic or biomechanical therapy capable of replacing the β cell mass has the potential
to positively impact the health and well being of millions of people with insulin-dependent
diabetes. Research in this area stands at a pivotal moment at which a number of viable
strategies exist or are under development. Broad application depends on achieving
both technical and financial feasibility. The ultimate goal of a “true cure,” in which
diabetic individuals achieve euglycemia with a single procedure associated with minimal
risk, without long-term toxic drugs, and unfettered by external devices and/or frequent
monitoring, appears to still be some years away. However, dramatic progress has been
achieved toward the more proximate objectives of improved glycemic control and elimination
of hypoglycemia and long-term vascular complications.
Long-term whole organ pancreas and isolated islet results have improved significantly
over the last decade, with the latter now approaching the success of the former in
insulin independence rates at the 5-year mark. It seems likely that allogeneic pancreas
and islet transplantation will remain a treatment of choice for the foreseeable future
in kidney recipients already obligated to lifelong immunosuppression until a more
complete and permanent restoration of euglycemia is available. Nascent tolerance promoting
protocols could aid in improving the risk-to-benefit balance for both islets and whole
organ pancreas. With the present supply of transplantable pancreases used optimally,
no more than 13% of the annual incident cases of T1D can be cured. In practical terms
though, today, fewer than 5% of the annual incident cases are transplanted. The reality
of the limited supply of deceased donor organs ultimately constrains the impact of
islet and pancreas transplantation and compels researchers to press forward to develop
broader strategies such as the AP, xenogeneic islets, and stem cell-derived β cells
for which the supply will be limitless; in these areas, recent progress has been most
impressive.
The AP continues to be refined with more sophisticated delivery algorithms, improved
sensors and exploration of mobile device control. For xenogeneic islets, dramatic
progress is evident in the long-term survival of porcine islets in primates using
genetically modified donors and/or improved biologic immunosuppressants. Microencapsulation
and macroencapsulation devices that exclude direct immunity by physical means may
further aid in fostering xenogeneic islet graft survival but will likely find their
primary place in the containment and protection of early versions of stem cell–derived
allogeneic β cells. Deriving functional β cells from stem cells has experienced the
most celebrated recent advances. Improved differentiation protocols that permit large
scale/unlimited production of IPCs are now available, and although “normal” β-cell
function has not yet been achieved, the ever quickening pace of progress suggests
they are not far off. Importantly, this therapeutic modality will ultimately need
to confront the likely requirement for a containment device and the need to be retransplanted
periodically. These blemishes notwithstanding, the tremendous perceived potential
of the approach for clinical application is evident in the huge venture capital investment
that was rapidly garnered after the report of the most recent advance in embryonic
stem cell differentiation into proper β cells. Consistent with the informal survey
we conducted, iPS-derived β cells, which currently suffer from regulatory hurdles
and the lack of a viable business model, and the seemingly more remote regeneration
of native β cells, may offer the ultimate chance for a personalized true cure of insulin-dependent
diabetes by avoidance of barrier devices and toxic immunosuppressive drugs.
The research agenda we have detailed is designed to facilitate full exploration of
the potential of each proposed β-cell replacement solution so the optimal therapy
is advanced as quickly as possible. Success in this endeavor will require broad and
deep financial support from philanthropic (JDRF, Diabetes Research and Wellness Foundation,
ADA, and so on) and public funding agencies worldwide; the investment needed is large
but the potential reward will be profound. It is imperative that high impact, scientifically
sound approaches are not overwhelmed by industry, private, or venture capital-supported
priorities just because they hold a more lucrative near-term business model; scientific
merit should dictate the course. The adherence of the historical funding agencies
to traditional peer-reviewed methodology will be the incubator of novel approaches.
This is a rapidly evolving landscape, and new data and novel ideas may radically divert
the path forward. However, the diverse recent progress is tangible and undeniable,
and the next decade is bound to witness a fascinating unfolding of competing solutions
to cure insulin-dependent diabetes.
Our assessment of the data presented creates the opportunity for IPITA/TTS to endorse
the following broad agenda for specific support by the peer-reviewed agencies.
(1) Allogeneic islet transplantation using novel strategies to facilitate engraftment,
enhance graft longevity and ultimately gain immunosuppression-free survival in adult
and pediatric patients.
(2) Xenogeneic islet-based approaches with and without encapsulation.
(3) Stem cell–based therapy of diabetes.
(4) Regeneration based therapy.
(5) Mobile device–based control of glucose sensing-insulin delivery: AP.