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      Use of Embryonic Stem Cells for Endocrine Disorders

      Hormone Research in Paediatrics

      S. Karger AG

      Preimplantation embryos, Embryonic stem cells, Diabetes, Pancreatic differentiation

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          Abstract

          Embryonic stem cells (ESCs) can be derived from unused cultured human embryos, provided by patients undergoing treatment for infertility, that would otherwise be disposed of. The inner cell mass cells form immortal pluripotent cell colonies that may be directed into a wide range of cell lineages and end-differentiated cell types. Endocrine lineages are now being actively explored, particularly for their potential to differentiate into pancreatic insulin-producing cells. The majority of published research involving stem cell-directed differentiation into pancreatic islet cells has been conducted using mouse ESCs. The evidence shows that cells that are glucose-responsive for insulin release can be produced using differentiation strategies involving a sequence of growth factor-directed steps. However, because of the close association between neural and pancreatic cell types and markers, it is possible that many of the cells produced by these strategies may be more neural than pancreatic. Thus, it is important to replicate the normal developmental pathways that include the sequential expression of appropriate endoderm and pancreatic progenitor markers. Human pancreatic differentiation from ESCs may require instruction from embryonic mesenchyme to enable the complete maturation of early pancreatic cells and the formation of true β-islet cells.

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          Most cited references 22

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          Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells.

          Previous reports have demonstrated the growth of undifferentiated human embryonic stem (HES) cells on mouse embryonic fibroblast (MEF) feeders and on laminin- or Matrigel-coated plastic surfaces supplemented with MEF-conditioned medium. These xenosupport systems run the risk of cross-transfer of animal pathogens from the animal feeder, matrix, or conditioned medium to the HES cells, thus compromising later clinical application. Here we show that human fetal and adult fibroblast feeders support prolonged undifferentiated HES cell growth of existing cell lines and are superior to cell-free matrices (collagen I, human extracellular matrix, Matrigel, and laminin) supplemented with human or MEF feeder-conditioned medium. Additionally, we report the derivation and establishment of a new HES cell line in completely animal-free conditions. Like HES cells cultured on MEF feeders, the HES cells grown on human feeders had normal karyotypes, tested positive for alkaline phosphatase activity, expressed Oct-4 and cell surface markers including SSEA-3, SSEA-4, Tra 1-60, and GCTM-2, formed teratomas in severely combined immunodeficient (SCID) mice, and retained all key morphological characteristics. Human feeder#150;supported HES cells should provide a safer alternative to existing HES cell lines in therapeutic applications.
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            A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells.

            Human embryonic stem (hES) cell lines were first cultured using fetal mouse fibroblasts as feeder cells. To avoid feeders and to reduce the amount of xeno-components, Matrigel- and laminin-coated dishes, and conditioned mouse feeder cell medium have been used, and hES cells have also been cultured on human fetal muscle and skin, and adult Fallopian tube epithelial cells. We used post-natal, commercially available human foreskin fibroblasts as feeder cells. Inner cell masses (ICM) were isolated from five supernumerary blastocysts, obtained as donations from couples undergoing IVF treatment. Two ICM showed continuous growth. One line, HS181, has been in culture for 41 weeks with a doubling time of 24-36 h. It continues to express stem cell markers alkaline phosphatase, Oct-4, stage-specific embryonic antigen (SSEA)-4 and tumour-related antigen (TRA)-1-60. The karyotype is 46,XX. Pluripotency was demonstrated by teratoma formation in immunodeficient mice. In high-density cultures, spontaneous differentiation to beating cells and neuron-like cells was seen. The second line, HS207, was cultured for 9 weeks and cryopreserved, as were samples of line HS181. Both lines began to grow after thawing. We used successfully human foreskin fibroblasts as feeder cells for derivation and continued undifferentiated growth of hES cells. These feeder cells are convenient for IVF units, because no fetal human tissues or tissue from operations are needed.
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              Characterization and culture of human embryonic stem cells.

              Human embryonic stem cells have been defined as self-renewing cells that can give rise to many types of cells of the body. How and whether these cells can be manipulated to replace cells in diseased tissues, used to screen drugs and toxins, or studied to better understand normal development, however, depends on knowing more about their fundamental properties. Many different human embryonic stem cell lines--which are pluripotent, proliferate indefinitely in vitro and maintain a normal, euploid karyotype over extended culture--have now been derived, but whether these cell lines are in fact equivalent remains unclear. It will therefore be important to define robust criteria for the assessment of both existing and newly derived cell lines and for the validation of new culture conditions.
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                Author and article information

                Journal
                HRE
                Horm Res Paediatr
                10.1159/issn.1663-2818
                Hormone Research in Paediatrics
                S. Karger AG
                978-3-8055-8255-1
                978-3-318-01446-4
                1663-2818
                1663-2826
                2007
                February 2007
                15 February 2007
                : 67
                : Suppl 1
                : 28-31
                Affiliations
                Monash Immunology and Stem Cell Laboratories, Monash University, Clayton, Vic., Australia
                Article
                97547 Horm Res 2007;67:28–31
                10.1159/000097547
                © 2007 S. Karger AG, Basel

                Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

                Page count
                References: 29, Pages: 4
                Categories
                Plenary Lecture 4

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