Approaches for corneal endothelium regenerative medicine

Differentiated cells can be reprogrammed to an embryonic-like state by transfer of nuclear contents into oocytes or by fusion with embryonic stem (ES) cells. Little is known about factors that induce this reprogramming. Here, we demonstrate induction of pluripotent stem cells from mouse embryonic or adult fibroblasts by introducing four factors, Oct3/4, Sox2, c-Myc, and Klf4, under ES cell culture conditions. Unexpectedly, Nanog was dispensable. These cells, which we designated iPS (induced pluripotent stem) cells, exhibit the morphology and growth properties of ES cells and express ES cell marker genes. Subcutaneous transplantation of iPS cells into nude mice resulted in tumors containing a variety of tissues from all three germ layers. Following injection into blastocysts, iPS cells contributed to mouse embryonic development. These data demonstrate that pluripotent stem cells can be directly generated from fibroblast cultures by the addition of only a few defined factors.

Many bacterial clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated (Cas) systems employ the dual RNA–guided DNA endonuclease Cas9 to defend against invading phages and conjugative plasmids by introducing site-specific double-stranded breaks in target DNA. Target recognition strictly requires the presence of a short protospacer adjacent motif (PAM) flanking the target site, and subsequent R-loop formation and strand scission are driven by complementary base pairing between the guide RNA and target DNA, Cas9–DNA interactions, and associated conformational changes. The use of CRISPR–Cas9 as an RNA-programmable DNA targeting and editing platform is simplified by a synthetic single-guide RNA (sgRNA) mimicking the natural dual trans-activating CRISPR RNA (tracrRNA)–CRISPR RNA (crRNA) structure. This review aims to provide an in-depth mechanistic and structural understanding of Cas9-mediated RNA-guided DNA targeting and cleavage. Molecular insights from biochemical and structural studies provide a framework for rational engineering aimed at altering catalytic function, guide RNA specificity, and PAM requirements and reducing off-target activity for the development of Cas9-based therapies against genetic diseases.

Current neural induction protocols in human ES cells (hESCs) rely on embryoid body formation, stromal feeder co-culture, or selective survival conditions; each strategy displaying significant drawbacks such as poorly defined culture conditions, protracted differentiation and low yield. Here we report that the synergistic action of two inhibitors of SMAD signaling, Noggin and SB431542, is sufficient for inducing rapid and complete neural conversion of hESCs under adherent culture conditions. Temporal fate analysis reveals a transient FGF5+ epiblast-like stage followed by PAX6+ neural cells competent of rosette formation. Initial cell density determines the ratio of CNS versus neural crest progeny. Directed differentiation of human iPSCs into midbrain dopamine and spinal motoneurons confirm robustness and general applicability of the novel induction protocol. Noggin/SB431542 based neural induction should greatly facilitate the use of hESC and hiPSCs in regenerative medicine and disease modeling and obviate the need for stromal feeder or embryoid body based protocols.