Many forms of blindness result from the dysfunction or loss of retinal photoreceptors. Induced pluripotent stem cells (iPSC) hold great potential for the modeling of these diseases or as potential therapeutic agents. However, to fulfill this promise, a remaining challenge is to induce human iPSC to recreate in vitro key structural and functional features of the native retina, in particular the presence of photoreceptors with outer-segment discs and light-sensitivity. Here we report that hiPSC can, in a highly autonomous manner, recapitulate spatiotemporally each of the main steps of retinal development observed in vivo and form 3-dimensional retinal cups that contain all major retinal cell types arranged in their proper layers. Moreover, the photoreceptors in our hiPSC-derived retinal tissue achieve advanced maturation, showing the beginning of outer-segment-disc formation and photosensitivity. This success brings us one step closer to the anticipated use of hiPSC for disease modeling and open possibilities for future therapies.

BackgroundThe production of cardiomyocytes from human induced pluripotent stem cells (hiPSC) holds great promise for patient-specific cardiotoxicity drug testing, disease modeling, and cardiac regeneration. However, existing protocols for the differentiation of hiPSC to the cardiac lineage are inefficient and highly variable. We describe a highly efficient system for differentiation of human embryonic stem cells (hESC) and hiPSC to the cardiac lineage. This system eliminated the variability in cardiac differentiation capacity of a variety of human pluripotent stem cells (hPSC), including hiPSC generated from CD34+ cord blood using non-viral, non-integrating methods.Methodology/Principal FindingsWe systematically and rigorously optimized >45 experimental variables to develop a universal cardiac differentiation system that produced contracting human embryoid bodies (hEB) with an improved efficiency of 94.7±2.4% in an accelerated nine days from four hESC and seven hiPSC lines tested, including hiPSC derived from neonatal CD34+ cord blood and adult fibroblasts using non-integrating episomal plasmids. This cost-effective differentiation method employed forced aggregation hEB formation in a chemically defined medium, along with staged exposure to physiological (5%) oxygen, and optimized concentrations of mesodermal morphogens BMP4 and FGF2, polyvinyl alcohol, serum, and insulin. The contracting hEB derived using these methods were composed of high percentages (64–89%) of cardiac troponin I+ cells that displayed ultrastructural properties of functional cardiomyocytes and uniform electrophysiological profiles responsive to cardioactive drugs.Conclusion/SignificanceThis efficient and cost-effective universal system for cardiac differentiation of hiPSC allows a potentially unlimited production of functional cardiomyocytes suitable for application to hPSC-based drug development, cardiac disease modeling, and the future generation of clinically-safe nonviral human cardiac cells for regenerative medicine.

We elucidate the cellular and molecular kinetics of the stepwise differentiation of human embryonic stem cells (hESCs) to primitive and definitive erythromyelopoiesis from human embryoid bodies (hEBs) in serum-free clonogenic assays. Hematopoiesis initiates from CD45 hEB cells with emergence of semiadherent mesodermalhematoendothelial (MHE) colonies that can generate endothelium and form organized, yolk sac-like structures that secondarily generate multipotent primitive hematopoietic stem progenitor cells (HSPCs), erythroblasts, and CD13 ؉ CD45 ؉ macrophages. A first wave of hematopoiesis follows MHE colony emergence and is predominated by primitive erythropoiesis characterized by a brilliant red hemoglobinization, CD71/CD325a (glycophorin A) expression, and exclusively embryonic/ fetal hemoglobin expression. A second wave of definitive-type erythroid burstforming units (BFU-e's), erythroid colonyforming units (CFU-e's), granulocytemacrophage colony-forming cells (GMCFCs), and multilineage CFCs follows next from hEB progenitors. These stages of hematopoiesis proceed spontaneously from hEB-derived cells without requirement for supplemental growth factors during hEB differentiation. Gene expression analysis of differentiating hEBs revealed that initiation of hematopoiesis correlated with increased levels of SCL/TAL1, GATA1, GATA2, CD34, CD31, and the homeobox gene-regulating factor CDX4 These data indicate that hematopoietic differentiation of hESCs models the earliest events of embryonic and definitive hematopoiesis in a manner resembling human yolk sac development, thus providing a valuable tool for dissecting the earliest events in human HSPC genesis. IntroductionClassic and contemporary anatomic studies of human embryos have revealed that human hematopoiesis begins in the second to third embryonic weeks with formation of mesoderm-derived blood islands in the extraembryonic mesoderm of the developing secondary yolk sac. 1,2 Blood islands develop foci of nucleated erythroblasts ("megaloblasts"), 3 intimately associated with and surrounded by endothelium. Yolk sac (primitive) blood cells consist of nucleated primitive erythrocytes expressing exclusively embryonic hemoglobins (eg, ⑀ 2 2 globin chains) and primitive macrophages that arise without detectable monocytic precursors. Following the onset of circulation at about 21 days of development, yolk sac cells are found in embryonic blood. The fetal liver subsequently replaces the yolk sac as the main hematopoietic organ 4,5 with appearance of definitive enucleate, macrocytic erythrocytes expressing fetal hemoglobins (eg, ␣ 2 ␥ 2 globin chains). Definitive blood cells and hematopoietic stem progenitor cells (HSPCs) can be detected in the fetal liver and embryo beginning at 5 to 6 weeks but have also been assayed as early as 4 to 5 weeks from human yolk sac, suggesting a gradual yolk sac/fetal liver HSPC transition. 1,6 The human adult long-term repopulating HSPC that ultimately seeds the fetal bone marrow and thymus is the legacy of fetal liver hematopoiesis.Both ...