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Objective Stem cell therapy for angiogenesis and vascular regeneration has been investigated using adult or embryonic stem cells. In the present study, we investigated the potential of endothelial cells (ECs) derived from human induced pluripotent stem cells (hiPSCs) to promote the perfusion of ischemic tissue in a murine model of peripheral arterial disease. Methods and Results Endothelial differentiation was initiated by culturing hiPSCs for 14 days in differentiation media supplemented with BMP-4 and VEGF. The hiPSC-ECs exhibited endothelial characteristics by forming capillary-like structures in matrigel and incorporating acetylated-LDL. They stained positively for EC markers such as KDR, CD31, CD144 and eNOS. In vitro exposure of hiPSC-ECs to hypoxia resulted in increased expression of various angiogenic related cytokines and growth factors. hiPSC-ECs were stably transduced with a double fusion construct encoded by the ubiquitin promoter, firefly luciferase for bioluminescence imaging (BLI) and green fluorescence protein (GFP) for fluorescent detection (pUb-Fluc-GFP). The hiPSC-ECs (5×105) were delivered by intramuscular injection into the ischemic hindlimb of SCID mice at day 0 and again on day 7 after femoral artery ligation (n=8). BLI showed that hiPSC-ECs survived in the ischemic limb for at least 2 weeks. In addition, laser Doppler imaging showed that the ratio of blood perfusion was increased by hiPSC-EC treatment by comparison to the saline-treated group (0.58±0.12 vs 0.44±0.04; P=0.005). The total number of capillaries in the ischemic limb of mice receiving hiPSC-EC injections was greater than those in the saline-treated group (1284 ±155 vs. 797±206 capillaries/mm2) (P<0.002). Conclusion This study is a first step toward development of a regenerative strategy for peripheral arterial disease based on the use of ECs derived from hiPSCs.

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Recent evidence suggests human embryonic stem (ES) and induced pluripotent stem (iPS) cell lines have differences in their epigenetics marks and transcriptomes, yet the impact of these differences on subsequent terminally differentiated cells is less well understood. Comparison of purified, homogeneous populations of somatic cells derived from multiple independent human iPS and ES lines will be required to address this critical question. Here, we report a differentiation protocol based on embryonic development that consistently yields large numbers of endothelial cells (EC) derived from multiple human ES or iPS cells. Mesoderm differentiation of embryoid bodies was maximized and defined growth factors were used to generate KDR+ EC progenitors. Magnetic purification of a KDR+ progenitor subpopulation resulted in an expanding, homogeneous pool of ECs that expressed EC markers and had functional properties of ECs. Comparison of the transcriptomes revealed limited gene expression variability between multiple lines of human iPS–derived ECs, or between lines of ES– and iPS–derived ECs. These results demonstrate a method to generate large numbers of pure human EC progenitors and differentiated ECs from pluripotent stem cells, and suggest individual lineages derived from human iPS cells may have significantly less variance than their pluripotent founders.

The vertebrae mesoderm is a source of cells that forms a variety of tissues, including the heart, vasculature, and blood. Consequently, the derivation of various mesoderm-specific cell types from human embryonic stem cells (hESCs) has attracted the interest of many investigators owing to their therapeutic potential in clinical applications. However, the need for efficient and reliable methods of differentiation into mesoderm lineage cell types remains a significant challenge. Here, we demonstrated that inhibition of glycogen synthase kinase-3 (GSK-3) is an essential first step toward efficient generation of the mesoderm. Under chemically defined conditions without additional growth factors/cytokines, short-term GSK inhibitor (GSKi) treatment effectively drives differentiation of hESCs into the primitive streak (PS), which can potentially commit toward the mesoderm when further supplemented with bone morphogenetic protein 4. Further analysis confirmed that the PS-like cells derived from GSKi treatment are bipotential, being able to specify toward the endoderm as well. Our findings suggest that the bipotential, PS/mesendoderm-like cell population exists only at the initial stages of GSK-3 inhibition, whereas long-term inhibition results in an endodermal fate. Lastly, we demonstrated that our differentiation approach could efficiently generate lateral plate (CD34 + KDR + ) and paraxial (CD34 -PDGFRa + ) mesoderm subsets that can be further differentiated along the endothelial and smooth muscle lineages, respectively. In conclusion, our study presents a unique approach for generating early mesoderm progenitors in a chemically directed fashion through the use of small-molecule GSK-3 inhibitor, which may be useful for future applications in regenerative medicine.

Background The generation of vascular progenitors (VP) from human induced pluripotent stem cells (hiPSC) has great potential for treating vascular disorders such as ischemic retinopathies. However, long-term in vivo engraftment of hiPSC-derived VP into retina has not yet been reported. This goal may be limited by the low differentiation yield, greater senescence, and poor proliferation of hiPSC-derived vascular cells. To evaluate the potential of hiPSC for treating ischemic retinopathies, we generated VP from a repertoire of viral-integrated and non-integrated fibroblast and cord blood (CB)-derived hiPSC lines, and tested their capacity for homing and engrafting into murine retina in an ischemia-reperfusion (I/R) model. Methods and Results VP from human embryonic stem cells (hESC) and hiPSC were generated with an optimized hemato-vascular differentiation system. FACS-purification of human embryoid body (hEB) cells differentially expressing endothelial/pericytic markers identified a CD31+ CD146+ VP population with high vascular potency. Episomal CB-iPSC generated these VP with higher efficiencies than fibroblast-iPSC. Moreover, in contrast to fibroblast-iPSC-VP, CB-iPSC-VP maintained expression signatures more comparable to hESC-VP, expressed higher levels of immature vascular markers, demonstrated less culture senescence and sensitivity to DNA damage, and possessed fewer transmitted reprogramming errors. Luciferase transgene-marked VP from hESC, CB-iPSC, and fibroblast-iPSC were injected systemically or directly into the vitreous of retinal I/R-injured adult NOD-SCID mice. Only hESC- and CB-iPSC-derived VP reliably homed and engrafted into injured retinal capillaries, with incorporation into damaged vessels for up to 45 days. Conclusions VP generated from CB-iPSC possessed augmented capacity to home, integrate into, and repair damaged retinal vasculature.

BackgroundHeterogeneity of endothelial cells (ECs) is a hallmark of the vascular system which may impact the development and management of vascular disorders. Despite the tremendous progress in differentiation of human embryonic stem cells (hESCs) towards endothelial lineage, differentiation into arterial and venous endothelial phenotypes remains elusive. Additionally, current differentiation strategies are hampered by inefficiency, lack of reproducibility, and use of animal-derived products.MethodsTo direct the differentiation of hESCs to endothelial subtypes, H1- and H9-hESCs were seeded on human plasma fibronectin and differentiated under chemically defined conditions by sequential modulation of glycogen synthase kinase-3 (GSK-3), basic fibroblast growth factor (bFGF), bone morphogenetic protein 4 (BMP4) and vascular endothelial growth factor (VEGF) signaling pathways for 5 days. Following the initial differentiation, the endothelial progenitor cells (CD34+CD31+ cells) were sorted and terminally differentiated under serum-free conditions to arterial and venous ECs. The transcriptome and secretome profiles of the two distinct populations of hESC-derived arterial and venous ECs were characterized. Furthermore, the safety and functionality of these cells upon in vivo transplantation were characterized.ResultsSequential modulation of hESCs with GSK-3 inhibitor, bFGF, BMP4 and VEGF resulted in stages reminiscent of primitive streak, early mesoderm/lateral plate mesoderm, and endothelial progenitors under feeder- and serum-free conditions. Furthermore, these endothelial progenitors demonstrated differentiation potential to almost pure populations of arterial and venous endothelial phenotypes under serum-free conditions. Specifically, the endothelial progenitors differentiated to venous ECs in the absence of VEGF, and to arterial phenotype under low concentrations of VEGF. Additionally, these hESC-derived arterial and venous ECs showed distinct molecular and functional profiles in vitro. Furthermore, these hESC-derived arterial and venous ECs were nontumorigenic and were functional in terms of forming perfused microvascular channels upon subcutaneous implantation in the mouse.ConclusionsWe report a simple, rapid, and efficient protocol for directed differentiation of hESCs into endothelial progenitor cells capable of differentiation to arterial and venous ECs under feeder-free and serum-free conditions. This could offer a human platform to study arterial–venous specification for various applications related to drug discovery, disease modeling and regenerative medicine in the future.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-015-0260-5) contains supplementary material, which is available to authorized users.

SummaryA major challenge in the widespread application of human embryonic stem (hES) cells in clinical therapy and basic scientific research is the development of efficient cryopreservation protocols. Conventional slow-cooling protocols utilizing standard cryoprotectant concentrations i.e. 10% (v/v) DMSO, yield extremely low survival rates of <5% as reported by previous studies. This study characterized cell death within frozen-thawed hES colonies that were cryopreserved under standard conditions. Surprisingly, our results showed that immediately after post-thaw washing, the overwhelming majority of hES cells were viable (%98%), as assessed by the trypan blue exclusion test. However, when the freshly-thawed hES colonies were incubated within a 37°C incubator, there was observed to be a gradual reduction in cell viability over time. The kinetics of cell death was drastically slowed-down by keeping the freshly-thawed hES colonies at 4°C, with >90% of cells remaining viable after 90 min of incubation at 4°C. This effect was reversible upon re-exposing the cells to physiological temperature. The vast majority of low temperature-exposed hES colonies gradually underwent cell death upon incubation for a further 90 min at 37°C. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end-labeling (TUNEL) assay confirmed apoptosis-induced nuclear DNA fragmentation in frozen-thawed hES cells after incubation at 37°C for 90 min. Expression of active caspase-3 enzyme, which is another prominent marker of apoptosis, was confirmed by immunocytochemical staining, while transmission electron microscopy showed typical ultrastructural features of apoptosis such as chromatin condensation and margination to the nuclear membrane. Hence, our results demonstrated that apoptosis instead of cellular necrosis, is the major mechanism of the loss of viability of cryopreserved hES cells during freeze-thawing with conventional slow-cooling protocols.