Megakaryocytes (MKs) are rare hematopoietic cells in the adult bone marrow and produce platelets that are critical to vascular hemostasis and wound healing. Ex vivo generation of MKs from human induced pluripotent stem cells (hiPSCs) provides a renewable cell source of platelets for treating thrombocytopenic patients and allows a better understanding of MK/platelet biology. The key requirements in this approach include developing a robust and consistent method to produce functional progeny cells, such as MKs from hiPSCs, and minimizing the risk and variation from the animal-derived products in cell cultures. In this study, we developed an efficient system to generate MKs from hiPSCs under a feeder-free and xeno-free condition, in which all animal-derived products were eliminated. Several crucial reagents were evaluated and replaced with Food and Drug Administration-approved pharmacological reagents, including romiplostim (Nplate, a thrombopoietin analog), oprelvekin (recombinant interleukin-11), and Plasbumin (human albumin). We used this method to induce MK generation from hiPSCs derived from 23 individuals in two steps: generation of CD34+CD45+ hematopoietic progenitor cells (HPCs) for 14 days; and generation and expansion of CD41+CD42a+ MKs from HPCs for an additional 5 days. After 19 days, we observed abundant CD41+CD42a+ MKs that also expressed the MK markers CD42b and CD61 and displayed polyploidy (≥16% of derived cells with DNA contents >4N). Transcriptome analysis by RNA sequencing revealed that megakaryocytic-related genes were highly expressed. Additional maturation and investigation of hiPSC-derived MKs should provide insights into MK biology and lead to the generation of large numbers of platelets ex vivo.

Objectives: Vitronectin (VTN) has been widely used for the maintenance and expansion of human pluripotent stem cells (hPSCs) as feeder‐free conditions. However, the effect of VTN on hPSC differentiation remains unclear. Here, we investigated the role of VTN in early haematopoietic development of hPSCs.Materials and Methods: A chemically defined monolayer system was applied to study the role of different matrix or basement membrane proteins in haematopoietic development of hPSCs. The role of integrin signalling in VTN‐mediated haematopoietic differentiation was investigated by integrin antagonists. Finally, small interfering RNA was used to knock down integrin gene expression in differentiated cells.Results: We found that the haematopoietic differentiation of hPSCs on VTN was far more efficient than that on Matrigel that is also often used for hPSC culture. VTN promoted the fate determination of endothelial‐haematopoietic lineage during mesoderm development to generate haemogenic endothelium (HE). Moreover, we demonstrated that the signals through αvβ3 and αvβ5 integrins were required for VTN‐promoted haematopoietic differentiation. Blocking αvβ3 and αvβ5 integrins by the integrin antagonists impaired the development of HE, but not endothelial‐to‐haematopoietic transition (EHT). Finally, both αvβ3 and αvβ5 were confirmed acting synergistically for early haematopoietic differentiation by knockdown the expression of αv, β3 or β5.Conclusion: The established VTN‐based monolayer system of haematopoietic differentiation of hPSCs presents a valuable platform for further investigating niche signals involved in human haematopoietic development.

Derived from mesoderm precursors, hemangioblasts are bipotential common progenitors of hematopoietic cells and endothelial cells. The regulatory events controlling hematopoietic and endothelial lineage specification are largely unknown, especially in humans. In this study, we establish a serum-free and feeder-free system with a high-efficient embryoid body (EB) generation to investigate the signals that direct differentiation of human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs). Consistent with previous studies, the CD34 ⁺ CD31 ⁺ VE-cadherin ⁺ (VEC ⁺ ) cells derived from hPSCs contain hematopoietic and endothelial progenitors. In the presence of hematopoietic and endothelial growth factors, some of CD34 ⁺ CD31 ⁺ VEC ⁺ cells give rise to blast colony-forming cells (BL-CFCs), which have been used to characterize bipotential hemangioblasts. We found that the level of the transforming growth factor beta (TGF-β) 1 protein is increased during hPSC differentiation, and that TGF-β signaling has the double-edged effect on hematopoietic and endothelial lineage differentiation in hPSCs. An addition of TGF-β to hPSC differentiation before mesoderm induction promotes the development of mesoderm and the generation of CD34 ⁺ CD31 ⁺ VEC ⁺ cells. An addition of TGF-β inhibitor, SB431542, before mesoderm induction downregulates the expression of mesodermal markers and reduces the number of CD34 ⁺ CD31 ⁺ VEC ⁺ progenitor cells. However, inhibition of TGF-β signaling after mesoderm induction increases CD34 ⁺ CD31 ⁺ VEC ⁺ progenitors and BL-CFCs. These data provide evidence that a balance of positive and negative effects of TGF-β signaling at the appropriate timing is critical, and potential means to improve hematopoiesis and vasculogenesis from hPSCs.

Transplantation of vascular cells derived from human pluripotent stem cells (hPSCs) offers an attractive noninvasive method for repairing the ischemic tissues and for preventing the progression of vascular diseases. Here, we found that in a serum-free condition, the proliferation rate of hPSC-derived endothelial cells is quickly decreased, accompanied with an increased cellular senescence, resulting in impaired gene expression of endothelial nitric oxide synthase (eNOS) and impaired vessel forming capability in vitro and in vivo. To overcome the limited expansion of hPSC-derived endothelial cells, we screened small molecules for specific signaling pathways and found that inhibition of transforming growth factor-β (TGF-β) signaling significantly retarded cellular senescence and increased a proliferative index of hPSC-derived endothelial cells. Inhibition of TGF-β signaling extended the life span of hPSC-derived endothelial and improved endothelial functions, including vascular network formation on Matrigel, acetylated low-density lipoprotein uptake, and eNOS expression. Exogenous transforming growth factor-β1 increased the gene expression of cyclin-dependent kinase inhibitors, p15^Ink4b, p16^Ink4a, and p21^CIP1, in endothelial cells. Conversely, inhibition of TGF-β reduced the gene expression of p15^Ink4b, p16^Ink4a, and p21^CIP1. Our findings demonstrate that the senescence of newly generated endothelial cells from hPSCs is mediated by TGF-β signaling, and manipulation of TGF-β signaling offers a potential target to prevent vascular aging. Significance Endothelial cells derived from human pluripotent stem cells (hPSCs) could provide a valuable cell source for tissue engineering and treatment of cardiovascular diseases, such as revascularization of ischemic tissue. To increase the life span of hPSC-derived endothelial cells, this study investigated expansion of hPSC-derived endothelial cells in a serum-free defined condition and their function for treatment of regional ischemia in an animal model. This study provides not only a reliable and unlimited source of endothelial cells for future clinical applications but also insights into molecular mechanisms of vascular aging.