PEAR1 is a potential regulator of early hematopoiesis of human pluripotent stem cells.

Hemogenic endothelial (HE) cells are specialized endothelial cells to give rise to hematopoietic stem/progenitor cells during hematopoietic development. The underlying mechanisms that regulate endothelial-to-hematopoietic transition (EHT) of human HE cells are not fully understand. Here, we identified platelet endothelial aggregation receptor-1 (PEAR1) as a novel regulator of early hematopoietic development in human pluripotent stem cells (hPSCs). We found that the expression of PEAP1 was elevated during hematopoietic development. A subpopulation of PEAR1+ cells overlapped with CD34+ CD144+ CD184+ CD73- arterial-type HE cells. Transcriptome analysis by RNA sequencing indicated that TAL1/SCL, GATA2, MYB, RUNX1 and other key transcription factors for hematopoietic development were mainly expressed in PEAR1+ cells, whereas the genes encoding for niche-related signals, such as fibronectin, vitronectin, bone morphogenetic proteins and jagged1, were highly expressed in PEAR1- cells. The isolated PEAR1+ cells exhibited significantly greater EHT capacity on endothelial niche, compared with the PEAR1- cells. Colony-forming unit (CFU) assays demonstrated the multilineage hematopoietic potential of PEAR1+ -derived hematopoietic cells. Furthermore, PEAR1 knockout in hPSCs by CRISPR/Cas9 technology revealed that the hematopoietic differentiation was impaired, resulting in decreased EHT capacity, decreased expression of hematopoietic-related transcription factors, and increased expression of niche-related signals. In summary, this study revealed a novel role of PEAR1 in balancing intrinsic and extrinsic signals for early hematopoietic fate decision.

Single-cell transcriptome of early hematopoiesis guides arterial endothelial-enhanced functional T cell generation from human PSCs.

Hematopoietic differentiation of human pluripotent stem cells (hPSCs) requires orchestration of dynamic cell and gene regulatory networks but often generates blood cells that lack natural function. Here, we performed extensive single-cell transcriptomic analyses to map fate choices and gene expression patterns during hematopoietic differentiation of hPSCs and showed that oxidative metabolism was dysregulated during in vitro directed differentiation. Applying hypoxic conditions at the stage of endothelial-to-hematopoietic transition in vitro effectively promoted the development of arterial specification programs that governed the generation of hematopoietic progenitor cells (HPCs) with functional T cell potential. Following engineered expression of the anti-CD19 chimeric antigen receptor, the T cells generated from arterial endothelium-primed HPCs inhibited tumor growth both in vitro and in vivo. Collectively, our study provides benchmark datasets as a resource to further understand the origins of human hematopoiesis and represents an advance in guiding in vitro generation of functional T cells for clinical applications.

Vitronectin-activated αvß3 and αvß5 integrin signalling specifies haematopoietic fate in human pluripotent stem cells.

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 αvß3 and αvß5 integrins were required for VTN-promoted haematopoietic differentiation. Blocking αvß3 and αvß5 integrins by the integrin antagonists impaired the development of HE, but not endothelial-to-haematopoietic transition (EHT). Finally, both αvß3 and αvß5 were confirmed acting synergistically for early haematopoietic differentiation by knockdown the expression of αv, ß3 or ß5. 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.

Defining early hematopoietic-fated primitive streak specification of human pluripotent stem cells by the orchestrated balance of Wnt, activin, and BMP signaling.

Distinct regions of the primitive streak (PS) have diverse potential to differentiate into several tissues, including the hematopoietic lineage originated from the posterior region of PS. Although various signaling pathways have been identified to promote the development of PS and its mesoderm derivatives, there is a large gap in our understanding of signaling pathways that regulate the hematopoietic fate of PS. Here, we defined the roles of Wnt, activin, and bone morphogenetic protein (BMP) signaling pathways in generating hematopoietic-fated PS from human pluripotent stem cells (hPSCs). We found that the synergistic balance of these signaling pathways was crucial for controlling the PS fate determination towards hematopoietic lineage via mesodermal progenitors. Although the induction of PS depends largely on the Wnt and activin signaling, the PS generated without BMP4 lacks the hematopoietic potential, indicating that the BMP signaling is necessary for the PS to acquire hematopoietic property. Appropriate levels of Wnt signaling is crucial for the development of PS and its specification to the hematopoietic lineage. Although the development of PS is less sensitive to activin or BMP signaling, the fate of PS to mesoderm progenitors and subsequent hematopoietic lineage is determined by appropriate levels of activin or BMP signaling. Collectively, our study demonstrates that Wnt, activin, and BMP signaling pathways play cooperative and distinct roles in regulating the fate determination of PS for hematopoietic development. Our understanding of the regulatory networks of hematopoietic-fated PS would provide important insights into early hematopoietic patterning and possible guidance for generating functional hematopoietic cells from hPSCs in vitro.

Suppression of Transforming Growth Factor-ß Signaling Delays Cellular Senescence and Preserves the Function of Endothelial Cells Derived from Human Pluripotent Stem Cells.

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-ß (TGF-ß) signaling significantly retarded cellular senescence and increased a proliferative index of hPSC-derived endothelial cells. Inhibition of TGF-ß 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-ß1 increased the gene expression of cyclin-dependent kinase inhibitors, p15Ink4b , p16Ink4a , and p21CIP1 , in endothelial cells. Conversely, inhibition of TGF-ß reduced the gene expression of p15Ink4b , p16Ink4a , and p21CIP1 . Our findings demonstrate that the senescence of newly generated endothelial cells from hPSCs is mediated by TGF-ß signaling, and manipulation of TGF-ß signaling offers a potential target to prevent vascular aging. Stem Cells Translational Medicine 2017;6:589-600.

Definitive Hematopoietic Multipotent Progenitor Cells Are Transiently Generated From Hemogenic Endothelial Cells in Human Pluripotent Stem Cells.

Generation of fully functional hematopoietic multipotent progenitor (MPP) cells from human pluripotent stem cells (hPSCs) has a great therapeutic potential to provide an unlimited cell source for treatment of hematological disorders. We previously demonstrated that CD34(+) CD31(+) CD144(+) population derived from hPSCs contain hemato-endothelial progenitors (HEPs) that give rise to hematopoietic and endothelial cells. Here, we report a differentiation system to generate definitive hematopoietic MPP cells from HEPs via endothelial monolayer. In the presence of angiogenic factors, HEPs formed an endothelial monolayer, from which hematopoietic clusters emerged through the process of endothelial-to-hematopoietic transition (EHT). EHT was significantly enhanced by hematopoietic growth factors. The definitive MPP cells generated from endothelial monolayer were capable of forming multilineage hematopoietic colonies, giving rise to T lymphoid cells, and differentiating into enucleated erythrocytes. Emergence of hematopoietic cells from endothelial monolayer occurred transiently. Hematopoietic potential was lost during prolonged culture of HEPs in endothelial growth conditions. Our study demonstrated that CD34(+) CD31(+) CD144(+) HEPs gave rise to hematopoietic MPP cells via hemogenic endothelial cells that exist transiently. The established differentiation system provides a platform for future investigation of regulatory factors involved in de novo generation of hematopoietic MPP cells and their applications in transplantation.

Cooperative Effect of Erythropoietin and TGF-ß Inhibition on Erythroid Development in Human Pluripotent Stem Cells.

Patient-specific human induced-pluripotent stem cells (hiPSCs) represent important cell sources to treat patients with acquired blood disorders. To realize the therapeutic potential of hiPSCs, it is crucial to understand signals that direct hiPSC differentiation to a hematopoietic lineage fate. Our previous study demonstrated that CD34(+)CD31(+) cells derived from human pluripotent stem cells (hPSCs) contain hemato-endothelial progenitors (HEPs) that give rise to hematopoietic cells and endothelial cells. Here, we established a serum-free and feeder-free system to induce the differentiation of hPSC-derived CD34(+)CD31(+) progenitor cells to erythroid cells. We show that extracellular matrix (ECM) proteins promote the differentiation of CD34(+)CD31(+) progenitor cells into CD235a(+) erythroid cells through CD41(+)CD235a(+) megakaryocyte-erythroid progenitors (MEP). Erythropoietin (EPO) is a predominant factor for CD34(+)CD31(+) progenitor differentiation to erythroid cells, whereas transforming growth factor beta (TGF-ß) inhibits the development of CD34(+)CD31(+) progenitor cells. Apoptosis of progenitor cells is induced by TGF-ß in early erythroid differentiation. Suppression of TGF-ß signaling by SB431542 at early stage of CD34(+)CD31(+) progenitor differentiation induces the erythroid cell generation. Together, these findings suggest that TGF-ß suppression and EPO stimulation promote erythropoiesis of CD34(+)CD31(+) progenitor cells derived from hPSCs.

EphB4 forward-signaling regulates cardiac progenitor development in mouse ES cells.

Eph receptor (Eph)-ephrin signaling plays an important role in organ development and tissue regeneration. Bidirectional signaling of EphB4-ephrinB2 regulates cardiovascular development. To assess the role of EphB4-ephrinB2 signaling in cardiac lineage development, we utilized two GFP reporter systems in embryonic stem (ES) cells, in which the GFP transgenes were expressed in Nkx2.5(+) cardiac progenitor cells and in α-MHC(+) cardiomyocytes, respectively. We found that both EphB4 and ephrinB2 were expressed in Nkx2.5-GFP(+) cardiac progenitor cells, but not in α-MHC-GFP(+) cardiomyocytes during cardiac lineage differentiation of ES cells. An antagonist of EphB4, TNYL-RAW peptides, that block the binding of EphB4 and ephrinB2, impaired cardiac lineage development in ES cells. Inhibition of EphB4-ephrinB2 signaling at different time points during ES cell differentiation demonstrated that the interaction of EphB4 and ephrinB2 was required for the early stage of cardiac lineage development. Forced expression of human full-length EphB4 or intracellular domain-truncated EphB4 in EphB4-null ES cells was established to investigate the role of EphB4-forward signaling in ES cells. Interestingly, while full-length EphB4 was able to restore the cardiac lineage development in EphB4-null ES cells, the truncated EphB4 that lacks the intracellular domain of tyrosine kinase and PDZ motif failed to rescue the defect of cardiomyocyte development, suggesting that EphB4 intracellular domain is essential for the development of cardiomyocytes. Our study provides evidence that receptor-kinase-dependent EphB4-forward signaling plays a crucial role in the development of cardiac progenitor cells.