Definitive Hematopoietic Multipotent Progenitor Cells Are Transiently Generated From Hemogenic Endothelial Cells in Human Pluripotent Stem Cells.
ABSTRACT: 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.
Project description:BACKGROUND:Human induced pluripotent stem cells (hiPSCs) offer a renewable source of cells for the generation of hematopoietic cells for cell-based therapy, disease modeling, and drug screening. However, current serum/feeder-free differentiation protocols rely on the use of various cytokines, which makes the process very costly or the generation of embryoid bodies (EBs), which are labor-intensive and can cause heterogeneity during differentiation. Here, we report a simple feeder and serum-free monolayer protocol for efficient generation of iPSC-derived multipotent hematoendothelial progenitors (HEPs), which can further differentiate into endothelial and hematopoietic cells including erythroid and T lineages. METHODS:Formation of HEPs from iPSCs was initiated by inhibition of GSK3 signaling for 2?days followed by the addition of VEGF and FGF2 for 3?days. The HEPs were further induced toward mature endothelial cells (ECs) in an angiogenic condition and toward T cells by co-culturing with OP9-DL1 feeder cells. Endothelial-to-hematopoietic transition (EHT) of the HEPs was further promoted by supplementation with the TGF-? signaling inhibitor. Erythroid differentiation was performed by culturing the hematopoietic stem/progenitor cells (HSPCs) in a three-stage erythroid liquid culture system. RESULTS:Our protocol significantly enhanced the number of KDR+ CD34+ CD31+ HEPs on day 5 of differentiation. Further culture of HEPs in angiogenic conditions promoted the formation of mature ECs, which expressed CD34, CD31, CD144, vWF, and ICAM-1, and could exhibit the formation of vascular-like network and acetylated low-density lipoprotein (Ac-LDL) uptake. In addition, the HEPs were differentiated into CD8+ T lymphocytes, which could be expanded up to 34-fold upon TCR stimulation. Inhibition of TGF-? signaling at the HEP stage promoted EHT and yielded a large number of HSPCs expressing CD34 and CD43. Upon erythroid differentiation, these HSPCs were expanded up to 40-fold and displayed morphological changes following stages of erythroid development. CONCLUSION:This protocol offers an efficient and simple approach for the generation of multipotent HEPs and could be adapted to generate desired blood cells in large numbers for applications in basic research including developmental study, disease modeling, and drug screening as well as in regenerative medicine.
Project description:Endothelial-to-hematopoietic transition (EHT) is an important stage in definitive hematopoietic development. However, the genetic mechanisms underlying human EHT remain poorly characterized. We performed single cell RNA-seq using 55 hemogenic endothelial cells (HECs: CD31+ CD144+ CD41- CD43- CD45- CD73- RUNX1c+ ), 47 vascular endothelial cells without hematopoietic potential (non-HE: CD31+ CD144+ CD41- CD43- CD45- CD73- RUNX1c- ), and 35 hematopoietic progenitor cells (HPCs: CD34+ CD43+ RUNX1c+ ) derived from human embryonic stem cells (hESCs). HE and HP were enriched in genes implicated in hemogenic endothelial transcriptional networks, such as ERG, GATA2, and FLI. We found transcriptional overlap between individual HECs and HPCs; however, these populations were distinct from non-HE. Further analysis revealed novel biomarkers for human HEC/HPCs, including TIMP3, ESAM, RHOJ, and DLL4. Collectively, we demonstrate that hESC-derived HE and HP share a common developmental pathway, while non-HE are more heterogeneous and transcriptionally distinct. Our findings provide a novel strategy to test new genetic targets and optimize the production of definitive hematopoietic cells from human pluripotent stem cells. Stem Cells 2018;36:206-217.
Project description:Hemogenic endothelium (HE) has been recognized as a source of hematopoietic stem cells (HSCs) in the embryo. Access to human HE progenitors (HEPs) is essential for enabling the investigation of the molecular determinants of HSC specification. Here, we show that HEPs capable of generating definitive hematopoietic cells can be obtained from human pluripotent stem cells (hPSCs) and identified precisely by a VE-cadherin(+)CD73(-)CD235a/CD43(-) phenotype. This phenotype discriminates true HEPs from VE-cadherin(+)CD73(+) non-HEPs and VE-cadherin(+)CD235a(+)CD41a(-) early hematopoietic cells with endothelial and FGF2-dependent hematopoietic colony-forming potential. We found that HEPs arise at the post-primitive-streak stage of differentiation directly from VE-cadherin-negative KDR(bright)APLNR(+)PDGFR?(low/-) hematovascular mesodermal precursors (HVMPs). In contrast, hemangioblasts, which are capable of forming endothelium and primitive blood cells, originate from more immature APLNR(+)PDGFR?(+) mesoderm. The demarcation of HEPs and HVMPs provides a platform for modeling blood development from endothelium with a goal of facilitating the generation of HSCs from hPSCs.
Project description:BACKGROUND:Strategies of generating functional blood cells from human pluripotent stem cells (hPSCs) remain largely unsuccessful due to the lack of a comprehensive understanding of hematopoietic development. Endothelial-to-hematopoietic transition (EHT) serves as the pivotal mechanism for the onset of hematopoiesis and is negatively regulated by TGF-? signaling. However, little is known about the underlying details of TGF-? signaling during EHT. METHODS:In this study, by applying genome-wide gene profiling, we identified muscle segment homeobox2 (MSX2) as a potential mediator of TGF-? signaling during EHT. We generated MSX2-deleted human embryonic stem cell (hESC) lines using the CRISPR/Cas9 technology and induced them to undergo hematopoietic differentiation. The role of MSX2 in hematopoiesis and functional regulation of TGF? signaling in EHT was studied. RESULTS:We identified MSX2 as a novel regulator of human hematopoiesis. MSX2 deletion promotes the production of hematopoietic cells from hESCs. Functional and bioinformatics studies further demonstrated that MSX2 deletion augments hematopoietic differentiation of hESCs by facilitating EHT. Mechanistically, MSX2 acts as a downstream target of TGF? signaling to mediate its function during EHT. CONCLUSIONS:Our results not only improve the understanding of EHT, but may also provide novel insight into the efficient production of functional blood cells from hPSCs for regenerative medicine.
Project description:Specification of endothelial cells (ECs) into arterial, venous, and lymphatic cells is a crucial process of vascular development, and expanding our knowledge about EC specification from human pluripotent stem cells (hPSCs) will aid the design of optimal strategies for producing desired types of ECs for therapies. In our prior studies, we revealed that hPSC-derived VE-cadherin(V)+CD31+CD34+ ECs are heterogeneous and include at least three major subsets with distinct hemogenic properties: V+CD43/235a-CD73- hemogenic endothelial progenitors (HEPs), V+CD43loCD235a+73- angiogenic hematopoietic progenitors (AHPs), and V+CD43/235a-73+ non-HEPs. In this study, using angiogenesis assays, we demonstrated that ECs within these subsets have distinct endothelial colony- and tube-forming properties, proliferative and migratory properties, and endothelial nitric oxide synthase and inflammatory cytokine production potentials. Culture of isolated subsets in arterial, venous, and lymphatic conditions revealed that AHPs are skewed toward lymphatic, HEPs toward arterial, and non-HEPs toward venous differentiation in vitro. These findings suggest that selection and enhancement of production of a particular EC subset may aid in generating desirable EC populations with arterial, venous, or lymphatic properties from hPSCs.
Project description:WNT/?-CATENIN signaling promotes the hematopoietic/endothelial differentiation of human embryonic stem cells and human induced pluripotent stem cells (hiPSCs). The transient addition of a GSK3? inhibitor (GSKi) has been found to facilitate in vitro endothelial cell differentiation from hESCs/hiPSCs. Because hematopoietic and endothelial cells are derived from common progenitors (hemogenic endothelial progenitors [HEPs]), we examined the effect of transient GSKi treatment on hematopoietic cell differentiation from hiPSCs. We found that transient GSKi treatment at the start of hiPSC differentiation induction altered the gene expression profile of the cells. Multiple CDX/HOX genes, which are expressed in the posterior mesoderm of developing embryos, were significantly upregulated by GSKi treatment. Further, inclusion of the GSKi in a serum- and stroma-free culture with chemically defined medium efficiently induced HEPs, and the HEPs gave rise to various lineages of hematopoietic and endothelial cells. Therefore, transient WNT/?-CATENIN signaling triggers activation of the CDX/HOX pathway, which in turn confers hemogenic posterior mesoderm identity to differentiating hiPSCs. These data enhance our understanding of human embryonic hematopoietic/endothelial cell development and provide a novel in vitro system for inducing the differentiation of hematopoietic cells from hiPSCs.
Project description:Induced pluripotent stem cells (iPSCs) offer a promising platform to model early embryonic developmental processes, to create disease models that can be evaluated by drug screens as well as proof-of-concept experiments for regenerative medicine. However, generation of iPSC-derived hemato-endothelial and hematopoietic progenitor cells for these applications is challenging due to variable and limited cell numbers, which necessitates enormous up-scaling or development of demanding protocols. Here, we unravel the function of key transcriptional regulators SCL, LMO2, GATA2, and ETV2 (SLGE) on early hemato-endothelial specification and establish a fully inducible and stepwise hemato-endothelial forward programming system based on SLGE-regulated overexpression. Regulated induction of SLGE in stable SLGE-iPSC lines drives very efficient generation of large numbers of hemato-endothelial progenitor cells (CD144+/CD73-), which produce hematopoietic progenitor cells (CD45+/CD34+/CD38-/CD45RA-/CD90+/CD49f+) through a gradual process of endothelial-to-hematopoietic transition (EHT).
Project description:Human pluripotent stem cells (hPSCs) provide an unlimited source for generating various kinds of functional blood cells. However, efficient strategies for generating large-scale functional blood cells from hPSCs are still lacking, and the mechanism underlying human hematopoiesis remains largely unknown. In this study, we identified myeloid ectopic viral integration site 1 homolog (MEIS1) as a crucial regulator of hPSC early hematopoietic differentiation. MEIS1 is vital for specification of APLNR+ mesoderm progenitors to functional hemogenic endothelial progenitors (HEPs), thereby controlling formation of hematopoietic progenitor cells (HPCs). TAL1 mediates the function of MEIS1 in HEP specification. In addition, MEIS1 is vital for megakaryopoiesis and thrombopoiesis from hPSCs. Mechanistically, FLI1 acts as a downstream gene necessary for the function of MEIS1 during megakaryopoiesis. Thus, MEIS1 controls human hematopoiesis in a stage-specific manner and can be potentially manipulated for large-scale generation of HPCs or platelets from hPSCs for therapeutic applications in regenerative medicine.
2018-01-01 | S-EPMC5830947 | BioStudies
Project description:Alternative splicing (AS) leads to transcriptome diversity in eukaryotic cells and is one of the key regulators driving cellular differentiation. Although AS is of crucial importance for normal hematopoiesis and hematopoietic malignancies, its role in early hematopoietic development is still largely unknown. Here, by using high-throughput transcriptomic analyses, we show that pervasive and dynamic AS takes place during hematopoietic development of human pluripotent stem cells (hPSCs). We identify a splicing factor switch that occurs during the differentiation of mesodermal cells to endothelial progenitor cells (EPCs). Perturbation of this switch selectively impairs the emergence of EPCs and hemogenic endothelial progenitor cells (HEPs). Mechanistically, an EPC-induced alternative spliced isoform of NUMB dictates EPC specification by precisely controlling NOTCH signaling. Furthermore, we demonstrate that the splicing factor SRSF2 regulates splicing of the EPC-induced NUMB isoform, and the SRSF2-NUMB-NOTCH splicing axis regulates EPC generation. The identification of this splicing factor switch provides a new molecular mechanism to control cell-fate and lineage specification.
Project description:Alternative splicing (AS) leads to transcriptome diversity in eukaryotic cells and is one of the key regulators driving cellular differentiation. Although AS is of crucial importance for normal hematopoiesis and hematopoietic malignancies, its role in early hematopoietic development is still largely unknown. Here, by using high-throughput transcriptomic analyses, we show that pervasive and dynamic AS takes place during hematopoietic development of human pluripotent stem cells (hPSCs). We identify a splicing factor switch that occurs during the differentiation of mesodermal cells to endothelial progenitor cells (EPCs). Perturbation of this switch selectively impairs the emergence of EPCs and hemogenic endothelial progenitor cells (HEPs). Mechanistically, an EPC-induced alternative spliced isoform of NUMB dictates EPC specification by controlling NOTCH signaling. Furthermore, we demonstrate that the splicing factor SRSF2 regulates splicing of the EPC-induced NUMB isoform, and the SRSF2-NUMB-NOTCH splicing axis regulates EPC generation. The identification of this splicing factor switch provides a new molecular mechanism to control cell fate and lineage specification.