Hematopoietic Stem and Progenitor Cells from Human Pluripotent Stem Cells Via Transcription Factor Conversion of Hemogenic Endothelium

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 371-371
Author(s):  
Ryohichi Sugimura ◽  
Areum Han ◽  
Deepak Jha ◽  
Yi-Fen Lu ◽  
Jeremy A Goettel ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
Progenitor Cells ◽  
Pluripotent Stem Cells ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Functional Characterization ◽  
Hemogenic Endothelium ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells

Abstract A variety of tissues can be differentiated from pluripotent stem cells (PSCs) in vitro through stepwise exposure to morphogens, or by conversion of one differentiated cell type into another by enforced expression of master transcription factors (TFs). Despite considerable effort, neither approach has yielded functional human hematopoietic stem cells (HSCs). Building upon recent evidence that HSCs derive from definitive hemogenic endothelium (HE), we performed morphogen-directed differentiation of human PSCs into HE followed by screening of 26 candidate HSC-specifying TFs for the capacity to promote multi-lineage hematopoietic engraftment in irradiated immune deficient murine hosts. From genomic PCR of engrafted cells, we recovered seven TFs (ERG, HOXA5, HOXA9, HOXA10, LCOR, RUNX1, SPI1) that were sufficient to convert HE into hematopoietic stem and progenitor cells (HSPCs) that engraft GLY-A+ erythrocytes, CD33+ myeloid, CD15+ CD31+ neutrophils, CD19+ IgM+ B and CD3+ T cells in primary and secondary murine recipients for 12-14 weeks. Limiting dilution analysis indicated that the frequency of repopulating cells generated by this method was 1 in 4,707-15,029, lower than the frequency in CD34+ cord blood cells (1 in 1,819-5,173). Functional characterization of terminally differentiated cells demonstrated features of definitive erythropoiesis (expression of adult beta globin and enucleation). Engrafted neutrophils responded to cytokine stimuli by activation of myeloperoxidase. Human IgM and IgG could be detected in the serum of engrafted mice, and titers of ovalbumin specific antibody increased in response to protein immunization, indicating boostable immunity. T-cells responded to PMA/Ionomycin stimuli by activation of IFNγ, and sequencing of the T cell receptor revealed a broad clonotype diversity. Proviral integration analysis demonstrated derivation of myeloid and lymphoid progeny from common clones in secondary animals, indicating generation of self-renewing, multipotential HSC-like cells from PSCs. Mechanistically, the seven TFs induced HOXA target genes (LMO2, SOX4, MEIS1 and ID2); upregulated expression of homing-related genes (CXCR4, VLA5 and S1PR1); and enhanced the endothelial to hematopoietic transition (EHT), as indicated by a 2.4-fold induction of a RUNX1c-reporter. Our combined approach of morphogen-driven differentiation and TF-mediated cell fate conversion produced HSPCs from PSCs that hold promise for modeling hematopoietic disease in humanized mice and for therapeutic strategies in genetic blood disorders. Disclosures No relevant conflicts of interest to declare.

Sociology of Normal Stem and Progenitor Cells in CML Niche

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1234-1234
Author(s):  
Robert S Welner ◽  
Giovanni Amabile ◽  
Deepak Bararia ◽  
Philipp B. Staber ◽  
Akos G. Czibere ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mouse Model ◽  
Progenitor Cells ◽  
Conflicts Of Interest ◽  
Hematopoietic Progenitor Cells ◽  
Quiescent State ◽  
Hematopoietic Progenitor ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells

Abstract Abstract 1234 Specialized bone marrow (BM) microenvironment niches are essential for hematopoietic stem and progenitor cell maintenance, and recent publications have focused on the leukemic stem cells interaction and placement within those sites. Surprisingly, little is known about how the integrity of this leukemic niche changes the normal stem and progenitor cells behavior and functionality. To address this issue, we started by studying the kinetics and differentiation of normal hematopoietic stem and progenitor cells in mice with Chronic Myeloid Leukemia (CML). CML accounts for ∼15% of all adult leukemias and is characterized by the BCR-ABL t(9;22) translocation. Therefore, we used a novel SCL-tTA BCR/ABL inducible mouse model of CML-chronic phase to investigate these issues. To this end, BM from leukemic and normal mice were mixed and co-transplanted into hosts. Although normal hematopoiesis was increasingly suppressed during the disease progression, the leukemic microenvironment imposed distinct effects on hematopoietic progenitor cells predisposing them toward the myeloid lineage. Indeed, normal hematopoietic progenitor cells from this leukemic environment demonstrated accelerated proliferation with a lack of lymphoid potential, similar to that of the companion leukemic population. Meanwhile, the leukemic-exposed normal hematopoietic stem cells were kept in a more quiescent state, but remained functional on transplantation with only modest changes in both engraftment and homing. Further analysis of the microenvironment identified several cytokines that were found to be dysregulated in the leukemia and potentially responsible for these bystander responses. We investigated a few of these cytokines and found IL-6 to play a crucial role in the perturbation of normal stem and progenitor cells observed in the leukemic environment. Interestingly, mice treated with anti-IL-6 monoclonal antibody reduced both the myeloid bias and proliferation defects of normal stem and progenitor cells. Results obtained with this mouse model were similarly validated using specimens obtained from CML patients. Co-culture of primary CML patient samples and GFP labeled human CD34+CD38- adult stem cells resulted in selective proliferation of the normal primitive progenitors compared to mixed cultures containing unlabeled normal bone marrow. Proliferation was blocked by adding anti-IL-6 neutralizing antibody to these co-cultures. Therefore, our current study provides definitive support and an underlying crucial mechanism for the hematopoietic perturbation of normal stem and progenitor cells during leukemogenesis. We believe our study to have important implications for cancer prevention and novel therapeutic approach for leukemia patients. We conclude that changes in cytokine levels and in particular those of IL-6 in the CML microenvironment are responsible for altered differentiation and functionality of normal stem cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals DLL4 and VCAM1 enhance the emergence of T cell-competent hematopoietic progenitors from human pluripotent stem cells

2021 ◽  
Author(s):  
Yale S Michaels ◽  
John M Edgar ◽  
Matthew C Major ◽  
Elizabeth L Castle ◽  
Carla Zimmerman ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
T Cell ◽  
Pluripotent Stem Cells ◽  
Adaptive Immune Response ◽  
Hematopoietic Progenitors ◽  
Cell Potential ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
The Impact

T cells are key mediators of the adaptive immune response and show tremendous efficacy as cellular therapeutics. However, obtaining primary T cells from human donors is expensive and variable. Pluripotent stem cells (PSCs) have the potential to serve as a consistent and renewable source of T cells, but differentiating PSCs into hematopoietic progenitors with T cell potential remains a significant challenge. Here, we developed an efficient serum- and feeder-free protocol for differentiating human PSCs into hematopoietic progenitors and T cells. This defined method allowed us to study the impact of individual recombinant proteins on blood emergence and lineage potential. We demonstrate that the presence of DLL4 and VCAM1 during the endothelial-to-hematopoietic transition (EHT) enhances downstream progenitor T cell output by >80-fold. Using single cell transcriptomics, we showed that these two proteins synergise to drive strong notch signalling in nascent hematopoietic stem and progenitor cells and that VCAM1 additionally drives a pro-inflammatory transcriptional program. Finally, we applied this differentiation method to study the impact of cytokine concentration dynamics on T cell maturation. We established optimised media formulations that enabled efficient and chemically defined differentiation of CD8αβ+, CD4-, CD3+, TCRαβ+ T cells from PSCs.

Blood Flow Is Required for the Release of Hematopoietic Stem- and Progenitor Cells From Hemogenic Endothelium in the Placenta.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 698-698
Author(s):  
Katrin E Rhodes ◽  
Ben Van Handel ◽  
Michele Wang ◽  
Yanling Wang ◽  
Akanksha Chhabra ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Blood Flow ◽  
Progenitor Cells ◽  
Conflicts Of Interest ◽  
Yolk Sac ◽  
Positive Staining ◽  
Hemogenic Endothelium ◽  
Hematopoietic Stem ◽  
Notch1 Signaling ◽  
Stem And Progenitor Cells

Abstract Abstract 698 Hematopoietic stem cells (HSCs) are required for continuous blood cell production throughout life. HSCs emerge only within a short developmental time window during embryogenesis. Mounting evidence posits that HSCs arise directly from hemogenic endothelial cells during midgestation within the large arteries of the conceptus, which include the dorsal aorta, the umbilical and vitelline arteries and the chorioallantoic vessels of the placenta. However, the microenvironmental signals that mediate this temporally regulated process remain unclear. Here we examine, by using Ncx1−/− embryos that lack heartbeat and circulation, how blood flow imparts instructive cues that ensure proper HSC development. Immunostaining revealed that CD41+ hematopoietic cells, although present, were markedly decreased in Ncx1-/-placentas as compared to wild-type controls. Furthermore, mutant placentas evidenced large clusters of round CD31+ cells protruding into the lumens of the chorioallantoic vessels. Based on these data, we hypothesized that lack of blood flow may impede the generation of hematopoietic stem and progenitor cells (HS/PCs) and that the endothelial clusters represent hemogenic intermediates. FACS analysis and colony forming assays confirmed a dramatic reduction in the number of clonogenic progenitors in the placenta and the embryo proper of Ncx mutants, while the yolk sac was unaffected. However, HS/PC generation in the placenta and embryo could be rescued by culturing explants on OP9 stroma before plating in colony forming assays, verifying intact hematopoietic potential. To determine if the rescue observed was due to expansion of existing progenitors or generation of new HS/PCs, we sorted CD41medckit+hematopoietic progenitors and CD31+CD41− endothelial cells from hematopoietic tissues and co-cultured them on stroma. These experiments demonstrated that endothelial cells from placenta, embryo proper and yolk sac can generate HS/PCs following stroma stimulation, confirming the presence of hemogenic endothelium in these organs. Immunostaining of Ncx−/− placentas revealed that although the development of the arterio-venous vascular network was impaired, Notch1 signaling, required for both arterial specification and HSC development, was robust in cells of the endothelial clusters. Furthermore, positive staining for Runx1 and c-myb indicated that cells in the clusters had activated the hematopoietic program. Interestingly, electron microscopy demonstrated that cells in the clusters were tethered to each other via adherens junctions, a characteristic of endothelial cells. In addition, they also maintained high levels of Flk1, expressed VEGF and were actively proliferating, consistent with exposure to extended hypoxia. These data suggest that although cells in the clusters have initiated hematopoietic commitment, they are unable to down-regulate their endothelial identity and complete hematopoietic emergence, resulting in the formation of clusters of hemogenic intermediates. These results imply that cues imparted via circulation are required to complete the commitment to a hematopoietic fate from hemogenic endothelium. Data from co-culture experiments suggest that prolonged Notch1 signaling impairs hematopoietic emergence from hemogenic endothelial cells, and may account for the HSC emergence defect in the absence of blood flow. Overall, these data suggest that blood flow and circulating primitive red blood cells are critical components of the dynamic microenvironment necessary to both relieve the hypoxia required for the specification and proliferation of hemogenic endothelium and provide important mechanical and/or molecular signals required by HSCs to fully commit to the hematopoietic fate and complete emergence. Disclosures: No relevant conflicts of interest to declare.

Latent Cardiogenic Potential in Endocardium and Hemogenic Endothelium Revealed in the Absence of Scl/tal1

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2362-2362
Author(s):  
Amelie Montel-Hagen ◽  
Ben Van Handel ◽  
Roberto Ferrari ◽  
Rajkumar Sasidharan ◽  
Tonis Org ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Progenitor Cells ◽  
Conflicts Of Interest ◽  
Lineage Tracing ◽  
Bhlh Transcription Factor ◽  
Wild Type ◽  
Transcriptional Program ◽  
Hemogenic Endothelium ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells

Abstract Abstract 2362 The endothelium in embryonic and extraembryonic hematopoietic tissues has the capacity to generate hematopoietic stem and progenitor cells (HS/PC). However, it is unknown how this unique endothelium is specified. Microarray analysis of endothelial cells from hematopoietic tissues of embryos deficient for the bHLH transcription factor Scl/tal1 revealed that Scl establishes a robust hematopoietic transcriptional program in the endothelium. Surprisingly, lack of Scl also induced an unexpected fate switching of the prospective hemogenic endothelium to the cardiac lineage. Scl deficient embryos displayed a dramatic upregulation of cardiac transcription factors and structural proteins within the yolk sac vasculature, resulting in the generation of spontaneously beating cardiomyocytes. Ectopic cardiac potential in Scl deficient embryos arose from endothelial-derived CD31+Pdgfrα+ cardiogenic progenitor cells (CPCs), which were present in all sites of HS/PC generation. Analysis of Runx1-deficient embryos revealed, that although Runx1 acts downstream of Scl during the emergence of definitive HS/PCs, it is not required for the suppression of the cardiac fate in the endothelium. The only wild type tissue that contained CD31+Pdgfrα+ putative CPCs was the heart, and this population was greatly expanded in Scl deficient embryos. Strikingly, endocardium in Scl−/− hearts also activated a robust cardiomyogenic transcriptional program and generated Troponin T+ cardiomyocytes both in vivo and in vitro. Although CD31+Pdgfrα+ CPCs from wild type hearts did not generate readily beating cells in culture, they produced cells expressing endothelial, smooth muscle and cardiomyocyte specific genes, implying multipotentiality of this novel CPC population. Furthermore, CD31+Pdgfrα+ CPCs were greatly reduced in Isl1−/− hearts, which fail to generate functional, multipotential CPCs. Lineage tracing using VE-cadherin Cre Rosa-YFP mouse strain demonstrated that, in addition to generating HS/PCs in hematopoietic tissues, endothelial cells are also the cell of origin for CD31+Pdgfrα+ CPCs in the heart. Together, these data suggest a broader role for embryonic endothelium as a potential source of tissue-specific stem and progenitor cells and implicate Scl/tal1 as an important regulator of endothelial fate choice. Disclosures: No relevant conflicts of interest to declare.

Single Cell Proteomics Reveals Novel Cytokine-Producing Function of Hematopoietic Stem and Progenitor Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 26-26
Author(s):  
Jimmy L. Zhao ◽  
Chao Ma ◽  
Ryan O'Connell ◽  
Dinesh S. Rao ◽  
James Heath ◽  
...  
Keyword(s):  
Stem Cells ◽  
Immune Response ◽  
Single Cell ◽  
Progenitor Cells ◽  
Cytokine Production ◽  
Conflicts Of Interest ◽  
Severe Neutropenia ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells

Abstract Abstract 26 During infection, hematopoietic stem and progenitor cells (HSPCs) are called upon to proliferate and differentiate to produce more innate and adaptive immune cells to combat infection. Traditionally, HSPCs are thought to respond to depletion of downstream hematopoietic cells during infection. More recent evidence suggests that HSPCs may respond directly to infection and pro-inflammatory cytokines. However, little is known about the direct immune response of HSPCs and the molecular signaling regulating this response upon sensing an infection. In this study, we have combined transgenic and genetic knockout mouse models with a novel single cell barcode proteomics microchip technology to tackle these questions. We show that although long-term hematopoietic stem cells (HSCs) (defined by Lineage-cKit+Sca1+CD150+CD48-) do not secrete cytokines upon toll-like receptor (TLR) stimulation, short-term HSCs and multipotent progenitor cells (MPPs) (defined by Lineage-cKit+Sca1+, referred to as LKS thereafter) can produce copious amounts of cytokines upon direct TLR-4 and TLR-2 stimulation, indicating that LKS cells can directly participate in an immune response by producing a myriad of cytokines, upon a bacterial infection. Within the population of LKS cells we detect multiple functional subsets of cells, specialized in producing myeloid-like, lymphoid-like or both types of cytokines. Moreover, we show that the cytokine production by LKS cells is regulated by the NF-κB activity, as p50-deficient LKS cells show reduced cytokine production while microRNA-146a (miR-146a)-deficient LKS cells show significantly increased cytokine production. As long-term HSCs differentiate, they start to gain effector immune function much earlier than we had originally anticipated. In light of this finding, we should start to view the stepwise differentiation scheme of HSCs, and perhaps all other stem cells, as a strategy to sequentially gain functional capacity, instead of simply losing stemness and self-renewal ability. The remarkable ability of LKS cells to produce copious amounts of cytokines in response to bacteria may provide some protective immunity during severe neutropenia and lymphopenia or in the early stage of HSC transplantation. This study further extends the functions of NF-κB to include the regulation of primitive hematopoietic stem and progenitor cells and provides direct evidence of the bacteria-responding ability of HSPCs through the TLR/NF-κB axis. The single cell barcode proteomics technology can be widely applied to study proteomics of other rare cells or heterogeneous cell population at a single cell level. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Human Definitive Hematopoietic Specification from Pluripotent Stem Cells Is Regulated By Mesodermal Expression of CDX4

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 883-883
Author(s):  
John Philip Creamer ◽  
Carissa Dege ◽  
Jolie T.K. Ho ◽  
Qihao Ren ◽  
Mark C Valentine ◽  
...  
Keyword(s):  
Stem Cells ◽  
Wnt Signaling ◽  
Pluripotent Stem Cells ◽  
Conflicts Of Interest ◽  
Genetic Regulation ◽  
Signal Pathways ◽  
Dependent Manner ◽  
Hemogenic Endothelium ◽  
Hematopoietic Stem ◽  
Aavs1 Locus

Abstract The generation of hematopoietic stem cells (HSCs) from human pluripotent stem cells (hPSCs) is a major goal for regenerative medicine, as it will provide an unlimited source of these cells for transplantation, and a unique platform for the study of both normal and disease hematopoietic processes. To reproducibly achieve this goal in all hPSC lines, we must first fully understand hematopoietic ontogeny. Understanding hematopoietic development is complicated by the existence of at least two distinct programs during development that are difficult to distinguish: a transient "primitive" extra-embryonic hematopoietic program that does not give rise to HSCs, and a "definitive" program that gives rise to HSCs and all hematopoietic lineages. We have recently developed a novel method to obtain and identify, from hPSCs, mesoderm harboring exclusively primitive or exclusively definitive hematopoietic potential, by the differential expression of CD235a within KDR+ mesoderm. With this, we were able to determine that stage-specific canonical Wnt signaling specified the definitive hematopoietic program, while simultaneously repressing the primitive hematopoietic program. Further, we have now found that definitive hematopoietic specification is also dependent on bFGF signaling during this same window of time. We then asked if this tractable system could help us understand the genetic regulation of definitive hematopoietic specification from hPSCs. We isolated Wnt-dependent KDR+CD235a- definitive hematopoietic mesoderm and Wnt-independent KDR+CD235a+ primitive hematopoietic mesoderm and performed whole-transcriptome gene expression analysis, which revealed strong CDX and HOX gene enrichment exclusively within KDR+CD235a- definitive hematopoietic mesoderm. Monitoring CDX expression over time in the differentiation cultures revealed that both CDX1 and CDX2 were expressed in a bFGF-independent manner, prior to Wnt-dependent definitive hematopoietic KDR+CD235a- mesoderm specification. In contrast, CDX4 was expressed exclusively within definitive hematopoietic KDR+CD235a- mesoderm in a Wnt- and bFGF-dependent manner. This expression pattern suggested that CDX4 expression is specific to definitive, but not primitive, hematopoietic specification. To determine whether CDX4 expression plays a role in definitive hematopoietic specification, we generated an inducible CDX4 expression hPSC line using the "safe-harbor" AAVS1 locus. We manipulated exogenous CDX4 expression during the same stage of the differentiation culture that Wnt signaling is critical for definitive hematopoietic specification. Interestingly, in the absence of Wnt stimulation, exogenous CDX4 expression caused a >90% repression in primitive hematopoietic potential. Critically, CDX4 expression during this same time conferred 10-fold greater definitive hematopoietic potential within CD34+CD73-CD184- hemogenic endothelium, giving rise to definitive erythroid-myeloid-lymphoid multilineage progenitors. This is consistent with CDX4 being the transcriptional effector of Wnt signaling during early hematopoietic specification within mesoderm. We next generated a CDX4 knockout hPSC line by CRISPR/Cas9, and a CDX4 knockdown hPSC line via shRNA expression from the AAVS1 locus. As expected, the absence of CDX4 expression did not reduce primitive hematopoietic potential in comparison to control lines. However, when Wnt signaling was stimulated to specify definitive hematopoiesis, hPSCs lacking CDX4 expression exhibited a 10-fold decrease in definitive CD34+CD73-CD184- hemogenic endothelium specification, indicating its expression is critical for definitive hematopoietic specification. Taken together, these findings indicate that CDX4 is the earliest identified transcription factor that is a critical regulator of human definitive hematopoietic specification, and provide a mechanistic basis for Wnt-mediated definitive hematopoietic specification from hPSCs. By understanding the genetic regulation of early definitive hematopoietic specification from hPSCs, we can now identify the additional signal pathways required for efficient HSC specification from hPSCs. Disclosures No relevant conflicts of interest to declare.

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