The Experts below are selected from a list of 1739109 Experts worldwide ranked by ideXlab platform
Dan S Kaufman - One of the best experts on this subject based on the ideXlab platform.
-
single cell resolution of human hematoendothelial cells defines transcriptional signatures of hemogenic endothelium
Stem Cells, 2018Co-Authors: Mathew G Angelos, Juan E Abrahante, Robert Blum, Dan S KaufmanAbstract:Author(s): Angelos, Mathew G; Abrahante, Juan E; Blum, Robert H; Kaufman, Dan S | Abstract: 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.
-
single cell resolution of human pluripotent stem cell derived hemato endothelial cells reveals distinct transcriptional signatures of hemogenic endothelium
Blood, 2016Co-Authors: Mathew G Angelos, Juan E Abrahante, Dan S KaufmanAbstract:Hemogenic endothelium is a rare and highly specialized subset of vascular endothelial cell that functions as a precursor cell population to definitive blood development. One of the mechanistic hallmarks of definitive hematopoiesis is the endothelial-to-hematopoietic transition (EHT), a process where hemogenic endothelial cells phenotypically switches to produce a detached and free-moving hematopoietic cell. While EHT has been visualized both in vitro and in vivo via lineage tracing studies, the regulation of this fate change at both a cellular and molecular level remains unclear. Human pluripotent stem cells, such as human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) can serve as a useful platform to characterize human hemogenic endothelium and to understand basic mechanisms underlying human EHT. We hypothesized that human hemogenic endothelium derived from hESCs is phenotypically and transcriptionally distinct from other vascular endothelial cells and hematopoietic progenitor cells. To identify human hemogenic endothelium, we used combined expression of endothelial cell surface antigens and RUNX1c expression via a fluorescent reporter previously validated in our lab. Specifically, we employed defined culture methods to differentiate hemogenic endothelial cells (HE) and vascular endothelial cells without hematopoietic potential (non-HE) from hESC harboring a RUNX1c-tdTomato reporter (hESC-RUNX1c-tdTomato). At Day 11 of differentiation, CD31+CD144+ endothelial cells were present, with approximately 40% of these cells tdTomato+. We next sorted HE (CD31+CD144+CD41-CD43-CD45-CD73-tdTomato+) and non-HE (CD31+CD144+CD41-CD43-CD45-CD73-tdTomato-) and cultured both populations in endothelial growth media (EGM) or hematopoietic growth media. HE retained characteristic cobblestone morphology and CD31 expression over the course of 5 days in EGM similar to control human umbilical vein endothelial cells. HE, but not non-HE, was able to generate non-adherent, tdTomato+ hematopoietic progenitor cells in hematopoietic growth media. Using these defined cell populations, we next performed single-cell RNASeq on HE, non-HE, and early hematopoietic progenitor cells (HP; CD34+CD43+tdTomato+). We captured a total of 55 HE, 47 non-HE, and 35 HP using the Fluidgm C1 single cell system and performed next generation sequencing of validated libraries. We analyzed single cell gene expression using Seurat, an R-based bioinformatics software package developed for the analysis of single cell NGS experiments. Populations were first validated based on expression of known genetic identifiers for vascular endothelium, hemogenic endothelium, and hematopoietic progenitor cells. HE and HP were both highly enriched for CDH5, ERG, ESAM, and FLI as compared to non-HE; these genes have been previously implicated in HE functionality. All single cell transcriptional profiles were similarly enriched for KDR, PECAM1, and LMO2, which are characteristic genes expressed in vascular endothelium. We next performed t-distributed stochastic neighbor embedding (t-SNE) using statistically significant principal components to distinguish groups of cells with similar transcriptional expression. Interestingly, we found overlap between individual HE and HP cells (Cluster 1); however, these cells were distinct from two, separate clusters of non-HE (Cluster 2 & 3; Figure 1). Further analysis of these clusters revealed novel biomarkers for HE/HP, such as TIMP3 (DGE: 2.06, Power: 0.91), ERG (DGE: 2.08, Power: 0.83), NOTCH4 (DGE: 2.35, Power: 0.78), and HEY1 (DGE: 2.47, Power: 0.74), Non-HE were found to cluster into two, distinct groups (Clusters 2 and 3), with Cluster 3 enriched for extracellular matrix genes (COL1A1 (DGE: 6.58, Power: 1.00), DCN (DGE: 6.09, Power: 1.00), VCAN (DGE: 3.55, Power: 1.00), FN1 (DGE: 2.19, Power: 0.96)). This profile suggests that some hESC-derived non-HE further differentiate into mesenchymal cells, in a process known as the endothelial-to mesenchymal transition (EndMT). Taken together, we demonstrate that hESC-derived HE and HP share a common developmental pathway, while non-HE is heterogeneous, but transcriptionally distinct. Our novel findings will be instrumental for testing new genetic targets to optimize the production of definitive hematopoietic cells. Disclosures Kaufman:Fate Therapeutics: Consultancy.
Mathew G Angelos - One of the best experts on this subject based on the ideXlab platform.
-
single cell resolution of human hematoendothelial cells defines transcriptional signatures of hemogenic endothelium
Stem Cells, 2018Co-Authors: Mathew G Angelos, Juan E Abrahante, Robert Blum, Dan S KaufmanAbstract:Author(s): Angelos, Mathew G; Abrahante, Juan E; Blum, Robert H; Kaufman, Dan S | Abstract: 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.
-
single cell resolution of human pluripotent stem cell derived hemato endothelial cells reveals distinct transcriptional signatures of hemogenic endothelium
Blood, 2016Co-Authors: Mathew G Angelos, Juan E Abrahante, Dan S KaufmanAbstract:Hemogenic endothelium is a rare and highly specialized subset of vascular endothelial cell that functions as a precursor cell population to definitive blood development. One of the mechanistic hallmarks of definitive hematopoiesis is the endothelial-to-hematopoietic transition (EHT), a process where hemogenic endothelial cells phenotypically switches to produce a detached and free-moving hematopoietic cell. While EHT has been visualized both in vitro and in vivo via lineage tracing studies, the regulation of this fate change at both a cellular and molecular level remains unclear. Human pluripotent stem cells, such as human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) can serve as a useful platform to characterize human hemogenic endothelium and to understand basic mechanisms underlying human EHT. We hypothesized that human hemogenic endothelium derived from hESCs is phenotypically and transcriptionally distinct from other vascular endothelial cells and hematopoietic progenitor cells. To identify human hemogenic endothelium, we used combined expression of endothelial cell surface antigens and RUNX1c expression via a fluorescent reporter previously validated in our lab. Specifically, we employed defined culture methods to differentiate hemogenic endothelial cells (HE) and vascular endothelial cells without hematopoietic potential (non-HE) from hESC harboring a RUNX1c-tdTomato reporter (hESC-RUNX1c-tdTomato). At Day 11 of differentiation, CD31+CD144+ endothelial cells were present, with approximately 40% of these cells tdTomato+. We next sorted HE (CD31+CD144+CD41-CD43-CD45-CD73-tdTomato+) and non-HE (CD31+CD144+CD41-CD43-CD45-CD73-tdTomato-) and cultured both populations in endothelial growth media (EGM) or hematopoietic growth media. HE retained characteristic cobblestone morphology and CD31 expression over the course of 5 days in EGM similar to control human umbilical vein endothelial cells. HE, but not non-HE, was able to generate non-adherent, tdTomato+ hematopoietic progenitor cells in hematopoietic growth media. Using these defined cell populations, we next performed single-cell RNASeq on HE, non-HE, and early hematopoietic progenitor cells (HP; CD34+CD43+tdTomato+). We captured a total of 55 HE, 47 non-HE, and 35 HP using the Fluidgm C1 single cell system and performed next generation sequencing of validated libraries. We analyzed single cell gene expression using Seurat, an R-based bioinformatics software package developed for the analysis of single cell NGS experiments. Populations were first validated based on expression of known genetic identifiers for vascular endothelium, hemogenic endothelium, and hematopoietic progenitor cells. HE and HP were both highly enriched for CDH5, ERG, ESAM, and FLI as compared to non-HE; these genes have been previously implicated in HE functionality. All single cell transcriptional profiles were similarly enriched for KDR, PECAM1, and LMO2, which are characteristic genes expressed in vascular endothelium. We next performed t-distributed stochastic neighbor embedding (t-SNE) using statistically significant principal components to distinguish groups of cells with similar transcriptional expression. Interestingly, we found overlap between individual HE and HP cells (Cluster 1); however, these cells were distinct from two, separate clusters of non-HE (Cluster 2 & 3; Figure 1). Further analysis of these clusters revealed novel biomarkers for HE/HP, such as TIMP3 (DGE: 2.06, Power: 0.91), ERG (DGE: 2.08, Power: 0.83), NOTCH4 (DGE: 2.35, Power: 0.78), and HEY1 (DGE: 2.47, Power: 0.74), Non-HE were found to cluster into two, distinct groups (Clusters 2 and 3), with Cluster 3 enriched for extracellular matrix genes (COL1A1 (DGE: 6.58, Power: 1.00), DCN (DGE: 6.09, Power: 1.00), VCAN (DGE: 3.55, Power: 1.00), FN1 (DGE: 2.19, Power: 0.96)). This profile suggests that some hESC-derived non-HE further differentiate into mesenchymal cells, in a process known as the endothelial-to mesenchymal transition (EndMT). Taken together, we demonstrate that hESC-derived HE and HP share a common developmental pathway, while non-HE is heterogeneous, but transcriptionally distinct. Our novel findings will be instrumental for testing new genetic targets to optimize the production of definitive hematopoietic cells. Disclosures Kaufman:Fate Therapeutics: Consultancy.
Juan E Abrahante - One of the best experts on this subject based on the ideXlab platform.
-
single cell resolution of human hematoendothelial cells defines transcriptional signatures of hemogenic endothelium
Stem Cells, 2018Co-Authors: Mathew G Angelos, Juan E Abrahante, Robert Blum, Dan S KaufmanAbstract:Author(s): Angelos, Mathew G; Abrahante, Juan E; Blum, Robert H; Kaufman, Dan S | Abstract: 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.
-
single cell resolution of human pluripotent stem cell derived hemato endothelial cells reveals distinct transcriptional signatures of hemogenic endothelium
Blood, 2016Co-Authors: Mathew G Angelos, Juan E Abrahante, Dan S KaufmanAbstract:Hemogenic endothelium is a rare and highly specialized subset of vascular endothelial cell that functions as a precursor cell population to definitive blood development. One of the mechanistic hallmarks of definitive hematopoiesis is the endothelial-to-hematopoietic transition (EHT), a process where hemogenic endothelial cells phenotypically switches to produce a detached and free-moving hematopoietic cell. While EHT has been visualized both in vitro and in vivo via lineage tracing studies, the regulation of this fate change at both a cellular and molecular level remains unclear. Human pluripotent stem cells, such as human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) can serve as a useful platform to characterize human hemogenic endothelium and to understand basic mechanisms underlying human EHT. We hypothesized that human hemogenic endothelium derived from hESCs is phenotypically and transcriptionally distinct from other vascular endothelial cells and hematopoietic progenitor cells. To identify human hemogenic endothelium, we used combined expression of endothelial cell surface antigens and RUNX1c expression via a fluorescent reporter previously validated in our lab. Specifically, we employed defined culture methods to differentiate hemogenic endothelial cells (HE) and vascular endothelial cells without hematopoietic potential (non-HE) from hESC harboring a RUNX1c-tdTomato reporter (hESC-RUNX1c-tdTomato). At Day 11 of differentiation, CD31+CD144+ endothelial cells were present, with approximately 40% of these cells tdTomato+. We next sorted HE (CD31+CD144+CD41-CD43-CD45-CD73-tdTomato+) and non-HE (CD31+CD144+CD41-CD43-CD45-CD73-tdTomato-) and cultured both populations in endothelial growth media (EGM) or hematopoietic growth media. HE retained characteristic cobblestone morphology and CD31 expression over the course of 5 days in EGM similar to control human umbilical vein endothelial cells. HE, but not non-HE, was able to generate non-adherent, tdTomato+ hematopoietic progenitor cells in hematopoietic growth media. Using these defined cell populations, we next performed single-cell RNASeq on HE, non-HE, and early hematopoietic progenitor cells (HP; CD34+CD43+tdTomato+). We captured a total of 55 HE, 47 non-HE, and 35 HP using the Fluidgm C1 single cell system and performed next generation sequencing of validated libraries. We analyzed single cell gene expression using Seurat, an R-based bioinformatics software package developed for the analysis of single cell NGS experiments. Populations were first validated based on expression of known genetic identifiers for vascular endothelium, hemogenic endothelium, and hematopoietic progenitor cells. HE and HP were both highly enriched for CDH5, ERG, ESAM, and FLI as compared to non-HE; these genes have been previously implicated in HE functionality. All single cell transcriptional profiles were similarly enriched for KDR, PECAM1, and LMO2, which are characteristic genes expressed in vascular endothelium. We next performed t-distributed stochastic neighbor embedding (t-SNE) using statistically significant principal components to distinguish groups of cells with similar transcriptional expression. Interestingly, we found overlap between individual HE and HP cells (Cluster 1); however, these cells were distinct from two, separate clusters of non-HE (Cluster 2 & 3; Figure 1). Further analysis of these clusters revealed novel biomarkers for HE/HP, such as TIMP3 (DGE: 2.06, Power: 0.91), ERG (DGE: 2.08, Power: 0.83), NOTCH4 (DGE: 2.35, Power: 0.78), and HEY1 (DGE: 2.47, Power: 0.74), Non-HE were found to cluster into two, distinct groups (Clusters 2 and 3), with Cluster 3 enriched for extracellular matrix genes (COL1A1 (DGE: 6.58, Power: 1.00), DCN (DGE: 6.09, Power: 1.00), VCAN (DGE: 3.55, Power: 1.00), FN1 (DGE: 2.19, Power: 0.96)). This profile suggests that some hESC-derived non-HE further differentiate into mesenchymal cells, in a process known as the endothelial-to mesenchymal transition (EndMT). Taken together, we demonstrate that hESC-derived HE and HP share a common developmental pathway, while non-HE is heterogeneous, but transcriptionally distinct. Our novel findings will be instrumental for testing new genetic targets to optimize the production of definitive hematopoietic cells. Disclosures Kaufman:Fate Therapeutics: Consultancy.
Moustapha Kassem - One of the best experts on this subject based on the ideXlab platform.
-
human stromal mesenchymal stem cells from bone marrow adipose tissue and skin exhibit differences in molecular phenotype and differentiation potential
Stem Cell Reviews and Reports, 2013Co-Authors: May Alnbaheen, Radhakrishnan Vishnubalaji, Dalia Ali, Amel Bouslimi, Fawzi F Aljassir, Matthias Megges, Alessandro Prigione, James Adjaye, Moustapha KassemAbstract:Human stromal (mesenchymal) stem cells (hMSCs) are multipotent stem cells with ability to differentiate into mesoderm-type cells e.g. osteoblasts and adipocytes and thus they are being introduced into clinical trials for tissue regeneration. Traditionally, hMSCs have been isolated from bone marrow, but the number of cells obtained is limited. Here, we compared the MSC-like cell populations, obtained from alternative sources for MSC: adipose tissue and skin, with the standard phenotype of human bone marrow MSC (BM-MSCs). MSC from human adipose tissue (human adipose stromal cells (hATSCs)) and human skin (human adult skin stromal cells, (hASSCs) and human new-born skin stromal cells (hNSSCs)) grew readily in culture and the growth rate was highest in hNSSCs and lowest in hATSCs. Compared with phenotype of hBM-MSC, all cell populations were CD34−, CD45−, CD14−, CD31−, HLA-DR−, CD13+, CD29+, CD44+, CD73+, CD90+,and CD105+. When exposed to in vitro differentiation, hATSCs, hASSCs and hNSSCs exhibited quantitative differences in their ability to differentiate into adipocytes and to osteoblastic cells. Using a microarray-based approach we have unveiled a common MSC molecular signature composed of 33 CD markers including known MSC markers and several novel markers e.g. CD165, CD276, and CD82. However, significant differences in the molecular phenotype between these different stromal cell populations were observed suggesting ontological and functional differences. In conclusion, MSC populations obtained from different tissues exhibit significant differences in their proliferation, differentiation and molecular phenotype, which should be taken into consideration when planning their use in clinical protocols.
Igor I Slukvin - One of the best experts on this subject based on the ideXlab platform.
-
identification of hemogenic endothelium and its direct precursor in human embryonic stem cell differentiation cultures
Blood, 2011Co-Authors: Kyungdal Choi, Maxim A Vodyanik, Shulan Tian, Ron Stewart, James A. Thomson, Igor I SlukvinAbstract:Abstract 1277 Animal studies demonstrated that the first HSCs are generated in the aorta-gonad-mesonephros from a unique population of endothelial cells, hemogenic endothelium (HE), through endothelial-hematopoietic transition. However, the identity of HE remains obscure and the specific features that distinguish HE from non-HE and the discrete stages of endothelial transition into hematopoietic cells are not characterized. Here we employed hESC differentiation system in coculture with OP9 to define the distinct population of HE, and to develop an experimental system for analysis of endothelial-hematopoietic transition in vitro. Previously, we demonstrated that endothelial cells could be distinguished from hematopoietic progenitors based on the lack of CD43 expression (Vodyanik et al., Blood 2006;108:2095). Here, we analyzed the earliest stages of endothelial development from hESCs before the typical hematopoietic CFCs could be detected. We found that the first CD144 + endothelial cells appear on day 4 of differentiation within a population of APLNR + cells expressing a high level of KDR. Based on expression of CD235a/CD43, CD73 and CD41a, we identified three major subsets within the emerging CD144 + cells: 1)CD235a/CD43 − CD73 + , 2)CD235a/CD43 − CD73 − , and 3)CD235a/CD43 + CD41a −. Although all three subsets formed monolayer endothelial cells when grown on fibronectin in endothelial media, only CD235a/CD43 + CD41a − cells displayed hematopoietic CFC potential, which required serum-free medium and were dependent on hematopoietic cytokines and FGF2. After culture with OP9, both CD144 + CD235a/CD43 − CD73 − and CD144 + CD235a/CD43 + CD41a − cells generated CD31 + CD43/45 − endothelial cells and a significant amount of CD43 + blood cells. In contrast CD144 + CD235a/CD43 − CD73 + cells formed almost exclusively endothelial cells. Single cell deposition experiments demonstrated that CD144 + CD235a/CD43 + CD41a − cells formed predominantly hematopoietic cells, while most CD144 + CD235a/CD43 − CD73 − cells gave rise to either hematopoietic or endothelial clusters with approximately 2.5% of cells forming hematoendothelial clusters, indicating the presence of bipotential progenitors within this population. Though all three CD144 + subsets had a very similar molecular signature and expressed typical endothelial genes and genes associated with angiohematopoietic and HSC development, CD235a/CD43 + CD41a − showed the higher expression of hematopoietic genes and lower expression of CDH5, CDH2, CAV1 , and APOLD1 typical endothelial genes. Based on the functional, phenotypic and molecular properties we designated the identity of subsets as follows: 1) CD144 + CD235a/CD43 + CD41a − angiogenic blood cells, i.e. cells that possessed primary hematopoietic characteristics but were also capable of generating endothelial cells; 2) CD144 + CD235a/CD43 − CD73 + endothelial progenitors, i.e. cells that had all functional and molecular features of endothelial cells and endothelial colony-forming potential on OP9; 3) CD144 + CD235a/CD43 − CD73 − HE cells, i.e. cells with primary endothelial characteristics lacking hematopoietic CFC potential and surface markers, but capable of generating blood and endothelial cells after coculture with stromal cells. To find out the direct precursors of HE and blood cells, we analyzed the differentiation potential of major subsets of mesodermal cells isolated on day 4 of hESC coculture with OP9. We found that only APLNR + KDR bright CD140a low/− mesodermal cells lacking expression of typical hematopoietic (CD43, CD45), endothelial (CD31, CD144), and mesenchymal (CD73, CD105) markers had the potential to generate both blood and endothelium. Moreover, single cell deposition experiments demonstrated that APLNR + KDR bright CD140a low/− cells formed HE clusters with a high frequency (about 1/10 cells), strongly indicating that these cells represent the direct precursors of HE. Because APLNR + KDR bright CD140a low/− cells upregulated expression LMO2, TAL1, CBFB, GATA2 , and FLI1 transcriptional regulators of hematopoietic and endothelial development, but were lacking expression of primitive streak genes, we designated these cells as angioblasts. Together these studies provide a hESC-based platform for identification of factors required for acquisition of HSC potential by blood cells following transition from endothelium. Disclosures: Thomson: CDI: Consultancy, Equity Ownership, Membership on an entity9s Board of Directors or advisory committees. Slukvin: CDI: Consultancy, Equity Ownership.
-
Hematopoietic differentiation and production of mature myeloid cells from human pluripotent stem cells
Nature Protocols, 2011Co-Authors: Kyungdal Choi, Maxim A Vodyanik, Igor I SlukvinAbstract:In this paper, we describe a protocol for hematopoietic differentiation of human pluripotent stem cells (hPSCs) and generation of mature myeloid cells from hPSCs through expansion and differentiation of hPSC-derived lin^−CD34^+CD43^+CD45^+ multipotent progenitors. The protocol comprises three major steps: (i) induction of hematopoietic differentiation by coculture of hPSCs with OP9 bone marrow stromal cells; (ii) short-term expansion of multipotent myeloid progenitors with a high dose of granulocyte-macrophage colony-stimulating factor; and (iii) directed differentiation of myeloid progenitors into neutrophils, eosinophils, dendritic cells, Langerhans cells, macrophages and osteoclasts. The generation of multipotent hematopoietic progenitors from hPSCs requires 9 d of culture and an additional 2 d to expand myeloid progenitors. Differentiation of myeloid progenitors into mature myeloid cells requires an additional 5–19 d of culture with cytokines, depending on the cell type.
-
leukosialin CD43 defines hematopoietic progenitors in human embryonic stem cell differentiation cultures
Blood, 2006Co-Authors: Maxim A Vodyanik, James A. Thomson, Igor I SlukvinAbstract:During hematopoietic differentiation of human embryonic stem cells (hESCs), early hematopoietic progenitors arise along with endothelial cells within the CD34+ population. Although hESC-derived hematopoietic progenitors have been previously identified by functional assays, their phenotype has not been defined. Here, using hESC differentiation in coculture with OP9 stromal cells, we demonstrate that early progenitors committed to hematopoietic development could be identified by surface expression of leukosialin (CD43). CD43 was detected on all types of emerging clonogenic progenitors before expression of CD45, persisted on differentiating hematopoietic cells, and reliably separated the hematopoietic CD34+ population from CD34+CD43–CD31+KDR+ endothelial and CD34+CD43–CD31–KDR– mesenchymal cells. Furthermore, we demonstrated that the first-appearing CD34+CD43+CD235a+CD41a+/–CD45– cells represent precommitted erythro-megakaryocytic progenitors. Multipotent lymphohematopoietic progenitors were generated later as CD34+CD43+CD41a–CD235a–CD45– cells. These cells were negative for lineage-specific markers (Lin–), expressed KDR, VE-cadherin, and CD105 endothelial proteins, and expressed GATA-2, GATA-3, RUNX1, C-MYB transcription factors that typify initial stages of definitive hematopoiesis originating from endothelial-like precursors. Acquisition of CD45 expression by CD34+CD43+CD45–Lin– cells was associated with progressive myeloid commitment and a decrease of B-lymphoid potential. CD34+CD43+CD45+Lin– cells were largely devoid of VE-cadherin and KDR expression and had a distinct FLT3highGATA3lowRUNX1lowPU1highMPOhighIL7RAhigh gene expression profile.