Project description:Transcription factors play critical roles in stem cell maintenance and differentiation. Using single cell RNA sequencing, we investigated transcription factors expressed in endothelial progenitors differentiated from human pluripotent stem cells (hPSCs) and identified upregulated differential expression of SOXF subgroup members SOX7, SOX17, and SOX18. To test whether overexpression of these factors increases differentiation efficiency, we established inducible hPSC lines and found only SOX17 improved differentiation of CD34+VEC+ cells. Temporal expression analysis of SOX17 and VEC revealed that SOX17 was turned on immediately before VEC, indicating SOX17 may be a causative factor in determining hemogenic endothelial differentiation. Upon Cas13d mediated repression of SOX17, differentiation was significantly abrogated. We found SOX17 forward programming is sufficient to generate more than 50% CD34+VEC+CD73- cells. Further differentiation of SOX17 forward programmed cells generated hematopoietic progenitors that emerged via an endothelial to hematopoietic transition and significantly upregulated definitive hematopoietic transcriptional programs. Our analyses reveal an uncharacterized function of SOX17 in directing hPSCs differentiation towards hematopoietic lineages.
Project description:Human embryonic stem cells (hESCs) are a powerful tool for modeling regenerative therapy. To search for the genes that promote hematopoietic development from human pluripotent stem cell, we overexpressed a list of hematopoietic regulator genes in human pluripotent stem cell-derived CD34+CD43- endothelial cells (ECs) enriched in hemogenic endothelium. Among genes tested, only SOX17, a gene encoding a transcription factor of the SOX family, promoted cell growth and supported expansion of CD34+CD43+CD45-/low cells expressing a hemogenic endothelial maker VE-cadherin. SOX17 was highly expressed in CD34+CD43- ECs but at a low level in CD34+CD43+CD45- pre-hematopoietic progenitor cells (pre-HPCs) and CD34+CD43+CD45+ HPCs. SOX17-overexpressing cells formed sphere-like colonies and generated few hematopoietic progenies. However, they retained hemogenic potential and gave rise to hematopoietic progenies upon inactivation of SOX17. Global gene expression analyses revealed that the CD34+CD43+CD45-/low cells expanded upon overexpression of SOX17 are hemogenic endothelium-like cells developmentally placed between ECs and pre-HPCs. Of interest, SOX17 also reprogrammed both pre-HPCs and HPCs into hemogenic endothelium-like cells. Genome-wide mapping of SOX17 revealed that SOX17 directly activates transcription of key regulator genes for vasculogenesis, hematopoiesis, and erythrocyte differentiation. Depletion of SOX17 in CD34+CD43- ECs severely compromised their hemogenic activity. These findings suggest that SOX17 plays a critical role in priming hemogenic potential in ECs, thereby regulates hematopoietic development from hESCs. This SuperSeries is composed of the SubSeries listed below.
Project description:Overexpression of transcription factor Sox17 in human ES cells-derived endothelial cells and hematopoietic cells enhances expansion of hemogenic endothelium-like cells.
Project description:Overexpression of transcription factor Sox17 in human ES cells-derived endothelial cells and hematopoietic cells enhances expansion of hemogenic endothelium-like cells. Human ES cells were differentiated for 6 days, 8 days or 12 days in EBs, then CD34+CD43-CD45- endothelial cells, CD34+CD43+CD45- pre-hematopoietic progenitor cells (HPCs) or CD34+CD43+CD45+ HPCs were isolated by fluorescence activated cell sorting (FACS) and subjected to a microarray analysis.M-cM-^@M-^@Some samples were plated onto OP9 cells after the isolation by FACS, and transduced with the 4OH-tamoxifen-inducible 1M-CM-^WFLAG-tagged Sox17-ERT retrovirus. The cells were cultured with 4OH-tamoxifen. CD34+CD43+CD45low hemogenic endothelium-like cells expanded by Sox17-ERT were collected by magnetic-activated cell sorting (MACS) and subjected to a ChIP-chip analysis.
Project description:Human embryonic stem cells (hESCs) are a powerful tool for modeling regenerative therapy. To search for the genes that promote hematopoietic development from human pluripotent stem cell, we overexpressed a list of hematopoietic regulator genes in human pluripotent stem cell-derived CD34+CD43- endothelial cells (ECs) enriched in hemogenic endothelium. Among genes tested, only SOX17, a gene encoding a transcription factor of the SOX family, promoted cell growth and supported expansion of CD34+CD43+CD45-/low cells expressing a hemogenic endothelial maker VE-cadherin. SOX17 was highly expressed in CD34+CD43- ECs but at a low level in CD34+CD43+CD45- pre-hematopoietic progenitor cells (pre-HPCs) and CD34+CD43+CD45+ HPCs. SOX17-overexpressing cells formed sphere-like colonies and generated few hematopoietic progenies. However, they retained hemogenic potential and gave rise to hematopoietic progenies upon inactivation of SOX17. Global gene expression analyses revealed that the CD34+CD43+CD45-/low cells expanded upon overexpression of SOX17 are hemogenic endothelium-like cells developmentally placed between ECs and pre-HPCs. Of interest, SOX17 also reprogrammed both pre-HPCs and HPCs into hemogenic endothelium-like cells. Genome-wide mapping of SOX17 revealed that SOX17 directly activates transcription of key regulator genes for vasculogenesis, hematopoiesis, and erythrocyte differentiation. Depletion of SOX17 in CD34+CD43- ECs severely compromised their hemogenic activity. These findings suggest that SOX17 plays a critical role in priming hemogenic potential in ECs, thereby regulates hematopoietic development from hESCs. This SuperSeries is composed of the SubSeries listed below. ChIP on chip analysis was carried out using the Mouse Promoter ChIP-on-chip Microarray Set (G4490A, Agilent, Palo Alto, Calif., USA). MEFs were subjected to ChIP assay using a Ring1B antibody. Purified immunoprecipitated and input DNA was subjected to T7 RNA polymerase-based amplification. Labeling, hybridization and washing were carried out according to the Agilent mammalian ChIP-on-chip protocol (ver.9.0). Scanned images were quantified with Agilent Feature Extraction software under standard conditions. Human ES cells were differentiated for 6 days in EBs, then CD34+CD43-CD45- endothelial cells were isolated, plated onto OP9 cells, and transduced with the 4OH-tamoxifen (4OHT)-inducible 3M-CM-^WFLAG-tagged Sox17-ERT retrovirus. The cells were seeded on OP9 stromal cells and cultured in the presence of 4OH-tamoxifen. At day 27 of the co-culture with OP9 cells, CD34+CD43+CD45low hemogenic endothelium-like cells overexpressing Sox17-ERT were collected by CD34 magnetic-activated cell sorting (MACS) and subjected to a ChIP-chip analysis. ChIP on chip analysis was carried out using the Mouse Promoter ChIP-on-chip Microarray Set (G4490A, Agilent, Palo Alto, Calif., USA). MEFs were subjected to ChIP assay using a Ring1B antibody. Purified immunoprecipitated and input DNA was subjected to T7 RNA polymerase-based amplification. Labeling, hybridization and washing were carried out according to the Agilent mammalian ChIP-on-chip protocol (ver.9.0). Scanned images were quantified with Agilent Feature Extraction software under standard conditions. Human ES cells were differentiated for 6 days in EBs, then CD34+CD43-CD45- endothelial cells were isolated, plated onto OP9 cells, and transduced with the 4OH-tamoxifen (4OHT)-inducible 3M-CM-^WFLAG-tagged Sox17-ERT retrovirus. The cells were seeded on OP9 stromal cells and cultured in the presence of 4OH-tamoxifen. At day 27 of the co-culture with OP9 cells, CD34+CD43+CD45low hemogenic endothelium-like cells overexpressing Sox17-ERT were collected by CD34 magnetic-activated cell sorting (MACS) and subjected to a ChIP-chip analysis.
Project description:The transcription factor Sox17 is expressed in early primitive endoderm-fated cells of the mouse embryo and in embryo-derived extraembryonic endoderm (ExEn) stem (XEN) cells. We have shown that overexpression of Sox17 in mouse embryonic stem cells (ESCs) drives cell fate to a committed XEN-like cell state (Sox17-XEN cells). When placed back into the embryo, Sox17-XEN cells contribute exclusively to the ExEn. Transient Sox17 expression is sufficient to drive this fate change during which time cells transit through distinct intermediate states prior to the generation of functional XEN-like cells. We identified dynamic regulatory networks driving Sox17-mediated XEN conversion by analyzing a dynamic regulatory map of gene expression bifurcation points throughout conversion, created using RNA-seq time series data. We found that Sox17 orchestrates this conversion process by acting in autoregulatory and feed-forward network motifs, regulating dynamic gene regulatory networks (GRNs) directing cell fate. We have shown that Sox17-mediated XEN conversion provides a powerful tool for understanding the regulation of cell fate changes and for the elucidation of GRNs regulating lineage decisions in the mouse embryo. Total RNA was extracted during a time course of Sox17 overexpression in mouse ESCs at 7 time points as well as from wild-type ESCs and wild-type XEN cells.
Project description:This SuperSeries is composed of the following subset Series: GSE30444: Retroviral Sox17 over-expression adult hematopoietic stem/progenitor cells microarray GSE30445: Sox17-transgenic hematopoietic stem cell microarray Refer to individual Series
Project description:Overexpression of transcription factor Sox17 in human ES cells-derived endothelial cells enhances expansion of hemogenic endothelium-like cells.
Project description:To determine the effect of Sox17 overexpression in mouse embryonic stem (ES) cells, we performed gain-of-function analysis by generating ES cell lines carrying a doxycycline inducible FLAG-tagged Sox17 transgene. We treated Sox17-inducible ES cells with doxycycline, collected RNA and performed genome-wide transcriptional analysis. We found that genes invovled in adhesion function and basement membrane establishment were transcriptionally upregulated in ES cells upon induction of Sox17. We also observed downregulation in the transcription of genes involved in pathways known to be functionally important for ES cell pluripotency and self-renewal. However, Sox17 expression was not sufficient to rapidly down-regulate Sox2, Nanog, and Oct4. Two independent doxycycline inducible Sox17-overexpressing mouse embryonic stem cells were derived. The genes expression changes in the Sox17-induced cells were compared to untreated (no doxycycline) controls and to control cells treated with or without doxycycline. The total RNA from these samples were amplified using Ambion Illumina TotalPrep RNA Amplification kit and arrayed on Illumina MouseRef8 v2 chips.
Project description:The transcription factor Sox17 is expressed in early primitive endoderm-fated cells of the mouse embryo and in embryo-derived extraembryonic endoderm (ExEn) stem (XEN) cells. We have shown that overexpression of Sox17 in mouse embryonic stem cells (ESCs) drives cell fate to a committed XEN-like cell state (Sox17-XEN cells). When placed back into the embryo, Sox17-XEN cells contribute exclusively to the ExEn. Transient Sox17 expression is sufficient to drive this fate change during which time cells transit through distinct intermediate states prior to the generation of functional XEN-like cells. We identified dynamic regulatory networks driving Sox17-mediated XEN conversion by analyzing a dynamic regulatory map of gene expression bifurcation points throughout conversion, created using RNA-seq time series data. We found that Sox17 orchestrates this conversion process by acting in autoregulatory and feed-forward network motifs, regulating dynamic gene regulatory networks (GRNs) directing cell fate. We have shown that Sox17-mediated XEN conversion provides a powerful tool for understanding the regulation of cell fate changes and for the elucidation of GRNs regulating lineage decisions in the mouse embryo.