Project description:Hematopoietic stem cells (HSCs) can regenerate the entire hematopoietic system in vivo, providing the most relevant criteria to measure candidate HSCs derived from human embryonic stem cell (hESC) or induced pluripotent stem cell (hiPSC) sources. Here, we show that unlike primitive hematopoietic cells derived from hESCs, phenotypically identical cells derived from hiPSC are more permissive to graft the bone marrow of xenotransplantation recipients. Despite establishment of bone marrow graft, hiPSC-derived cells fail to demonstrate hematopoietic differentiation in vivo. However, once removed from recipient bone marrow, hiPSC-derived grafts were capable of in vitro multilineage hematopoietic differentiation, indicating that xenograft imparts a restriction to in vivo hematopoietic progression. This failure to regenerate multilineage hematopoiesis in vivo was attributed to the inability to downregulate key microRNAs involved in hematopoiesis. Based on these analyses, our study indicates that hiPSCs provide a beneficial source of pluripotent stem cell-derived hematopoietic cells for transplantation compared with hESCs. Since use of the human-mouse xenograft models prevents detection of putative hiPSC-derived HSCs, we suggest that new preclinical models should be explored to fully evaluate cells generated from hiPSC sources. Human pluripotent stem cell-derived hematopoietic cells were isolated and qPCR-based microRNA profiling was performed.
Project description:Sclerotomal progenitors derived from pluripotent stem cells hold promises for modeling skeletal development and recreating embryonic bone microenvironment to restore hematopoiesis and immunity in ageing. Current strategy to derive mouse sclerotomal progenitors suffered from low differentiation efficiency and heterogenous cell progenies. Here, we developed a fast and efficient strategy to generate sclerotomal progenitors from murine embryonic stem cells (mESCs). Naïve mESCs were differentiated through epiblast-like cells (EpiLCs) and primitive streak, and the sclerotomal fate was directly induced from primitive streak by modulating BMP and SHH signaling, achiving an 85.8% differentiation efficiency. Moreover, the resulting progenitors showed similar global gene expression profiles as primary sclerotome, possessed strong osteochondral bipotential and could recapitulate key features of endochondral ossification upon micromass-mediated osteogenic induction, including bone marrow niche regeneration. To investigate the cellular component of the bone marrow derived from scl-progenitors micromass , we performed droplet-based scRNAseq (10X Genomics) of the differentiated bone tissue.
Project description:Hematopoietic stem cells (HSCs) can regenerate the entire hematopoietic system in vivo, providing the most relevant criteria to measure candidate HSCs derived from human embryonic stem cell (hESC) or induced pluripotent stem cell (hiPSC) sources. Here, we show that unlike primitive hematopoietic cells derived from hESCs, phenotypically identical cells derived from hiPSC are more permissive to graft the bone marrow of xenotransplantation recipients. Despite establishment of bone marrow graft, hiPSC-derived cells fail to demonstrate hematopoietic differentiation in vivo. However, once removed from recipient bone marrow, hiPSC-derived grafts were capable of in vitro multilineage hematopoietic differentiation, indicating that xenograft imparts a restriction to in vivo hematopoietic progression. This failure to regenerate multilineage hematopoiesis in vivo was attributed to the inability to downregulate key microRNAs involved in hematopoiesis. Based on these analyses, our study indicates that hiPSCs provide a beneficial source of pluripotent stem cell-derived hematopoietic cells for transplantation compared with hESCs. Since use of the human-mouse xenograft models prevents detection of putative hiPSC-derived HSCs, we suggest that new preclinical models should be explored to fully evaluate cells generated from hiPSC sources.
Project description:The role of the INV16 genetic translocation in acute myeloid leukemia may be to alter expression in primitive hematopoietic progenitors of genes important for regulating hematopoiesis. To identify transcriptional targets of INV16 in primitive hematopoietic progenitors, FACS-purified progenitors from murine bone marrow were transduced with retrovirus encoding INV16 and analyzed for alterations in gene expression using whole transcriptome expression arrays. Normal murine bone marrow cells of the Lineage-negative, c-Kit+, Sca-1+, Flt3-negative phenotype (KSLF) were FACS-purified, transduced with retrovirus encoding INV16 (INV), the non-leukemogenic INV16 mutant deleted of the assembly competent domain (ACD) or empty retroviral vector control (MIB). Productively transduced, GFP-positive cells were FACS-sorted 24 hours later, and RNA isolated and analyzed using Affymetrix whole transcriptome expression arrays. Replicate numbers of sorts/transductions/analyses of 4, 3, and 5 were performed for INV, ACD, and MIB, respectively.
Project description:The role of the INV16 genetic translocation in acute myeloid leukemia may be to alter expression in primitive hematopoietic progenitors of genes important for regulating hematopoiesis. To identify transcriptional targets of INV16 in primitive hematopoietic progenitors, FACS-purified progenitors from murine bone marrow were transduced with retrovirus encoding INV16 and analyzed for alterations in gene expression using whole transcriptome expression arrays.
Project description:EML cells are a multipotent murine hematopoietic cell line derived in a simple process from normal bone marrow. These cells offer a model system with several advantages for the study of the early steps in hematopoietic differentiation. Cultured EML cells
Project description:To investigate DNA methylation variations at single-base resolution in bone marrow primitive hematopoietic stem cells from Myelodysplastic syndromes patients using whole genome bisulfite sequencing (WGBS)
Project description:To explore the mechanisms by which DCAF8 deficiency induces functional defects in hematopoietic stem cells with an aging-like phenotype, and given DCAF8’s role as a substrate receptor in the E3 ubiquitin ligase complex, we conducted proteomic analysis on Lineage negative bone marrow cells from wild-type and Dcaf8 knockout mice. This analysis aimed to identify protein alterations, providing insights into potential substrates of DCAF8 in murine hematopoietic cells.
Project description:To explore the mechanisms by which DCAF8 deficiency induces functional defects in hematopoietic stem cells with an aging-like phenotype, and given DCAF8’s role as a substrate receptor in the E3 ubiquitin ligase complex, we conducted ubiquitin proteomic analysis on bone marrow cells from wild-type and Dcaf8 knockout mice. This analysis aimed to identify ubiquitinated proteins and assess changes in ubiquitination, providing insights into potential substrates of DCAF8 in murine hematopoietic cells.
Project description:To explore the mechanisms by which DCAF8 deficiency induces functional defects in hematopoietic stem cells with an aging-like phenotype, and given DCAF8’s role as a substrate receptor in the E3 ubiquitin ligase complex, we conducted ubiquitin proteomic analysis on bone marrow cells from wild-type and Dcaf8 knockout mice. This analysis aimed to identify ubiquitinated proteins and assess changes in ubiquitination, providing insights into potential substrates of DCAF8 in murine hematopoietic cells.