Project description:Single cell RNA-seq is a powerful tool for studying complex biological systems such as HSPCs cultured in vitro. We detected 11, 3, 4, 8 and 8 subpopulations in uncultured CD34+ cells,control cells, CD34+ cells with the treatment of CFO, cytokines and CFO + cytokines using t-Distributed Stochastic Neighbor Embedding (t-SNE) analysis.
Project description:Neural progenitor cells (NPCs) can be induced from somatic cells by defined factors. Here we report that NPCs can be generated from mouse embryonic fibroblasts by a chemical cocktail, namely VCR (V, VPA, an inhibitor of HDACs; C, CHIR99021, an inhibitor of GSK-3 kinases and R, Repsox, an inhibitor of TGF-β pathways), under a physiological hypoxic condition. These chemical-induced NPCs (ciNPCs) resemble mouse brain-derived NPCs regarding their proliferative and self-renewing abilities, gene expression profiles, and multipotency for different neuroectodermal lineages in vitro and in vivo. Further experiments reveal that alternative cocktails with inhibitors of histone deacetylation, glycogen synthase kinase, and TGF-β pathways show similar efficacies for ciNPC induction. Moreover, ciNPCs can also be induced from mouse tail-tip fibroblasts and human urinary cells with the same chemical cocktail VCR. Thus our study demonstrates that lineage-specific conversion of somatic cells to NPCs could be achieved by chemical cocktails without introducing exogenous factors. To access the exact identity of ciNPCs, we extracted mRNA from mouse brain-derived NPCs (as control NPCs), MEFs, ciNPCs at passage 5 and passage 13 and compared the global gene expression patterns of these cells by microarray analysis.
Project description:Neural progenitor cells (NPCs) can be induced from somatic cells by defined factors. Here we report that NPCs can be generated from mouse embryonic fibroblasts by a chemical cocktail, namely VCR (V, VPA, an inhibitor of HDACs; C, CHIR99021, an inhibitor of GSK-3 kinases and R, Repsox, an inhibitor of TGF-β pathways), under a physiological hypoxic condition. These chemical-induced NPCs (ciNPCs) resemble mouse brain-derived NPCs regarding their proliferative and self-renewing abilities, gene expression profiles, and multipotency for different neuroectodermal lineages in vitro and in vivo. Further experiments reveal that alternative cocktails with inhibitors of histone deacetylation, glycogen synthase kinase, and TGF-β pathways show similar efficacies for ciNPC induction. Moreover, ciNPCs can also be induced from mouse tail-tip fibroblasts and human urinary cells with the same chemical cocktail VCR. Thus our study demonstrates that lineage-specific conversion of somatic cells to NPCs could be achieved by chemical cocktails without introducing exogenous factors.
Project description:The direct conversion, or trans-differentiation, of non-cardiac cells into cardiomyocytes by forced expression of transcription factors and microRNAs provide promising ways of cardiac regeneration. However, genetic manipulations are still not desirable in real clinical applications. we report the generation of automatically beating cardiomyocyte-like cells from mouse fibroblasts with only chemical cocktails. These chemical-induced cardiomyocyte-like cells (CiCMs) express cardiomyocyte-specific markers, exhibit sarcomeric organization, and possess typical cardiac calcium flux and electrophysiological features. Microarray-bassed gene expression patterns of Mouse embryonic fibroblasts (MEFs), CiCMs, and cardiomyocytes(CMs) indicated a clear transition from dividing MEFs to differentiated cardiomyocyte-like state in CiCM samples. Mouse embryonic fibroblasts were treated with a small-molecule combination CRFVPT (10 μM CHIR99021 (C); 10 μM RepSox (R); 50 μM Forskolin (F); 0.5 mM VPA (V); 5 μM Parnate, (P); 1 μM TTNPB (T)) to induce transdifferentiation to chemical-induced cardiomyocyte-like cells. CiCMs beating clusters were picked at day 24 for analysis. MEFs were isolated from mouse embryos, and CMs were isolated from mouse hearts. Total RNA of MEFs, CiCMs and CMs were extracted and hybridization on Affymetrix microarrays.
Project description:The direct conversion, or trans-differentiation, of non-cardiac cells into cardiomyocytes by forced expression of transcription factors and microRNAs provide promising ways of cardiac regeneration. However, genetic manipulations are still not desirable in real clinical applications. we report the generation of automatically beating cardiomyocyte-like cells from mouse fibroblasts with only chemical cocktails. These chemical-induced cardiomyocyte-like cells (CiCMs) express cardiomyocyte-specific markers, exhibit sarcomeric organization, and possess typical cardiac calcium flux and electrophysiological features. Microarray-bassed gene expression patterns of Mouse embryonic fibroblasts (MEFs), CiCMs, and cardiomyocytes(CMs) indicated a clear transition from dividing MEFs to differentiated cardiomyocyte-like state in CiCM samples.
Project description:Functional maintenance of terminally differentiated cells outside the in vivo microenvironment has proved challenging. Current strategies that manipulate cell-cell or cell-matrix connections are difficult to constitute complex regulatory networks for cell function maintenance. Small molecules are easily combined for flexible spatiotemporal modulations, theoretically favorable for synergetic regulation of cell-innate signaling pathways to maintain cell function in vitro. Here, we developed small-molecule cocktails enabling robust maintenance of primary human hepatocytes (PHHs) longer than four weeks, with gene expression profiles, resembling those of freshly isolated PHHs; and prolong-cultured PHHs, for the first time, could maintain drug-metabolizing activities of enzymes accounting for over 80% of drug-oxidation and support hepatitis B virus infection in vitro for over one month. Our study demonstrates that this chemical approach effectively maintains terminally differentiated hepatocytes in vitro, which could be extended to various cell types.
Project description:Functional maintenance of terminally differentiated cells outside the in vivo microenvironment has proved challenging. Current strategies that manipulate cell-cell or cell-matrix connections are difficult to constitute complex regulatory networks for cell function maintenance. Small molecules are easily combined for flexible spatiotemporal modulations, theoretically favorable for synergetic regulation of cell-innate signaling pathways to maintain cell function in vitro. Here, we developed small-molecule cocktails enabling robust maintenance of primary human hepatocytes (PHHs) longer than four weeks, with gene expression profiles, resembling those of freshly isolated PHHs; and prolong-cultured PHHs, for the first time, could maintain drug-metabolizing activities of enzymes accounting for over 80% of drug-oxidation and support hepatitis B virus infection in vitro for over one month. Importantly, this cocktail also promotes functional maturation of human pluripotent stem cell-derived hepatocytes. Our study demonstrates that this chemical approach effectively maintains terminally differentiated hepatocytes in vitro, which could be extended to various cell types.
Project description:We used ChIP-Seq to map Ldb1, Scl and Gata2 binding sites in mouse hematopoietic progenitor cells (HPCs). Together with functional studies using conventional and conditional Ldb1 deficient mouse models and bioinformatics analysis, we systematically determined a transcriptional program controlled by Ldb1 complexes in HSC maintenance. To evaluate the role of Ldb1 in hematopoietic stem cell maintenance.