Project description:Recent advances in direct reprogramming using cell type-specific transcription factors provide an unprecedented opportunity for rapid generation of desired human cell types from easily accessible tissues. However, due to the diversity of conversion factors that facilitate the process, an arduous screening step is inevitable to find the appropriate combination(s). Here, we show that under chemically defined conditions minimal pluripotency factors are sufficient to directly reprogram human fibroblasts into stably self-renewing neural progenitor/stem cells (NSCs), but without passing through a pluripotent intermediate stage. These NSCs can be expanded and propagated in vitro without losing their potential to differentiate into various neuronal subtypes and glia. Our direct reprogramming strategy represents a simple and advanced paradigm of direct conversion that will provide an unlimited source of human neural cells for cell therapy, disease modeling, and drug screening. We used microarray to compare the global gene expression pattern between human fibroblasts and human neural epitheliums from human ESCs or directly from fibroblasts. We cultured cells and harvested them and then extracted total RNA for microarray.
Project description:Recent advances in direct reprogramming using cell type-specific transcription factors provide an unprecedented opportunity for rapid generation of desired human cell types from easily accessible tissues. However, due to the diversity of conversion factors that facilitate the process, an arduous screening step is inevitable to find the appropriate combination(s). Here, we show that under chemically defined conditions minimal pluripotency factors are sufficient to directly reprogram human fibroblasts into stably self-renewing neural progenitor/stem cells (NSCs), but without passing through a pluripotent intermediate stage. These NSCs can be expanded and propagated in vitro without losing their potential to differentiate into various neuronal subtypes and glia. Our direct reprogramming strategy represents a simple and advanced paradigm of direct conversion that will provide an unlimited source of human neural cells for cell therapy, disease modeling, and drug screening. We used microarray to compare the global gene expression pattern between human fibroblasts and human neural epitheliums from human ESCs or directly from fibroblasts.
Project description:Recent advances have suggested that direct induction of neural stem cells could provide an alternative to derivation from somatic tissues or pluripotent cells. Here we show direct derivation of stably expandable NS cells from mouse fibroblasts through a curtailed version of reprogramming to pluripotency. By constitutively inducing Sox2, Klf4, and c-Myc while strictly limiting Oct4 activity to the initial phase of reprogramming, we generated neurosphere-like colonies that could be expanded for more than 50 passages and do not depend on sustained expression of the reprogramming factors. These induced NS (iNS) cells uniformly display morphological and molecular features of NS cells such as the expression of Nestin, Pax6, and Olig2 and have a similar genome-wide transcriptional profile to brain-derived NSCs. iNS cells can differentiate into neurons, astrocytes and oligodendrocytes in vitro and in vivo. Our results demonstrate that functional neural stem cells can be generated from somatic cells by factor-driven induction. mRNA extracted from Murine Embryonic Fibroblasts (MEF), murine Embryonic Stem Cell (ES), murine Neuronal Stem Cell (NS) and three murine induces Neuronal Stem Cell clones 2, 3 and 5 (iNS2, iNS3, iNS5) has been hybridized on Illumina MouseWG6 V2 arrays for genome wide expression analysis. Samples were run at least as triple, MEF, iNS3, iNS5 as quadruple technical replicates. Differential gene expression analysis has been performed on the grouped expression data with the Murine Embryonic Fibroblasts group as the reference.
Project description:Recent studies have demonstrated direct reprogramming of fibroblasts into a range of somatic cell types, but to date stem/progenitor cells have only been reprogrammed for the blood and neuronal lineages. We previously reported generation of induced hepatocyte-like (iHep) cells by transduction of Gata4, Hnf1α, and Foxa3 in p19 Arf null mouse embryonic fibroblasts (MEFs). Here, we show that Hnf1β and Foxa3, liver organogenesis transcription factors, are sufficient to reprogram MEFs into induced hepatic stem cells (iHepSCs). iHepSCs can be stably expanded in vitro and possess the potential of bi-directional differentiation into both hepatocytic and cholangiocytic lineages. In the injured liver of fumarylacetoacetate hydrolase (Fah)-deficient mice, repopulating iHepSCs become hepatocyte-like cells. They also engraft as cholangiocytes into bile ducts of mice with DDC-induced bile ductular injury. Lineage-conversion into bi-potential expandable iHepSCs provides a strategy to enable efficient derivation of both hepatocytes and cholangiocytes for use in disease modeling and tissue engineering. iHepSCs were converted form fibroblasts by transduction of Hnf1β and Foxa3. iHepSCs were induced to differentiate into hepatocyte-like cells and cholangiocytes in vitro. Totally, 9 samples including four clones of iHepSCS, one clone of LEPCs, two samples of MEFs and two samples of iHepSCs-derived cholangocytes were analyzed.
Project description:Recent advances have suggested that direct induction of neural stem cells could provide an alternative to derivation from somatic tissues or pluripotent cells. Here we show direct derivation of stably expandable NS cells from mouse fibroblasts through a curtailed version of reprogramming to pluripotency. By constitutively inducing Sox2, Klf4, and c-Myc while strictly limiting Oct4 activity to the initial phase of reprogramming, we generated neurosphere-like colonies that could be expanded for more than 50 passages and do not depend on sustained expression of the reprogramming factors. These induced NS (iNS) cells uniformly display morphological and molecular features of NS cells such as the expression of Nestin, Pax6, and Olig2 and have a similar genome-wide transcriptional profile to brain-derived NSCs. iNS cells can differentiate into neurons, astrocytes and oligodendrocytes in vitro and in vivo. Our results demonstrate that functional neural stem cells can be generated from somatic cells by factor-driven induction.