Similar Datasets
Project description:Direct lineage reprogramming provides a unique system to study cell fate transitions and unearth molecular mechanisms that safeguard cellular identity. We previously reported on direct conversion of mouse fibroblasts into induced myogenic progenitor cells (iMPCs) by means of transient MyoD overexpression in concert with small molecules treatment. Here we employed integrative multi-omic assays to delineate the molecular landscape of fibroblast reprogramming into iMPCs in comparison to transdifferentiation into myogenic cells solely by MyoD overexpression. Utilizing bulk RNA-sequencing and mass spectrometry we characterized molecular regulators and pathways that endow a myogenic stem cell identity on fibroblasts only in the presence of small molecule treatment. We further demonstrate that iMPC reprogramming is a stepwise process, commencing with the appearance of myofibers and committed myogenic progenitors prior to the formation of satellite cell-like myogenic progenitors that express a suite of stem cell markers including Pax7, Dek, Myf5, Sox8, and Dmrt2. To directly compare iMPCs to satellite cell-derived primary myoblasts, we employed a fluorescent Pax7-GFP reporter to purify Pax7+ cells from the heterogenous iMPC cultures and assess their equivalency to FACS-purified Pax7-GFP+ myoblasts. We demonstrate that Pax7+ iMPCs share molecular attributes with myoblasts, however also express genes and signaling pathways that are indicative of activated satellite cells including Carm1, Dlk1, Lgr5, Fos, Dek, Calcr and Pitx3. We further establish that iMPC formation and maintenance is dependent on the Notch pathway, as small molecule inhibition of Notch abrogates iMPC formation and derails stable iMPC cultures via depletion of Pax7 expressing cells. Lastly, using single cell RNA-sequencing we determine the cell populations that comprise iMPCs in addition to reconstructing the differentiation trajectory present in these heterogenous cultures. We demonstrate that a highly proliferative activated satellite cell-like population differentiates into committed myoblasts and myocytes that further give rise to myofibers that express mature muscle markers, thus capturing a dynamic differentiation program in vitro. Collectively, this study charts a molecular blueprint for reprogramming fibroblasts into myogenic stem and progenitor cells and further establishes the fidelity of stable iMPC cultures in capturing skeletal muscle regeneration in vitro for disease modeling and basic research applications.
2022-01-06 | GSE186271 | GEO
Project description:Direct lineage reprogramming provides a unique system to study cell fate transitions and unearth molecular mechanisms that safeguard cellular identity. We previously reported on direct conversion of mouse fibroblasts into induced myogenic progenitor cells (iMPCs) by means of transient MyoD overexpression in concert with small molecules treatment. Here we employed integrative multi-omic assays to delineate the molecular landscape of fibroblast reprogramming into iMPCs in comparison to transdifferentiation into myogenic cells solely by MyoD overexpression. Utilizing bulk RNA-sequencing and mass spectrometry we characterized molecular regulators and pathways that endow a myogenic stem cell identity on fibroblasts only in the presence of small molecule treatment. We further demonstrate that iMPC reprogramming is a stepwise process, commencing with the appearance of myofibers and committed myogenic progenitors prior to the formation of satellite cell-like myogenic progenitors that express a suite of stem cell markers including Pax7, Dek, Myf5, Sox8, and Dmrt2. To directly compare iMPCs to satellite cell-derived primary myoblasts, we employed a fluorescent Pax7-GFP reporter to purify Pax7+ cells from the heterogenous iMPC cultures and assess their equivalency to FACS-purified Pax7-GFP+ myoblasts. We demonstrate that Pax7+ iMPCs share molecular attributes with myoblasts, however also express genes and signaling pathways that are indicative of activated satellite cells including Carm1, Dlk1, Lgr5, Fos, Dek, Calcr and Pitx3. We further establish that iMPC formation and maintenance is dependent on the Notch pathway, as small molecule inhibition of Notch abrogates iMPC formation and derails stable iMPC cultures via depletion of Pax7 expressing cells. Lastly, using single cell RNA-sequencing we determine the cell populations that comprise iMPCs in addition to reconstructing the differentiation trajectory present in these heterogenous cultures. We demonstrate that a highly proliferative activated satellite cell-like population differentiates into committed myoblasts and myocytes that further give rise to myofibers that express mature muscle markers, thus capturing a dynamic differentiation program in vitro. Collectively, this study charts a molecular blueprint for reprogramming fibroblasts into myogenic stem and progenitor cells and further establishes the fidelity of stable iMPC cultures in capturing skeletal muscle regeneration in vitro for disease modeling and basic research applications.
2022-01-06 | GSE169053 | GEO
Project description:Direct lineage reprogramming provides a unique system to study cell fate transitions and unearth molecular mechanisms that safeguard cellular identity. We previously reported on direct conversion of mouse fibroblasts into induced myogenic progenitor cells (iMPCs) by means of transient MyoD overexpression in concert with small molecules treatment. Here we employed integrative multi-omic assays to delineate the molecular landscape of fibroblast reprogramming into iMPCs in comparison to transdifferentiation into myogenic cells solely by MyoD overexpression. Utilizing bulk RNA-sequencing and mass spectrometry we characterized molecular regulators and pathways that endow a myogenic stem cell identity on fibroblasts only in the presence of small molecule treatment. We further demonstrate that iMPC reprogramming is a stepwise process, commencing with the appearance of myofibers and committed myogenic progenitors prior to the formation of satellite cell-like myogenic progenitors that express a suite of stem cell markers including Pax7, Dek, Myf5, Sox8, and Dmrt2. To directly compare iMPCs to satellite cell-derived primary myoblasts, we employed a fluorescent Pax7-GFP reporter to purify Pax7+ cells from the heterogenous iMPC cultures and assess their equivalency to FACS-purified Pax7-GFP+ myoblasts. We demonstrate that Pax7+ iMPCs share molecular attributes with myoblasts, however also express genes and signaling pathways that are indicative of activated satellite cells including Carm1, Dlk1, Lgr5, Fos, Dek, Calcr and Pitx3. We further establish that iMPC formation and maintenance is dependent on the Notch pathway, as small molecule inhibition of Notch abrogates iMPC formation and derails stable iMPC cultures via depletion of Pax7 expressing cells. Lastly, using single cell RNA-sequencing we determine the cell populations that comprise iMPCs in addition to reconstructing the differentiation trajectory present in these heterogenous cultures. We demonstrate that a highly proliferative activated satellite cell-like population differentiates into committed myoblasts and myocytes that further give rise to myofibers that express mature muscle markers, thus capturing a dynamic differentiation program in vitro. Collectively, this study charts a molecular blueprint for reprogramming fibroblasts into myogenic stem and progenitor cells and further establishes the fidelity of stable iMPC cultures in capturing skeletal muscle regeneration in vitro for disease modeling and basic research applications.
2022-01-06 | GSE169054 | GEO
Project description:Integrative molecular roadmap for reprogramming mouse fibroblasts into induced myogenic stem and progenitor cells
| PRJNA714970 | ENA
Project description:Integrative molecular roadmap for reprogramming mouse fibroblasts into induced myogenic stem and progenitor cells [ATAC-Seq]
| PRJNA773099 | ENA
Project description:Integrative molecular roadmap for reprogramming mouse fibroblasts into induced myogenic stem and progenitor cells [bulk RNA-seq]
| PRJNA714972 | ENA
Project description:Integrative molecular roadmap for reprogramming mouse fibroblasts into induced myogenic stem and progenitor cells [single-cell RNA-seq]
| PRJNA714973 | ENA
Project description:MicroRNAs play a pivotal role in cellular maintenance, proliferation, and differentiation. They have also been implicated to play a key role in disease pathogenesis, and more recently, cellular reprogramming. Certain microRNA clusters can enhance or even directly induce reprogramming, while repressing key proteins involved in microRNA processing decreases reprogramming efficiency. Although microRNAs clearly play important roles in cellular reprogramming, it remains unknown whether microRNAs are absolutely necessary. We endeavored to answer this fundamental question by attempting to reprogram Dicer-null mouse embryonic fibroblasts (MEFs) that lack almost all functional microRNAs using a defined set of transcription factors. Transduction of reprogramming factors using either lentiviral or piggyBac transposon vector into two, independently derived lines of Dicer-null MEFs failed to produce cells resembling embryonic stem cells (ESCs). However, expression of human Dicer in the Dicer-null MEFs restored their reprogramming potential. Our study demonstrates for the first time that microRNAs are indispensable for dedifferentiation reprogramming.
| S-EPMC3380844 | biostudies-literature
Project description:Somatic cells can be reprogrammed into induced pluripotent stem (iPS) cells by the transcription factors Oct4, Sox2, and Klf4 in combination with c-Myc. Recently, Sox2 plus Oct4 was shown to reprogram fibroblasts and Oct4 alone was able to reprogram mouse and human neural stem cells (NSCs) into iPS cells. Here, we report that Bmi1 leads to the transdifferentiation of mouse fibroblasts into NSC-like cells, and, in combination with Oct4, can replace Sox2, Klf4 and c-Myc during the reprogramming of fibroblasts into iPS cells. Furthermore, activation of sonic hedgehog signaling (by Shh, purmorphamine, or oxysterol) compensates for the effects of Bmi1, and, in combination with Oct4, reprograms mouse embryonic and adult fibroblasts into iPS cells. One- and two-factor iPS cells are similar to mouse embryonic stem cells in their global gene expression profile, epigenetic status, and in vitro and in vivo differentiation into all three germ layers, as well as teratoma formation and germline transmission in vivo. These data support that converting fibroblasts with Bmi1 or activation of the sonic hedgehog pathway to an intermediate cell type that expresses Sox2, Klf4, and N-Myc allows iPS generation via the addition of Oct4.
| S-EPMC3193470 | biostudies-literature
Project description:Pluripotent stem cells represent important tools for both basic and translational science as they enable to study mechanisms of development, model diseases in vitro and provide a potential source of tissue-specific progenitors for cell therapy. Concomitantly with the increasing knowledge of the molecular mechanisms behind activation of the skeletal myogenic program during embryonic development, novel findings in the stem cell field provided the opportunity to begin recapitulating in vitro the events occurring during specification of the myogenic lineage. In this review, we will provide a perspective of the molecular mechanisms responsible for skeletal myogenic commitment in the embryo and how this knowledge was instrumental for specifying this lineage from pluripotent stem cells. In addition, we will discuss the current limitations for properly recapitulating skeletal myogenesis in the petri dish, and we will provide insights about future applications of pluripotent stem cell-derived myogenic cells.
| S-EPMC5723218 | biostudies-other