Direct reprogramming of mouse fibroblasts into functional skeletal muscle progenitors
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE92336 | GEO | 2018/05/01
REPOSITORIES: GEO
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Project description:Skeletal muscle harbors quiescent stem cells termed satellite cells and proliferative progenitors termed myoblasts, which play pivotal roles during muscle regeneration. However, current technology does not allow permanent capture of these cell populations in vitro. Here, we show that ectopic expression of the myogenic transcription factor MyoD, combined with exposure to small molecules, reprograms mouse fibroblasts into expandable induced myogenic progenitor cells (iMPCs). iMPCs express key skeletal muscle stem and progenitor cell markers including Pax7 and Myf5 and give rise to Dystrophin-expressing myofibers upon transplantation, a subset of which maintain Pax7 expression in vivo and sustain serial regenerative responses. Similar to satellite cells, iMPCs originate from Pax7+ cells and require Pax7 itself for maintenance. Finally, we show that iMPCs can be established from muscle tissue following small molecule exposure alone. This study thus reports on a robust approach to derive expandable myogenic stem/progenitor-like cells from multiple differentiated cell types.
2018-05-01 | GSE108543 | 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 transient MyoD overexpression in concert with small molecules treatment. Here we employed integrative multi-omic approaches 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 uncovered molecular regulators and pathways that endow a myogenic stem cell identity on fibroblasts only in the presence of small molecule treatment. In addition, we demonstrate that Pax7+ cells in iMPCs share molecular attributes with myoblasts, however in addition express unique genes, proteins and pathways that are indicative of a more activated satellite cell-like state in vitro. Collectively, this study charts a molecular blueprint for reprogramming fibroblasts into muscle 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.
2022-05-19 | PXD029379 | Pride
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 | GSE169054 | 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:Genetic mutations in dystrophin manifest in Duchenne muscular dystrophy (DMD), the most prevalent form of a genetically inherited muscle disease. Dystrophin is expressed in skeletal muscle stem cells and fibers, playing a critical role in maintaining skeletal muscle structure, regeneration and function. Here, we report on direct reprogramming of fibroblasts from the Dmdmdx mouse model into induced myogenic progenitor cells (Dmdmdx iMPCs) utilizing transient MyoD overexpression in concert with small molecule treatment. Dmdmdx iMPCs proliferate extensively in vitro and express canonical skeletal muscle stem and progenitor cell markers including Pax7, Sox8 and Myf5. Furthermore, Dmdmdx iMPCs readily give rise to highly contractile and multinucleated myofibers that express a suite of mature skeletal muscle genes however lack dystrophin expression. Utilizing an exon-skipping based approach with CRISPR/Cas9 we report on a genetic correction of the dystrophin mutation in Dmdmdx-iMPCs and subsequent restoration of dystrophin protein expression in vitro. Furthermore, engraftment of genetically-corrected Dmdmdx iMPCs into dystrophic limb muscles of Dmdmdx mice restored dystrophin expression in vivo. Collectively, our findings report on a novel in vitro stem cell-based model for DMD and further establish a new approach to treat this disease via a combination of direct cellular reprogramming, genome engineering and stem cell transplantation methods.
2021-12-15 | GSE164599 | GEO
Project description:Transcription factor-based reprogramming provides an attractive approach to produce desired cell types for regenerative medicine purposes. Such cellular conversions are widely dependent on viral vectors to efficiently deliver and express defined factors in target cells. However, use of viral vectors is associated with unfavorable genomic integrations that can trigger deleterious molecular consequences, rendering this method a potential impediment to clinical applications. Here, we report on a highly efficient transgene-free approach to directly convert mouse fibroblasts into induced myogenic progenitor cells (iMPCs) by overexpression of synthetic MyoD-mRNA in concert with an enhanced small molecule cocktail. First, we performed a candidate compound screen and identified two molecules that enhance fibroblast reprogramming into iMPCs by suppression of the JNK and JAK/STAT pathways. Simultaneously, we developed an optimal transfection protocol to transiently overexpress synthetic MyoD-mRNA in fibroblasts. Combining these two techniques enabled robust and rapid reprogramming of fibroblasts into Pax7 positive iMPCs in as little as 10 days. Nascent transgene-free iMPCs proliferated extensively in vitro, expressed a suite of myogenic stem cell markers, and could differentiate into highly multinucleated and contractile myotubes. Furthermore, using global and single-cell transcriptome assays, we delineated gene expression changes associated with JNK and JAK/STAT pathway inhibition during reprogramming, and identified in iMPCs a Pax7+ stem cell subpopulation resembling satellite cells. Last, transgene-free iMPCs robustly engrafted skeletal muscles of a Duchenne muscular dystrophy mouse model, restoring dystrophin expression in hundreds of myofibers. In summary, this study reports on an improved and clinically safer approach to convert fibroblasts into myogenic stem cells that can efficiently contribute to muscle regeneration in vivo.
2023-07-24 | GSE208063 | GEO
Project description:Transcription factor-based reprogramming provides an attractive approach to produce desired cell types for regenerative medicine purposes. Such cellular conversions are widely dependent on viral vectors to efficiently deliver and express defined factors in target cells. However, use of viral vectors is associated with unfavorable genomic integrations that can trigger deleterious molecular consequences, rendering this method a potential impediment to clinical applications. Here, we report on a highly efficient transgene-free approach to directly convert mouse fibroblasts into induced myogenic progenitor cells (iMPCs) by overexpression of synthetic MyoD-mRNA in concert with an enhanced small molecule cocktail. First, we performed a candidate compound screen and identified two molecules that enhance fibroblast reprogramming into iMPCs by suppression of the JNK and JAK/STAT pathways. Simultaneously, we developed an optimal transfection protocol to transiently overexpress synthetic MyoD-mRNA in fibroblasts. Combining these two techniques enabled robust and rapid reprogramming of fibroblasts into Pax7 positive iMPCs in as little as 10 days. Nascent transgene-free iMPCs proliferated extensively in vitro, expressed a suite of myogenic stem cell markers, and could differentiate into highly multinucleated and contractile myotubes. Furthermore, using global and single-cell transcriptome assays, we delineated gene expression changes associated with JNK and JAK/STAT pathway inhibition during reprogramming, and identified in iMPCs a Pax7+ stem cell subpopulation resembling satellite cells. Last, transgene-free iMPCs robustly engrafted skeletal muscles of a Duchenne muscular dystrophy mouse model, restoring dystrophin expression in hundreds of myofibers. In summary, this study reports on an improved and clinically safer approach to convert fibroblasts into myogenic stem cells that can efficiently contribute to muscle regeneration in vivo.
2023-07-24 | GSE208059 | GEO
Project description:Numerous studies have been conducted to improve the supply of myogenic stem cells for patients with muscle disease, including Duchenne muscular dystrophy. Collecting muscle samples from patients in order to obtain myoblasts or satellite cells is very invasive. Thus, a new method for obtaining myogenic stem cells is required. Here, we established stably expandable induced myogenic stem cells (iMSCs) with defined factors. The iMSCs showed high expression levels of Pax7, Myf5, and MyoD. Also, the iMSCs differentiate into myotubes which are multinucleated fiber. Additionally, the iMSCs differentiated into myotubes, which are multinucleated fibers. We confirmed its myogenic differentiation capacity in mdx mice by detecting dystrophin-positive cells in iMSCs-injected TA muscles. The iMSCs showed higher proliferation capacity than MDSCs in both in vitro and vivo. This study suggests the possibility to apply directly converted expandable myogenic stem cells to muscle disease patients.
2018-11-06 | GSE94506 | GEO
Project description:The satellite cell is considered the major tissue-resident stem cell underlying muscle regeneration, however, multiple non-satellite cell myogenic progenitors have been identified. PW1/Peg3 is expressed in satellite cells as well as a subset of interstitial cells with myogenic potential termed PICs (PW1+ Interstitial Cells). PICs differ from satellite cells by their anatomical location (satellite cells are sublaminal and PICs are interstitial), they do not express any myogenic marker and arise from a Pax3-independent lineage. Upon isolation from juvenile muscle (1 to 3 weeks old), PICs are capable to form both skeletal and smooth muscle suggesting they constitute a more plastic population compared to satellite cells. We used microarrays to gain insight into the relantionship between PICs and satellite cells. PICs and satellite cells were isolated from 1-week old mouse muscle and subsequent RNA extraction was performed.
2012-09-01 | E-GEOD-40523 | biostudies-arrayexpress