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Artikel ; Online: Transgene-free direct conversion of murine fibroblasts into functional muscle stem cells.

Qabrati, Xhem / Kim, Inseon / Ghosh, Adhideb / Bundschuh, Nicola / Noé, Falko / Palmer, Andrew S / Bar-Nur, Ori

NPJ Regenerative medicine

2023 Band 8, Heft 1, Seite(n) 43

Abstract: Transcription factor-based cellular 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 ...

Abstract	Transcription factor-based cellular 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
Sprache	Englisch
Erscheinungsdatum	2023-08-08
Erscheinungsland	United States
Dokumenttyp	Journal Article
ISSN	2057-3995
ISSN (online)	2057-3995
DOI	10.1038/s41536-023-00317-z
Datenquelle	MEDical Literature Analysis and Retrieval System OnLINE

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Artikel ; Online: Integrative molecular roadmap for direct conversion of fibroblasts into myocytes and myogenic progenitor cells.

Kim, Inseon / Ghosh, Adhideb / Bundschuh, Nicola / Hinte, Laura / Petrosyan, Eduard / von Meyenn, Ferdinand / Bar-Nur, Ori

Science advances

2022 Band 8, Heft 14, Seite(n) eabj4928

Abstract: Transient MyoD overexpression in concert with small molecule treatment reprograms mouse fibroblasts into induced myogenic progenitor cells (iMPCs). However, the molecular landscape and mechanisms orchestrating this cellular conversion remain unknown. ... ...

Abstract	Transient MyoD overexpression in concert with small molecule treatment reprograms mouse fibroblasts into induced myogenic progenitor cells (iMPCs). However, the molecular landscape and mechanisms orchestrating this cellular conversion remain unknown. Here, we undertook an integrative multiomics approach to delineate the process of iMPC reprogramming in comparison to myogenic transdifferentiation mediated solely by MyoD. Using transcriptomics, proteomics, and genome-wide chromatin accessibility assays, we unravel distinct molecular trajectories that govern the two processes. Notably, only iMPC reprogramming is characterized by gradual up-regulation of muscle stem cell markers, unique signaling pathways, and chromatin remodelers in conjunction with exclusive chromatin opening in core myogenic promoters. In addition, we determine that the Notch pathway is indispensable for iMPC formation and self-renewal and further use the Notch ligand Dll1 to homogeneously propagate iMPCs. Collectively, this study charts divergent molecular blueprints for myogenic transdifferentiation or reprogramming and underpins the heightened capacity of iMPCs for capturing myogenesis ex vivo.
Sprache	Englisch
Erscheinungsdatum	2022-04-06
Erscheinungsland	United States
Dokumenttyp	Journal Article
ZDB-ID	2810933-8
ISSN	2375-2548 ; 2375-2548
ISSN (online)	2375-2548
ISSN	2375-2548
DOI	10.1126/sciadv.abj4928
Datenquelle	MEDical Literature Analysis and Retrieval System OnLINE

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Artikel ; Online: CRISPR/Cas9 editing of directly reprogrammed myogenic progenitors restores dystrophin expression in a mouse model of muscular dystrophy.

Domenig, Seraina A / Bundschuh, Nicola / Lenardič, Ajda / Ghosh, Adhideb / Kim, Inseon / Qabrati, Xhem / D'Hulst, Gommaar / Bar-Nur, Ori

Stem cell reports

2022 Band 17, Heft 2, Seite(n) 321–336

Abstract: Genetic mutations in dystrophin manifest in Duchenne muscular dystrophy (DMD), the most commonly inherited muscle disease. Here, we report on reprogramming of fibroblasts from two DMD mouse models into induced myogenic progenitor cells (iMPCs) by MyoD ... ...

Abstract	Genetic mutations in dystrophin manifest in Duchenne muscular dystrophy (DMD), the most commonly inherited muscle disease. Here, we report on reprogramming of fibroblasts from two DMD mouse models into induced myogenic progenitor cells (iMPCs) by MyoD overexpression in concert with small molecule treatment. DMD iMPCs proliferate extensively, while expressing myogenic stem cell markers including Pax7 and Myf5. Additionally, DMD iMPCs readily give rise to multinucleated myofibers that express mature skeletal muscle markers; however, they lack DYSTROPHIN expression. Utilizing an exon skipping-based approach with CRISPR/Cas9, we report on genetic correction of the dystrophin mutation in DMD iMPCs and restoration of protein expression in vitro. Furthermore, engraftment of corrected DMD iMPCs into the muscles of dystrophic mice restored DYSTROPHIN expression and contributed to the muscle stem cell reservoir. Collectively, our findings report on a novel in vitro cellular model for DMD and utilize it in conjunction with gene editing to restore DYSTROPHIN expression in vivo.
Mesh-Begriff(e)	Animals ; CRISPR-Cas Systems/genetics ; Cell Differentiation ; Cellular Reprogramming/genetics ; Disease Models, Animal ; Dystrophin/genetics ; Dystrophin/metabolism ; Fibroblasts/cytology ; Fibroblasts/metabolism ; Gene Editing/methods ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Muscle Development ; Muscular Dystrophy, Duchenne/genetics ; Muscular Dystrophy, Duchenne/metabolism ; Muscular Dystrophy, Duchenne/pathology ; Mutation ; MyoD Protein/genetics ; MyoD Protein/metabolism ; Myoblasts/cytology ; Myoblasts/metabolism ; Stem Cells/cytology ; Stem Cells/metabolism
Chemische Substanzen	Dystrophin ; MyoD Protein
Sprache	Englisch
Erscheinungsdatum	2022-01-06
Erscheinungsland	United States
Dokumenttyp	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2720528-9
ISSN	2213-6711 ; 2213-6711
ISSN (online)	2213-6711
ISSN	2213-6711
DOI	10.1016/j.stemcr.2021.12.003
Datenquelle	MEDical Literature Analysis and Retrieval System OnLINE

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Artikel ; Online: Generation of allogenic and xenogeneic functional muscle stem cells for intramuscular transplantation.

Lenardič, Ajda / Domenig, Seraina A / Zvick, Joel / Bundschuh, Nicola / Tarnowska-Sengül, Monika / Furrer, Regula / Noé, Falko J / Trautmann, Christine Ling Li / Ghosh, Adhideb / Bacchin, Giada / Gjonlleshaj, Pjeter / Qabrati, Xhem / Masschelein, Evi / De Bock, Katrien / Handschin, Christoph / Bar-Nur, Ori

The Journal of clinical investigation

2024

Abstract: Satellite cells, the stem cells of skeletal muscle tissue, hold a remarkable regeneration capacity and therapeutic potential in regenerative medicine. However, low satellite cell yield from autologous or donor-derived muscles hinders the adoption of ... ...

Abstract	Satellite cells, the stem cells of skeletal muscle tissue, hold a remarkable regeneration capacity and therapeutic potential in regenerative medicine. However, low satellite cell yield from autologous or donor-derived muscles hinders the adoption of satellite cell transplantation for the treatment of muscle diseases, including Duchenne muscular dystrophy (DMD). To address this limitation, here we investigated whether satellite cells can be derived in allogeneic or xenogeneic animal hosts. First, injection of CRISPR/Cas9-corrected mouse DMD-induced pluripotent stem cells (iPSCs) into mouse blastocysts carrying an ablation system of host satellite cells gave rise to intraspecies chimeras exclusively carrying iPSC-derived satellite cells. Furthermore, injection of genetically corrected DMD-iPSCs into rat blastocysts resulted in the formation of interspecies rat-mouse chimeras harboring mouse satellite cells. Remarkably, iPSC-derived satellite cells or derivative myoblasts produced in intraspecies or interspecies chimeras restored dystrophin expression in DMD mice following intramuscular transplantation, and contributed to the satellite cell pool. Collectively, this study demonstrates the feasibility of producing therapeutically competent stem cells across divergent animal species, raising the possibility of generating human muscle stem cells in large animals for regenerative medicine purposes.
Sprache	Englisch
Erscheinungsdatum	2024-05-07
Erscheinungsland	United States
Dokumenttyp	Journal Article
ZDB-ID	3067-3
ISSN	1558-8238 ; 0021-9738
ISSN (online)	1558-8238
ISSN	0021-9738
DOI	10.1172/JCI166998
Datenquelle	MEDical Literature Analysis and Retrieval System OnLINE

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Artikel: Voluntary Resistance Running as a Model to Induce mTOR Activation in Mouse Skeletal Muscle.

D'Hulst, Gommaar / Palmer, Andrew S / Masschelein, Evi / Bar-Nur, Ori / De Bock, Katrien

Frontiers in physiology

2019 Band 10, Seite(n) 1271

Abstract: Long-term voluntary resistance running has been shown to be a valid model to induce muscle growth in rodents. Moreover, the mammalian target of rapamycin complex 1 (mTORC1) is a key signaling complex regulating exercise/nutrient-induced alterations in ... ...

Abstract	Long-term voluntary resistance running has been shown to be a valid model to induce muscle growth in rodents. Moreover, the mammalian target of rapamycin complex 1 (mTORC1) is a key signaling complex regulating exercise/nutrient-induced alterations in muscle protein synthesis. How acute resistance running affects mTORC1 signaling in muscle and if resistance applied to the wheel can modulate mTORC1 activation has not yet been fully elucidated. Here, we show that both acute resistance running and acute free running activated mTORC1 signaling in the
Sprache	Englisch
Erscheinungsdatum	2019-10-04
Erscheinungsland	Switzerland
Dokumenttyp	Journal Article
ZDB-ID	2564217-0
ISSN	1664-042X
ISSN	1664-042X
DOI	10.3389/fphys.2019.01271
Datenquelle	MEDical Literature Analysis and Retrieval System OnLINE

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Artikel: Screening method to identify hydrogel formulations that facilitate myotube formation from encapsulated primary myoblasts.

Deshmukh, Dhananjay V / Pasquero, Nils / Rathore, Gajraj / Zvick, Joel / Bar-Nur, Ori / Dual, Jurg / Tibbitt, Mark W

Bioengineering & translational medicine

2020 Band 5, Heft 3, Seite(n) e10181

Abstract: Hydrogel-based three-dimensional (3D) cellular models are attractive for bioengineering and pharmaceutical development as they can more closely resemble the cellular function of native tissue outside of the body. In general, these models are composed of ... ...

Abstract	Hydrogel-based three-dimensional (3D) cellular models are attractive for bioengineering and pharmaceutical development as they can more closely resemble the cellular function of native tissue outside of the body. In general, these models are composed of tissue specific cells embedded within a support material, such as a hydrogel. As hydrogel properties directly affect cell function, hydrogel composition is often tailored to the cell type(s) of interest and the functional objective of the model. Here, we develop a parametric analysis and screening method to identify suitable encapsulation conditions for the formation of myotubes from primary murine myoblasts in methacryloyl gelatin (GelMA) hydrogels. The effect of the matrix properties on the myotube formation was investigated by varying GelMA weight percent (wt%, which controls gel modulus), cell density, and Matrigel concentration. Contractile myotubes form via myoblast fusion and are characterized by myosin heavy chain (MyHC) expression. To efficiently screen the gel formulations, we developed a fluorescence-based plate reader assay to quantify MyHC staining in the gel samples, as a metric of myotube formation. We observed that lower GelMA wt% resulted in increased MyHC staining (myotube formation). The cell density did not significantly affect MyHC staining, while the inclusion of Matrigel increased MyHC staining, however, a concentration dependent effect was not observed. These findings were supported by the observation of spontaneously contracting myotubes in samples selected in the initial screen. This work provides a method to rapidly screen hydrogel formulations for the development of 3D cellular models and provides specific guidance on the formulation of gels for myotube formation from primary murine myoblasts in 3D.
Sprache	Englisch
Erscheinungsdatum	2020-09-03
Erscheinungsland	United States
Dokumenttyp	Journal Article
ISSN	2380-6761
ISSN	2380-6761
DOI	10.1002/btm2.10181
Datenquelle	MEDical Literature Analysis and Retrieval System OnLINE

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Artikel ; Online: Exclusive generation of rat spermatozoa in sterile mice utilizing blastocyst complementation with pluripotent stem cells.

Zvick, Joel / Tarnowska-Sengül, Monika / Ghosh, Adhideb / Bundschuh, Nicola / Gjonlleshaj, Pjeter / Hinte, Laura C / Trautmann, Christine L / Noé, Falko / Qabrati, Xhem / Domenig, Seraina A / Kim, Inseon / Hennek, Thomas / von Meyenn, Ferdinand / Bar-Nur, Ori

Stem cell reports

2022 Band 17, Heft 9, Seite(n) 1942–1958

Abstract: Blastocyst complementation denotes a technique that aims to generate organs, tissues, or cell types in animal chimeras via injection of pluripotent stem cells (PSCs) into genetically compromised blastocyst-stage embryos. Here, we report on successful ... ...

Abstract	Blastocyst complementation denotes a technique that aims to generate organs, tissues, or cell types in animal chimeras via injection of pluripotent stem cells (PSCs) into genetically compromised blastocyst-stage embryos. Here, we report on successful complementation of the male germline in adult chimeras following injection of mouse or rat PSCs into mouse blastocysts carrying a mutation in Tsc22d3, an essential gene for spermatozoa production. Injection of mouse PSCs into Tsc22d3-Knockout (KO) blastocysts gave rise to intraspecies chimeras exclusively embodying PSC-derived functional spermatozoa. In addition, injection of rat embryonic stem cells (rESCs) into Tsc22d3-KO embryos produced interspecies mouse-rat chimeras solely harboring rat spermatids and spermatozoa capable of fertilizing oocytes. Furthermore, using single-cell RNA sequencing, we deconstructed rat spermatogenesis occurring in a mouse-rat chimera testis. Collectively, this study details a method for exclusive xenogeneic germ cell production in vivo, with implications that may extend to rat transgenesis, or endangered animal species conservation efforts.
Mesh-Begriff(e)	Animals ; Blastocyst ; Chimera ; Embryonic Stem Cells ; Male ; Mice ; Mice, Knockout ; Pluripotent Stem Cells ; Rats ; Spermatozoa
Sprache	Englisch
Erscheinungsdatum	2022-08-04
Erscheinungsland	United States
Dokumenttyp	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2720528-9
ISSN	2213-6711 ; 2213-6711
ISSN (online)	2213-6711
ISSN	2213-6711
DOI	10.1016/j.stemcr.2022.07.005
Datenquelle	MEDical Literature Analysis and Retrieval System OnLINE

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Artikel ; Online: Molecular analysis of FMR1 reactivation in fragile-X induced pluripotent stem cells and their neuronal derivatives.

Bar-Nur, Ori / Caspi, Inbal / Benvenisty, Nissim

Journal of molecular cell biology

2012 Band 4, Heft 3, Seite(n) 180–183

Mesh-Begriff(e)	Animals ; Antimetabolites, Antineoplastic/pharmacology ; Azacitidine/pharmacology ; Cell Differentiation ; Cells, Cultured ; Chromatin Assembly and Disassembly ; Epigenesis, Genetic ; Fragile X Mental Retardation Protein/genetics ; Fragile X Mental Retardation Protein/metabolism ; Fragile X Syndrome/genetics ; Histone Deacetylase Inhibitors/pharmacology ; Humans ; Hydroxamic Acids/pharmacology ; Induced Pluripotent Stem Cells/cytology ; Induced Pluripotent Stem Cells/drug effects ; Induced Pluripotent Stem Cells/metabolism ; Mice ; Neurons/drug effects ; Neurons/metabolism
Chemische Substanzen	Antimetabolites, Antineoplastic ; FMR1 protein, human ; Histone Deacetylase Inhibitors ; Hydroxamic Acids ; Fragile X Mental Retardation Protein (139135-51-6) ; trichostatin A (3X2S926L3Z) ; Azacitidine (M801H13NRU)
Sprache	Englisch
Erscheinungsdatum	2012-06
Erscheinungsland	United States
Dokumenttyp	Letter ; Research Support, Non-U.S. Gov't
ZDB-ID	2500949-7
ISSN	1759-4685 ; 1674-2788
ISSN (online)	1759-4685
ISSN	1674-2788
DOI	10.1093/jmcb/mjs007
Datenquelle	MEDical Literature Analysis and Retrieval System OnLINE

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Artikel ; Online: Exercise promotes satellite cell contribution to myofibers in a load-dependent manner.

Masschelein, Evi / D'Hulst, Gommaar / Zvick, Joel / Hinte, Laura / Soro-Arnaiz, Inés / Gorski, Tatiane / von Meyenn, Ferdinand / Bar-Nur, Ori / De Bock, Katrien

Skeletal muscle

2020 Band 10, Heft 1, Seite(n) 21

Abstract: Background: Satellite cells (SCs) are required for muscle repair following injury and are involved in muscle remodeling upon muscular contractions. Exercise stimulates SC accumulation and myonuclear accretion. To what extent exercise training at ... ...

Abstract	Background: Satellite cells (SCs) are required for muscle repair following injury and are involved in muscle remodeling upon muscular contractions. Exercise stimulates SC accumulation and myonuclear accretion. To what extent exercise training at different mechanical loads drive SC contribution to myonuclei however is unknown. Results: By performing SC fate tracing experiments, we show that 8 weeks of voluntary wheel running increased SC contribution to myofibers in mouse plantar flexor muscles in a load-dependent, but fiber type-independent manner. Increased SC fusion however was not exclusively linked to muscle hypertrophy as wheel running without external load substantially increased SC fusion in the absence of fiber hypertrophy. Due to nuclear propagation, nuclear fluorescent fate tracing mouse models were inadequate to quantify SC contribution to myonuclei. Ultimately, by performing fate tracing at the DNA level, we show that SC contribution mirrors myonuclear accretion during exercise. Conclusions: Collectively, mechanical load during exercise independently promotes SC contribution to existing myofibers. Also, due to propagation of nuclear fluorescent reporter proteins, our data warrant caution for the use of existing reporter mouse models for the quantitative evaluation of satellite cell contribution to myonuclei.
Mesh-Begriff(e)	Animals ; Cell Fusion ; Cell Nucleus/physiology ; Cells, Cultured ; Mice ; Mice, Inbred C57BL ; Muscle Fibers, Skeletal/cytology ; Muscle Fibers, Skeletal/physiology ; Running ; Satellite Cells, Skeletal Muscle/cytology ; Satellite Cells, Skeletal Muscle/physiology
Sprache	Englisch
Erscheinungsdatum	2020-07-09
Erscheinungsland	England
Dokumenttyp	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2595637-1
ISSN	2044-5040 ; 2044-5040
ISSN (online)	2044-5040
ISSN	2044-5040
DOI	10.1186/s13395-020-00237-2
Datenquelle	MEDical Literature Analysis and Retrieval System OnLINE

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Artikel: Global indiscriminate methylation in cell-specific gene promoters following reprogramming into human induced pluripotent stem cells.

Nissenbaum, Jonathan / Bar-Nur, Ori / Ben-David, Eyal / Benvenisty, Nissim

Stem cell reports

2013 Band 1, Heft 6, Seite(n) 509–517

Abstract: Molecular reprogramming of somatic cells into human induced pluripotent stem cells (iPSCs) is accompanied by extensive changes in gene expression patterns and epigenetic marks. To better understand the link between gene expression and DNA methylation, we ...

Abstract	Molecular reprogramming of somatic cells into human induced pluripotent stem cells (iPSCs) is accompanied by extensive changes in gene expression patterns and epigenetic marks. To better understand the link between gene expression and DNA methylation, we have profiled human somatic cells from different embryonic cell types (endoderm, mesoderm, and parthenogenetic germ cells) and the iPSCs generated from them. We show that reprogramming is accompanied by extensive DNA methylation in CpG-poor promoters, sparing CpG-rich promoters. Intriguingly, methylation in CpG-poor promoters occurred not only in downregulated genes, but also in genes that are not expressed in the parental somatic cells or their respective iPSCs. These genes are predominantly tissue-specific genes of other cell types from different lineages. Our results suggest a role of DNA methylation in the silencing of the somatic cell identity by global nonspecific methylation of tissue-specific genes from all lineages, regardless of their expression in the parental somatic cells.
Mesh-Begriff(e)	Cell Culture Techniques ; Cell Differentiation/genetics ; Cellular Reprogramming/genetics ; CpG Islands ; DNA Methylation ; Gene Expression Regulation ; Humans ; Oligonucleotide Array Sequence Analysis ; Pluripotent Stem Cells/cytology ; Promoter Regions, Genetic
Sprache	Englisch
Erscheinungsdatum	2013-12-12
Erscheinungsland	United States
Dokumenttyp	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2720528-9
ISSN	2213-6711
ISSN	2213-6711
DOI	10.1016/j.stemcr.2013.11.007
Datenquelle	MEDical Literature Analysis and Retrieval System OnLINE

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