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Generation of allogenic and xenogeneic functional muscle stem cells for intramuscular transplantation

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.

ORGANISM(S): Rattus norvegicus

PROVIDER: GSE254371 | GEO | 2024/04/25

REPOSITORIES: GEO

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Generation of allogenic and xenogeneic functional muscle stem cells for intramuscular transplantation

Project description: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.

2024-04-25 | GSE215061 | GEO

ERBB3 and NGFR mark distinct skeletal muscle progenitor cells in human development enabling enrichment and maturation of hPSC muscle

Project description:Abstract: Human pluripotent stem cells (hPSCs) can be directed to differentiate into skeletal muscle progenitor cells (SMPCs). However, the myogenic potential of hPSC-SMPCs compared to human fetal or adult satellite cells (Scs) remains unclear. This study demonstrates hPSC-SMPCs derived by commonly used protocols are functionally less mature than freshly-isolated human fetal or adult Scs. We utilized RNA-SEQ of human fetal Scs to identify differentially expressed genes including NGFR, ERBB3, which enriched for MYOD+ or PAX7+ cells. Furthermore, inhibition of TGFβ (TGFβi) improved hPSC-muscle maturation in three independent lines as measured by TEM and expression of myosins. Engraftment of CRISPR/Cas9-corrected Duchenne Muscular Dystrophy (DMD) hiPSC-SMPC subpopulations treated with TGFβi in vivo, increased dystrophin-positive fibers to levels of engrafted cultured fetal Scs in mdx mouse models of DMD. This work provides the first characterization of the developmental status of hPSC-SMPCs and identifies candidates to enable robust myogenic activity in vitro and in vivo.

2016-09-27 | GSE87365 | GEO

Transcriptome analysis of human DMD-K2957fs and CRISPR-corrected (CORR-K2957fs ) myogenic cultures during secondary myogenic differentiation.

Project description:To investigate transcriptomes of skeletal muscle cells in health and Duchene muscular dystrophy (DMD), we performed RNA sequencing of DMD-K2957fs and CRISPR-corrected (CORR-K2957fs ) myogenic cultures during secondary differentiation at 5 time points.

2022-11-16 | GSE189053 | GEO

Correction of the Exon 2, Exon 2-8 and Exons 8-9 duplications in DMD patient myogenic cells by a Single CRISPR/Cas9 system

Project description:Duchenne Muscular dystrophy (DMD), a yet-incurable X-linked recessive disorder that results in muscle wasting and loss of ambulation is due to mutations in the dystrophin gene. Exonic duplications of dystrophin gene are a common type of mutations found in DMD patients. In this study, we utilized a single guideRNA CRISPR strategy targeting intronic regions to delete the extra duplicated regions in patient myogenic cells carrying duplication of exon 2, exons 2 to 9, and exons 8 to 9 in the DMD gene. Immunostaining on CRISPR corrected derived myotubes demonstrated the rescue of dystrophin protein. RNA sequencing of the corrected differentiated myogenic derivatives indicated rescue of dystrophin related molecular pathways. Examination of predicted close-match off-targets evidenced no aberrant gene editing at these loci. Here, we demonstrated a single guide CRISPR strategy that can be utilized to delete multi-exons duplication in patient DMD genes without significant off target effect, leading to restoration of transcriptome in the corrected patient derived cells. This study further expands the application of single guide CRISPR strategy as a potential therapeutic approach for patients with larger DNA region duplication in DMD patients.

2024-01-01 | GSE248257 | GEO

Long-term maintenance of dystrophin expression and resistance to injury of skeletal muscle in gene-edited mice

Project description:Duchenne Muscular Dystrophy (DMD) is a lethal muscle disease caused by mutations in the dystrophin gene. CRISPR/Cas9 genome-editing has been used to correct DMD mutations in animal models at young ages. However, the longevity and durability of CRISPR/Cas9 editing remained to be determined. To address these issues, we subjected DEx44 DMD mice to systemic delivery of AAV9 expressing CRISPR/Cas9 gene editing components to reframe exon 45 of the dystrophin gene, allowing robust dystrophin expression and maintenance of muscle structure and function. We showed that genome correction by CRISPR/Cas9 confers lifelong expression of dystrophin in mice and that corrected skeletal muscle is highly durable and resistant to myofiber necrosis and fibrosis, even in response to chronic injury. In contrast, when muscle fibers were ablated by barium chloride injection, satellite cells were unable to restore dystrophin expression due to restriction of gene editing components. Analysis of on-target and off-target gene editing in aged mice confirmed the stability of gene correction and the lack of significant off-target editing at 18 months of age. These findings demonstrate the long-term durability of CRISPR/Cas9 genome editing as a therapy for maintaining the integrity and function of DMD muscle, even under conditions of stress.

2021-07-27 | GSE180771 | GEO

The integrated single cell RNAseq analysis of bone marrow cells produced by mouse-mouse intraspecies blastocyst complementation and mouse-rat interspecies blastocyst complementation

Project description:We report the application of single cell RNAseq analysis of bone marrow cells from mouse-mouse intraspecies blastocyst complementation and mouse-rat interspecies blastocyst complementation.

2022-10-14 | GSE184940 | GEO

Generation of functional rat spermatozoa in sterile mice utilizing blastocyst complementation with pluripotent stem cells [scRNAseq_chimera]

Project description:Pluripotent stem cells (PSCs) provide a powerful tool to produce transgenic animals for biomedical research. However, impaired PSC contribution to chimerism and most notably the germline oftentimes impedes production of genetically modified animals, rendering techniques that expediate PSC contribution to the germline highly desirable. Blastocyst complementation denotes a technique which purposes to generate organs, tissues or cells in animal chimeras via microinjection of PSCs into genetically compromised blastocyst-stage embryos. Here we report on successful blastocyst complementation of the male germline in adult chimeras following microinjection of mouse or rat PSCs into mouse blastocysts mutated for Tsc22d3, an essential gene for spermatozoa production. Microinjection of mouse embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) into Tsc22d3-KnockOut (KO) blastocyst-stage embryos gave rise to intraspecies chimeras embodying functional spermatozoa, which were solely derived from microinjected PSCs. Furthermore, microinjection of rat ESCs into Tsc22d3-KO mouse embryos gave rise to viable mouse-rat chimeras that exhibited extensive contribution of rat cells to various organs. Notably, multiple mouse-rat chimeras showed contribution of rat ESCs to the male germline, solely harboring rat spermatids and spermatozoa that were rat ESC-derived and could fertilize rat oocytes. Collectively, this study reports a method for exclusive production of functional germ cells of one species in another via blastocyst complementation with PSCs. Implications of this study extend to development of transgenic rats via sterile mice, and may further assist efforts to generate xenogeneic gametes from endangered animal species.

2022-08-01 | GSE202699 | GEO

Generation of functional rat spermatozoa in sterile mice utilizing blastocyst complementation with pluripotent stem cells [bulk RNA-seq]

2022-08-01 | GSE167433 | GEO

Generation of functional rat spermatozoa in sterile mice utilizing blastocyst complementation with pluripotent stem cells [scRNAseq_mouse]

2022-08-01 | GSE167434 | GEO

Generation of functional rat spermatozoa in sterile mice utilizing blastocyst complementation with pluripotent stem cells [scRNAseq_rat]

Project description:Pluripotent stem cells (PSCs) provide a powerful tool to produce transgenic animals for biomedical research. However, impaired PSC contribution to chimerism and most notably to the germline oftentimes impedes production of genetically modified animals, rendering techniques that expediate PSC contribution to the germline highly desirable. Blastocyst complementation denotes a technique which purposes to generate organs, tissues or cells in animal chimeras following microinjection of PSCs into genetically compromised blastocyst-stage embryos. Here, we report on successful blastocyst complementation of the male germline in adult chimeras via microinjection of mouse or rat PSCs into mouse blastocysts mutated for Tsc22d3, an essential gene for spermatozoa production. Microinjection of mouse embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) into Tsc22d3-KnockOut (KO) blastocyst-stage embryos gave rise to intraspecies chimeras embodying functional spermatozoa, which were solely derived from the microinjected PSCs. Furthermore, microinjection of rat ESCs into Tsc22d3-KO mouse embryos gave rise to viable mouse-rat chimeras that exhibited extensive contribution of rat cells to various tissues and organs. Notably, multiple mouse-rat chimeras showed contribution of rat ESCs to the male germline, solely harboring rat spermatids and spermatozoa that were rat ESC-derived and could fertilize rat oocytes. Collectively, this study reports on a method for exclusive germ cell production of one species in another via blastocyst complementation with PSCs. Implications of this study may extend to development of transgenic rat gametes in sterile mice and could further assist efforts to generate germ cells from endangered animal species.

2022-08-01 | GSE203350 | GEO

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