RESUMEN
Muscular dystrophies are among major inherited muscle disorders characterized by progressive muscle damage and fibrosis with no definitive cure. Recently, gene or cell based therapies have been developed to restore the missing gene expression or replace the damaged tissues. In order to test the efficiency of these therapies in mice models of muscular dystrophies, the arterial route of delivery is very advantageous as it provides uniform muscle exposure to the therapeutic agents or cells. Although there are few reports of arterial delivery of the therapeutic agents or cells in mice, there is no in-depth description and evaluation of its efficacy in perfusion of downstream muscles. This study is aimed to develop a practical method for intra-femoral artery perfusion in mice and to evaluate perfusion efficiency using near-infrared-fluorescence (NIRF) imaging as well as histology following stem cell delivery. Our results provide a practical guide to perform this delicate method in mice. By using a sensitive fluorescent dye, different muscle groups of the hindlimb have been evaluated for proper perfusion. As the final step, we have validated the efficiency of arterial cell delivery into muscles using human iPS-derived myogenic cells in an immunodeficient mouse model for Duchenne muscular dystrophy (NSG-mdx(4cv)).
Asunto(s)
Arteria Femoral/cirugía , Músculo Esquelético/citología , Distrofia Muscular Animal/terapia , Distrofia Muscular de Duchenne/terapia , Perfusión , Trasplante de Células Madre , Animales , Diferenciación Celular , Células Cultivadas , Distrofina/deficiencia , Arteria Femoral/metabolismo , Miembro Posterior , Humanos , Procesamiento de Imagen Asistido por Computador , Técnicas para Inmunoenzimas , Ratones , Ratones Endogámicos mdx , Músculo Esquelético/metabolismoRESUMEN
Volumetric muscle defect, caused by trauma or combat injuries, is a major health concern leading to severe morbidity. It is characterized by partial or full thickness loss of muscle and its bio-scaffold, resulting in extensive fibrosis and scar formation. Therefore, the ideal therapeutic option is to use stem cells combined with bio-scaffolds to restore muscle. For this purpose, muscle-derived stem cells (MDSCs) are a great candidate due to their unique multi-lineage differentiation potential. In this study, we evaluated the regeneration potential of MDSCs for muscle loss repair using a novel in situ fibrin gel casting. Muscle defect was created by a partial thickness wedge resection in the tibialis anterior (TA) muscles of NSG mice which created an average of 25% mass loss. If untreated, this defect leads to severe muscle fibrosis. Next, MDSCs were delivered using a novel in situ fibrin gel casting method. Our results demonstrated MDSCs are able to engraft and form new myofibers in the defect when casted along with fibrin gel. LacZ labeled MDSCs were able to differentiate efficiently into new myofibers and significantly increase muscle mass. This was also accompanied by significant reduction of fibrotic tissue in the engrafted muscles. Furthermore, transplanted cells also contributed to new vessel formation and satellite cell seeding. These results confirmed the therapeutic potential of MDSCs and feasibility of direct in situ casting of fibrin/MDSC mixture to repair muscle mass defects.
Asunto(s)
Fibrina/uso terapéutico , Músculo Esquelético/patología , Células Madre/fisiología , Animales , Diferenciación Celular/fisiología , Células Cultivadas , Fibrosis/dietoterapia , Fibrosis/terapia , Ratones , Músculo Esquelético/lesiones , Regeneración , Células Madre/citologíaRESUMEN
Recently, a new type of limb-girdle muscular dystrophy (LGMD type 2Z) has been identified due to a missense mutation in POGLUT1 (protein O-glucosyltransferase-Rumi), an enzyme capable of adding glucose to a distinct serine residue of epidermal growth factor-like repeats containing a C-X-S-X-(P/A)-C consensus sequence such as Notch receptors. Affected patients demonstrate reduced Notch signaling, decreased muscle stem cell pool and hypoglycosylation of α-dystroglycan, leading to LGMD phenotype. Here we report the generation and characterization of an iPSC line (CSCRMi001-A) from a LGMD-2Z patient with missense mutation in POGLUT1 which can be used for in vitro disease modeling.
Asunto(s)
Glucosiltransferasas/genética , Células Madre Pluripotentes Inducidas/metabolismo , Distrofia Muscular de Cinturas/genética , Adulto , Línea Celular , Glucosiltransferasas/metabolismo , Humanos , Masculino , Distrofia Muscular de Cinturas/metabolismo , Distrofia Muscular de Cinturas/patología , Mutación , Mutación MissenseRESUMEN
Human iPS cells hold great promise for disease modeling and treatment of degenerative disorders including muscular dystrophies. Although a few research groups have used them for skeletal muscle differentiation, most were based on gene over-expression or long-term mesenchymal differentiation and retrospective identification of myogenic cells. Therefore, this study was aimed to generate a knock-in reporter human iPS cell line for MYF5, as an early myogenic specification gene, to allow prospective identification and purification of myogenic progenitors from human iPS cells. By using a CRISPR/Cas9 double nickase strategy, a 2A-GFP reporter was inserted before the stop codon of the MYF5 gene using homologous recombination. This approach allowed for highly efficient in-frame targeting of MYF5 in human iPS cells. Furthermore, in order to prove the reporter function, endogenous MYF5 expression was induced using a novel dead Cas9-VP160 transcriptional activator. Induced clones demonstrated appropriate MYF5-GFP co-expression. Finally, to confirm the differentiation potential, reporter human iPS clones were differentiated through embryoid body method and MYF5-GFP(+) myogenic cells were sorted and characterized. These data provides valuable guidelines for generation of knock-in reporter human iPS cell lines for myogenic genes which can be used for disease modeling, drug screening, gene correction and future in vivo applications.
Asunto(s)
Sistemas CRISPR-Cas , Expresión Génica , Marcación de Gen , Genes Reporteros , Recombinación Homóloga , Células Madre Pluripotentes Inducidas/metabolismo , Factor 5 Regulador Miogénico/genética , Biomarcadores , Diferenciación Celular , Línea Celular , Transdiferenciación Celular , Reprogramación Celular/genética , Técnicas de Reprogramación Celular , Cuerpos Embrioides , Técnicas de Sustitución del Gen , Orden Génico , Sitios Genéticos , Vectores Genéticos/genética , Células HEK293 , Humanos , Células Madre Pluripotentes Inducidas/citología , Análisis de Secuencia de ADNRESUMEN
Directed differentiation of iPS cells toward various tissue progenitors has been the focus of recent research. Therefore, generation of tissue-specific reporter iPS cell lines provides better understanding of developmental stages in iPS cells. This technical report describes an efficient strategy for generation and validation of knock-in reporter lines in human iPS cells using the Cas9-nickase system. Here, we have generated a knock-in human iPS cell line for the early myogenic lineage specification gene of PAX7. By introduction of site-specific double-stranded breaks (DSB) in the genomic locus of PAX7 using CRISPR/Cas9 nickase pairs, a 2A-GFP reporter with selection markers has been incorporated before the stop codon of the PAX7 gene at the last exon. After positive and negative selection, single cell-derived human iPS clones have been isolated and sequenced for in-frame positioning of the reporter construct. Finally, by using a nuclease-dead Cas9 activator (dCas9-VP160) system, the promoter region of PAX7 has been targeted for transient gene induction to validate the GFP reporter activity. This was confirmed by flow cytometry analysis and immunostaining for PAX7 and GFP. This technical report provides a practical guideline for generation and validation of knock-in reporters using CRISPR/Cas9 system.