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Premyogenic progenitors derived from human pluripotent stem cells expand in floating culture and differentiate into transplantable myogenic progenitors.

ABSTRACT: Human induced pluripotent stem cells (hiPSCs) are a potential source for cell therapy of Duchenne muscular dystrophy. To reliably obtain skeletal muscle progenitors from hiPSCs, we treated hiPS cells with a Wnt activator, CHIR-99021 and a BMP receptor inhibitor, LDN-193189, and then induced skeletal muscle cells using a previously reported sphere-based culture. This protocol greatly improved sphere formation efficiency and stably induced the differentiation of myogenic cells from hiPS cells generated from both healthy donors and a patient with congenital myasthenic syndrome. hiPSC-derived myogenic progenitors were enriched in the CD57(-) CD108(-) CD271(+) ERBB3(+) cell fraction, and their differentiation was greatly promoted by TGF-? inhibitors. TGF-? inhibitors down-regulated the NFIX transcription factor, and NFIX short hairpin RNA (shRNA) improved the differentiation of iPS cell-derived myogenic progenitors. These results suggest that NFIX inhibited differentiation of myogenic progenitors. hiPSC-derived myogenic cells differentiated into myofibers in muscles of NSG-mdx 4Cv mice after direct transplantation. Our results indicate that our new muscle induction protocol is useful for cell therapy of muscular dystrophies.

SUBMITTER: Sakai-Takemura F

PROVIDER: S-EPMC5920060 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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Premyogenic progenitors derived from human pluripotent stem cells expand in floating culture and differentiate into transplantable myogenic progenitors.

Sakai-Takemura Fusako F Narita Asako A Masuda Satoru S Wakamatsu Toshifumi T Watanabe Nobuharu N Nishiyama Takashi T Nogami Ken'ichiro K Blanc Matthias M Takeda Shin'ichi S Miyagoe-Suzuki Yuko Y

Scientific reports 20180426 1

Human induced pluripotent stem cells (hiPSCs) are a potential source for cell therapy of Duchenne muscular dystrophy. To reliably obtain skeletal muscle progenitors from hiPSCs, we treated hiPS cells with a Wnt activator, CHIR-99021 and a BMP receptor inhibitor, LDN-193189, and then induced skeletal muscle cells using a previously reported sphere-based culture. This protocol greatly improved sphere formation efficiency and stably induced the differentiation of myogenic cells from hiPS cells gene ...[more]

PMID: 29700358

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Project description:The mammalian heart has little capacity to regenerate, and following injury the myocardium is replaced by non-contractile scar tissue. Consequently, increased wall stress and workload on the remaining myocardium leads to chamber dilation, dysfunction, and heart failure. Cell-based therapy with an autologous, epigenetically reprogrammed, and cardiac-committed progenitor cell source could potentially reverse this process by replacing the damaged myocardium with functional tissue. However, it is unclear whether cardiac progenitor cell-derived cardiomyocytes are capable of attaining levels of structural and functional maturity comparable to that of terminally-fated cardiomyocytes. Here, we first describe the derivation of mouse induced pluripotent stem (iPS) cells, which once differentiated allow for the enrichment of Nkx2-5(+) cardiac progenitors, and the cardiomyocyte-specific expression of the red fluorescent protein. We show that the cardiac progenitors are multipotent and capable of differentiating into endothelial cells, smooth muscle cells and cardiomyocytes. Moreover, cardiac progenitor selection corresponds to cKit(+) cell enrichment, while cardiomyocyte cell-lineage commitment is concomitant with dual expression of either cKit/Flk1 or cKit/Sca-1. We proceed to show that the cardiac progenitor-derived cardiomyocytes are capable of forming electrically and mechanically coupled large-scale 2D cell cultures with mature electrophysiological properties. Finally, we examine the cell progenitors' ability to form electromechanically coherent macroscopic tissues, using a physiologically relevant 3D culture model and demonstrate that following long-term culture the cardiomyocytes align, and form robust electromechanical connections throughout the volume of the biosynthetic tissue construct. We conclude that the iPS cell-derived cardiac progenitors are a robust cell source for tissue engineering applications and a 3D culture platform for pharmacological screening and drug development studies.

Project description:BACKGROUND:Duchenne muscular dystrophy (DMD) is a devastating genetic muscular disorder with no effective treatment that is caused by the loss of dystrophin. Human induced pluripotent stem cells (hiPSCs) offer a promising unlimited resource for cell-based therapies of muscular dystrophy. However, their clinical applications are hindered by inefficient myogenic differentiation, and moreover, the engraftment of non-transgene hiPSC-derived myogenic progenitors has not been examined in the mdx mouse model of DMD. METHODS:We investigated the muscle regenerative potential of myogenic progenitors derived from hiPSCs in mdx mice. The hiPSCs were transfected with enhanced green fluorescent protein (EGFP) vector and defined as EGFP hiPSCs. Myogenic differentiation was performed on EGFP hiPSCs with supplementary of basic fibroblast growth factor, forskolin, 6-bromoindirubin-3'-oxime as well as horse serum. EGFP hiPSCs-derived myogenic progenitors were engrafted into mdx mice via both intramuscular and intravenous injection. The restoration of dystrophin expression, the ratio of central nuclear myofibers, and the transplanted cells-derived satellite cells were accessed after intramuscular and systemic transplantation. RESULTS:We report that abundant myogenic progenitors can be generated from hiPSCs after treatment with these three small molecules, with consequent terminal differentiation giving rise to mature myotubes in vitro. Upon intramuscular or systemic transplantation into mdx mice, these myogenic progenitors engrafted and contributed to human-derived myofiber regeneration in host muscles, restored dystrophin expression, ameliorated pathological lesions, and seeded the satellite cell compartment in dystrophic muscles. CONCLUSIONS:This study demonstrates the muscle regeneration potential of myogenic progenitors derived from hiPSCs using non-transgenic induction methods. Engraftment of hiPSC-derived myogenic progenitors could be a potential future therapeutic strategy to treat DMD in a clinical setting.

Project description:Human myogenic progenitors can be derived from pluripotent stem cells (PSCs) for use in modeling natural and pathological myogenesis, as well as treating muscle diseases. Transgene-free methods of deriving myogenic progenitors from different PSC lines often produce mixed populations that are heterogeneous in myogenic differentiation potential, yet detailed and accurate characterization of human PSC-derived myogenic progenitors remains elusive in the field. The isolation and purification of human PSC-derived myogenic progenitors is thus an important methodological consideration when we investigate the properties and behaviors of these cells in culture. We previously reported a transgene-free, serum-free floating sphere culture method for the derivation of myogenic progenitors from human PSCs. In this study, we first performed comprehensive cell surface protein profiling of the sphere culture cells through the screening of 255 antibodies. Next, we used magnetic activated cell sorting and enriched the cells according to the expression of specific surface markers. The ability of muscle differentiation in the resulting cells was characterized by immunofluorescent labeling and quantification of positively stained cells. Our results revealed that myotube-forming cells resided in the differentiated cultures of CD29+, CD56+, CD271+, and CD15- fractions, while thick and multinucleated myotubes were identified in the differentiated cultures from CD9+ and CD146+ fractions. We found that PAX7 localization to the nucleus correlates with myotube-forming ability in these sorted populations. We also demonstrated that cells in unsorted, CD271+, and CD15- fractions responded differently to cryopreservation and prolonged culture expansion. Lastly, we showed that CD271 expression is essential for terminal differentiation of human PSC-derived myogenic progenitors. Taken together, these cell surface proteins are not only useful markers to identify unique cellular populations in human PSC-derived myogenic progenitors but also functionally important molecules that can provide valuable insight into human myogenesis.

Dataset Information

Premyogenic progenitors derived from human pluripotent stem cells expand in floating culture and differentiate into transplantable myogenic progenitors.

Publications

Premyogenic progenitors derived from human pluripotent stem cells expand in floating culture and differentiate into transplantable myogenic progenitors.

Similar Datasets

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