Abstract
The current protocols for generation of induced pluripotent stem (iPS) cells involve genome integrating viral vectors which may induce tumorgenesis. The aim of this study was to develop and optimize a non-viral method without genetic manipulation for reprogramming of skeletal myoblasts (SMs) using small molecules.Methods and ResultsSMs from young male Oct3/4-GFP+ transgenic mouse were treated with DNA methyltransferase (DNMT) inhibitor, RG108. Two weeks later, GFP+ colonies of SM derived iPS cells (SiPS) expressing GFP and with morphological similarity of mouse embryonic stem (ESCs) were formed and propagated in vitro. SiPS were positive for alkaline phosphatase activity, expressed SSEA1, displayed ES cell specific pluripotency markers and formed teratoma in nude mice. Optimization of culture conditions for embryoid body (EBs) formation yielded spontaneously contracting EBs having morphological, molecular, and ultra-structural similarities with cardiomyocytes and expressed early and late cardiac markers. miR profiling showed abrogation of let-7 family and upregulation of ESCs specific miR-290-295 cluster thus indicating that SiPS were similar to ESCs in miR profile. Four weeks after transplantation into the immunocompetent mice model of acute myocardial infarction (n = 12 per group), extensive myogenesis was observed in SiPS transplanted hearts as compared to DMEM controls (n = 6 per group). A significant reduction in fibrosis and improvement in global heart function in the hearts transplanted with SiPS derived cardiac progenitor cells were observed.ConclusionsReprogramming of SMs by DNMT inhibitor is a simple, reproducible and efficient technique more likely to generate transgene integration-free iPS cells. Cardiac progenitors derived from iPS cells propagated extensively in the infarcted myocardium without tumorgenesis and improved cardiac function.
Highlights
The introduction of four transcription factors, Oct3/4, Sox2, Klf4, and c-Myc can successfully reprogram mouse and human somatic cells into induced pluripotent stem cells [1,2]
Cardiac progenitors derived from induced pluripotent stem (iPS) cells propagated extensively in the infarcted myocardium without tumorgenesis and improved cardiac function
We further report that cardiac progenitors (SiPS-CPs) derived from beating EBs obtained from SM derived iPS cells (SiPS) showed remarkable regeneration of myocardium and formed gap junctions with the resident cardiomyocytes when transplanted in an infarcted mouse heart
Summary
The introduction of four transcription factors, Oct3/4, Sox, Klf, and c-Myc can successfully reprogram mouse and human somatic cells into induced pluripotent stem (iPS) cells [1,2]. Given the molecular pathways underlying iPS cell generation, the use of lesser number of transcription factors combined with small molecules is being promoted for pluripotency [9,11]. We report here for the first time that mouse skeletal myoblasts (SMs) can be efficiently reprogrammed into iPS cells (SiPs) with DNMT inhibitor by induction of a single transcription factor Oct3/4. These SiPS cells resemble ES cells in their molecular behavior and differentiation characteristics. Our purely chemical approach is superior and safest in efficient reprogramming of SMs for generation of cardiac progenitors
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
