Transgene-free direct conversion of murine fibroblasts into functional muscle stem cells

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+ stem cell subpopulation resembling satellite cells. Last, transgene-free iMPCs robustly engrafted skeletal muscles of a Duchenne muscular dystrophy mouse model, restoring dystrophin expression in hundreds of myofibers. In summary, this study reports on an improved and clinically safer approach to convert fibroblasts into myogenic stem cells that can efficiently contribute to muscle regeneration in vivo.

Cell Reprogramming and Differentiation Utilizing Messenger RNA for Regenerative Medicine

“…The generated iMPCs were shown to express a series of myogenic stem cell markers and to differentiate into contractile myotubes. Furthermore, in a mouse model of Duchenne muscular dystrophy, iMPCs strongly engrafted into skeletal muscle and restored dystrophin expression in hundreds of myofibers [ 147 ].…”

A Self‐Renewing Biomimetic Skeletal Muscle Construct Engineered using Induced Myogenic Progenitor Cells

“…[ 35–41 ] Several recent studies reported on production of iMPCs by transient expression of the canonical myogenic transcription factor MyoD in conjunction with the small molecules Forskolin, RepSox, and CHIR99021 (F/R/C). [ 35,42–44 ] Notably, iMPC cultures are composed of disorganized multinucleated and contractile myotubes, in addition to self‐renewing muscle stem cells that can expand extensively while maintaining differentiation capacity. [ 35 ] Furthermore, unlike conventional myoblasts, muscle stem cells present in iMPCs share several attributes with in vivo proliferating SCs, thus serving as a potential superior cell source for skeletal muscle engineering.…”