Assessment of different strategies for scalable production and proliferation of human myoblasts

Abstract

Objectives: Myoblast transfer therapy (MTT) is a technique to replace muscle satellite cells with genetically repaired or healthy myoblasts, to treat muscular dystrophies. However, clinical trials with human myoblasts were ineffective, showing almost no benefit with MTT. One important obstacle is the rapid senescence of human myoblasts. The main purpose of our study was to compare the various methods for scalable generation of proliferative human myoblasts. Methods:We compared the immortalization of primary myoblasts with hTERT, cyclin D1 and CDK4 R24C , two chemically defined methods for deriving myoblasts from pluripotent human embryonic stem cells (hESCs), and introduction of viral MyoD into hESC-myoblasts. Results:Our results show that, while all the strategies above are suboptimal at generating bona fide human myoblasts that can both proliferate and differentiate robustly, chemically defined hESC-monolayer-myoblasts show the most promise in differentiation potential.Conclusions: Further efforts to optimize the chemically defined differentiation of hESCmonolayer-myoblasts would be the most promising strategy for the scalable generation of human myoblasts, for applications in MTT and high-throughput drug screening.

Functional skeletal muscle constructs from transdifferentiated human fibroblasts

“…Transduction of human myoblasts with human telomerase reverse transcriptase (hTERT) is not sufficient to immortalize these cells 7 . Even though overexpression of cyclin-dependent kinase 4 (CDK4) or cyclin D1 with hTERT could result in myoblasts capable of extensive proliferation, these cells have compromised myogenic differentiation potential 7 , 8 . Recent advances in deriving myogenic cells from induced human pluripotent stem cells (hiPSCs) may provide opportunities to address the limited availability of human myogenic cells 9 – 12 .…”

Adult progenitor rejuvenation with embryonic factors

“…To address this question, we used young and old primary adult human skeletal muscle (HSKM) progenitors to screen for a variety of embryonically regulated factors that are not lineage‐specific, to biomimic the foetal growth phase. We defined ‘young’ as <5 population doublings, and ‘old’ as more than 20 population doublings, based on prior experience with replicative senescence 46 . First, we found that old progenitors, compared with progenitors, showed significantly higher levels of cell cycle inhibitors such as p21 WAF1 , p27 KIP1 , and p16 INK4a (Figure 1A).…”

“…Differentiation was initiated by replacing growth media with differentiation medium, comprising of DMEM/F‐12, 2% KnockOut Serum Replacement (Gibco), 1% L‐glutamine (Gibco) and 1% penicillin‐streptomycin (Gibco), when the young adult HSKM progenitors were 80%–100% confluent. These young adult HSKM progenitors were previously validated to be 100% MYOD1 + during the proliferative stage, with robust expression of myogenic markers after differentiation 46 …”

Transcriptomic Profile of Primary Culture of Skeletal Muscle Cells Isolated from Semitendinosus Muscle of Beef and Dairy Bulls

“…It is a known fact that in the early stages of myoblast differentiation, the MyoD+/MYOG+ myocytes begin to accumulate muscle-specific ACTA1 and MYH3. Subsequently, myocytes may fuse, forming α-actinin+/MYOG+ multinucleated myotubes, and finally, muscle fibers [ 65 ].…”