A Myogenic Double-Reporter Human Pluripotent Stem Cell Line Allows Prospective Isolation of Skeletal Muscle Progenitors

Abstract

SUMMARY Myogenic differentiation of human pluripotent stem cells (hPSCs) has been done by gene overexpression or directed differentiation. However, viral integration, long-term culture, and the presence of unwanted cells are the main obstacles. By using CRISPR/Cas9n, a double-reporter human embryonic stem cell (hESC) line was generated for PAX7/ MYF5, allowing prospective readout. This strategy allowed pathway screen to define efficient myogenic induction in hPSCs. Next, surface marker screen allowed identification of CD10 and CD24 for purification of myogenic progenitors and exclusion of non-myogenic cells. CD10 expression was also identified on human satellite cells and skeletal muscle progenitors. In vitro and in vivo studies using transgene and/or reporter-free hPSCs further validated myogenic potential of the cells by formation of new fibers expressing human dystrophin as well as donor-derived satellite cells in NSG-mdx4Cv mice. This study provides biological insights for myogenic differentiation of hPSCs using a double-reporter cell resource and defines an improved myogenic differentiation and purification strategy.

Towards stem cell therapies for skeletal muscle repair

“…In support of these findings, NGFR has been shown to be enriched in ES-derived PAX7:eGFP+ cells and was used to isolate progenitors with enhanced myogencity 69 . Similarly, using a PAX7/MYF5 double reporter hPSC line Wu et al 70 described an optimized 15-day differentiation protocol to generate expandable hPSC derived myogenic progenitors via enrichment of CD24 neg / CD10 + cells. These cells possessed enhanced myotube formation and robust in vivo engraftment, generating h-dystrophin+ myofibers when transplanted into mdx/nsg mice.…”

Probing pluripotency gene regulatory networks with quantitative live cell imaging

“…So far, a direct measure of correlations in the fluctuations in multiple transcription factors in mammalian cells has not been reported to our knowledge. However, dual reporter cell lines that might be used for such an analysis where FPs are associated with two different genes associated with pluripotency and differentiation networks have been reported [7] , [23] , [24] , [26] , [27] , [80] . Fig.…”

“…Current methods incorporating genomic editing aided by TALENs, Zn fingers or CRISPR has greatly improved specific gene targeting efficiencies by several orders of magnitude [85] . These approaches have made it practical to engineer stem cell lines with single [14] , [86] , [87] , [88] , [89] , [90] , [91] , [92] , and dual [7] , [24] , [26] , [27] , [93] gene reporter systems. Dual gene reporters have also been generated in mouse cells through crossing of mice containing a single gene engineered reporter [23] , [25] , [80] .…”

“…Incomplete characterization of cell lines and insufficient reporting of details for creating and validating cell lines is common and may be an impediment to their adoption by others. While cell line characterization is quite extensive in some reports [14] , [17] , [26] , [96] too few are sufficiently documented. This failure leads to a lack of confidence in the biological significance of the data generated using these cell lines.…”

“…Quantifying fluctuations in the expression of a single gene in individual cells over time directly by live cell microscopy has been reported [7] , [10] , [22] . However, although dual-reporter cell lines have been engineered [7] , [23] , [24] , [25] , [26] , [27] , measuring trajectories of expression of more than one gene in individual cells has not been reported to our knowledge. Correlations in expression of multiple gene products can indicate mutual control of those genes by upstream factors [28] , and correlated responses between network components provides a higher degree of confidence in the nature and strength of the relationship between gene products such as transcription factors [29] than can be achieved by static measurements such as flow cytometry or transcriptomics.…”