Expansion and Purification Are Critical for the Therapeutic Application of Pluripotent Stem Cell-Derived Myogenic Progenitors

Abstract

SummaryRecent reports have documented the differentiation of human pluripotent stem cells toward the skeletal myogenic lineage using transgene- and cell purification-free approaches. Although these protocols generate myocytes, they have not demonstrated scalability, safety, and in vivo engraftment, which are key aspects for their future clinical application. Here we recapitulate one prominent protocol, and show that it gives rise to a heterogeneous cell population containing myocytes and other cell types. Upon transplantation, the majority of human donor cells could not contribute to myofiber formation. As a proof-of-principle, we incorporated the inducible PAX7 lentiviral system into this protocol, which then enabled scalable expansion of a homogeneous population of skeletal myogenic progenitors capable of forming myofibers in vivo. Our findings demonstrate the methods for scalable expansion of PAX7+ myogenic progenitors and their purification are critical for practical application to cell replacement treatment of muscle degenerative diseases.

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

“…These efforts employed by tuning major signaling pathways governing stem cell differentiation toward mesoderm and muscle, such as WNT, BMP, and transforming growth factor β (TGF-β) (Barberi et al, 2007; Borchin et al, 2013; Caron et al, 2016; Chal et al, 2015; Hicks et al, 2018; Shelton et al, 2014; Xi et al, 2017; Xu et al, 2013). Although these methods were successful in inducing skeletal myogenesis, parallel generation of unwanted cell types hindered the purity of the myogenic cells, as recently reported by our collaborators (Kim et al, 2017). Even in the most recent methods (Hicks et al, 2018; Shelton et al, 2016), the need for a long-term culture (up to 50 days) and associated difficulties (such as mixed cultures and difficulties in maintenance and harvest) are among major obstacles.…”

“…So far, previous studies have suffered from the lack of prospective readout to guide the differentiation path and subsequently purify myogenic progenitors and therefore carrying unwanted or mixed cell populations (Kim et al, 2017). Here, we have taken the advantage of generating a double-reporter hESC line for muscle lineage.…”

“…During the past decade, several efforts have been made to differentiate PSCs toward skeletal myogenic lineage (Barberi et al, 2007; Borchin et al, 2013; Chal et al, 2015; Choi et al, 2016; Darabi et al, 2008, 2011, 2012; Kim et al, 2017; Magli et al, 2017; Shelton et al, 2014; Tanaka et al, 2013; Tedesco et al, 2012; Xi et al, 2017; Xu et al, 2013). Initial reports by collaborators using myogenic gene overexpression during differentiation provided proof of the concept, i.e., myogenic potential of murine and human PSCs (Darabi et al, 2008, 2011, 2012; Darabi and Perlingeiro, 2016; Tanaka et al, 2013).…”

Myogenic progenitor specification from pluripotent stem cells

“…Although it would be expected that cells capable of differentiating in vitro are able to fuse to existing myofibers in vivo, the cell-to-cell interactions occurring in the petri dish combined with a still heterogeneous composition of the population under investigation (particularly in the case of protocols not involving cell-sorting) represent a limit for the direct translation of these PS cell-derived myogenic cell preparations. Indeed, this is supported by recent findings demonstrating the need of cell-purification strategies and robust lineage commitment for the successful engraftment of muscle progenitors derived using directed differentiation of PS cells [138]. …”

“…An obvious example of the variation among lines is the requirement of variable concentration of the GSK3i inhibitor for mesoderm induction [124]. In the case of skeletal myogenesis, it has been shown that human PS cell lines display a variable capability to differentiate using a chemically defined protocol, thus implying the need of further improvements before the generalization of current protocols [138]. As in the case of maturation, detailed analysis of the epigenetic mechanisms behind cell differentiation will definitively help to understand why some PS cell lines are more permissive than others.…”