Differentiation of the human PAX7-positive myogenic precursors/satellite cell lineage in vitro

Abstract

Satellite cells (SC) are muscle stem cells which can regenerate adult muscles upon injury. Most SC originate from PAX7-positive myogenic precursors set aside during development. While myogenesis has been studied in mouse and chicken embryos, little is known about human muscle development. Here, we report the generation of human induced Pluripotent Stem (iPS) cell reporter lines in which fluorescent proteins have been introduced into the PAX7 and MYOG loci. We use single cell RNA sequencing to analyze the developmental trajectory of the iPS-derived PAX7-positive myogenic precursors. We show that the PAX7-positive cells generated in culture can produce myofibers and self-renew in vitro and in vivo. Together, we demonstrate that cells exhibiting characteristics of human fetal satellite cells can be produced in vitro from iPS cells, opening interesting avenues for muscular dystrophy cell therapy. This work provides significant insights into the development of the human myogenic lineage.

Generation of PAX7 Reporter Cells to Investigate Skeletal Myogenesis from Human Pluripotent Stem Cells

“…Therefore, not all GFP + sorted reporter cells are PAX7 + . This is reflected by post-sort PAX7 Cytospin-IF microscopy and well-illustrated in singe cell RNA-seq studies from our and other groups ( Al Tanoury et al., 2020 ; Xi et al., 2020 ). To enrich for 100% PAX7 + cells, variants of GFP or other fluorescent proteins with shorter half-lives ( Andersen et al., 1998 ; Hackett et al., 2006 ; Li et al., 1998 ; Mateus and Avery, 2000 ) could be used to construct the reporter cells.…”

“…In addition, the efficiency of IRES-mediated translation has been reported to be lower compared to the regular 5′ cap dependent mechanism and could be susceptible to preceding mRNA secondary structures ( Hennecke et al., 2001 ). In this regard, insertion of a fluorescent reporter to the 5′ ( Al Tanoury et al., 2020 ) or 3′ ( Wu et al., 2016 ) of PAX7 through the 2A self-cleaving peptides allows the endogenous translational machinery to drive more efficient reporter expression as standalone proteins. Moreover, using homozygous knockin cells could also increase GFP signals.…”

Prednisolone rescues Duchenne Muscular Dystrophy phenotypes in human pluripotent stem cells-derived skeletal musclein vitro

“…Here, we describe a novel method which significantly increases myofiber maturation over existing protocols. While current strategies can result in differentiation of myogenic cells up to the embryonic to fetal transition (Al Tanoury et al, 2020; Xi et al, 2020), our improved method results in well-organized myofibers with significant activation of the fast myosin IIa ( MYH1 ), IIx (MYH2) and IIb (MYH4), which are respectively first expressed during fetal, late fetal, and early post-natal stages (Schiaffino et al, 2015). While most of DMD pathological landmarks have been defined during post-natal stages, the primary events leading to these defects likely happen during fetal development.…”

hiPSC-derived 3D Bioprinted Skeletal Muscle Tissue Implants Regenerate Skeletal Muscle Following Volumetric Muscle Loss

“…Among the printable gel mixtures inside a 5% GelMA supporting matrix (previously discussed in Figure 2d), 7.5% (w/v) GelMA bioink (stiffness = 40±8 kPa, further reduced to 22±2 kPa after 3 weeks of incubation in standard culture medium) provided a 3D microenvironment that was mechanically ideal for the myogenic induction of hiPSC-MPCs (i.e., 10-50 kPa and >30% elastic strain) (Figure S10). 41,46 To this end, 3-to 4-week old hiPSC-derived progenitor cells were obtained following our previously described serum-free differentiation protocol (Figure S11) 11,47 and were encapsulated in a bulk supporting matrix or bioink for 3D culture studies. Further steps of myogenic induction were carried out by delivering differentiation factors to the hiPSC-MPC-laden constructs.…”

“…hiPSC-derived myogenic progenitors were generated following a serum-free differentiation method. 11,47 Three-to four-week-old wild-type hiPSC-derived primary myogenic populations were detached to replate (cell density = 3.5-4 x 10 4 cm 2 ) myogenic progenitors on Matrigelcoated plates (Corning) and the cells were then cultured in skeletal muscle growth medium (SKGM-2, Lonza) supplemented with 10 μM ROCK inhibitor for 24 hours. Afterwards, the ROCK inhibitor was removed and the culture was continued with SKGM-2 for ~2 days until they were dissociated using Accutase (Stemcell Technologies) and were frozen or prepared for bioprinting.…”