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PAX7 Targets, CD54, Integrin ?9?1, and SDC2, Allow Isolation of Human ESC/iPSC-Derived Myogenic Progenitors.

ABSTRACT: Pluripotent stem (PS)-cell-derived cell types hold promise for treating degenerative diseases. However, PS cell differentiation is intrinsically heterogeneous; therefore, clinical translation requires the development of practical methods for isolating progenitors from unwanted and potentially teratogenic cells. Muscle-regenerating progenitors can be derived through transient PAX7 expression. To better understand the biology, and to discover potential markers for these cells, here we investigate PAX7 genomic targets and transcriptional changes in human cells undergoing PAX7-mediated myogenic commitment. We identify CD54, integrin ?9?1, and Syndecan2 (SDC2) as surface markers on PAX7-induced myogenic progenitors. We show that these markers allow for the isolation of myogenic progenitors using both fluorescent- and CGMP-compatible magnetic-based sorting technologies and that CD54+?9?1+SDC2+ cells contribute to long-term muscle regeneration in vivo. These findings represent a critical step toward enabling the translation of PS-cell-based therapies for muscle diseases.

SUBMITTER: Magli A

PROVIDER: S-EPMC5528177 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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PAX7 Targets, CD54, Integrin α9β1, and SDC2, Allow Isolation of Human ESC/iPSC-Derived Myogenic Progenitors.

Magli Alessandro A Incitti Tania T Kiley James J Swanson Scott A SA Darabi Radbod R Rinaldi Fabrizio F Selvaraj Sridhar S Yamamoto Ami A Tolar Jakub J Yuan Ce C Stewart Ron R Thomson James A JA Perlingeiro Rita C R RCR

Cell reports 20170601 13

Pluripotent stem (PS)-cell-derived cell types hold promise for treating degenerative diseases. However, PS cell differentiation is intrinsically heterogeneous; therefore, clinical translation requires the development of practical methods for isolating progenitors from unwanted and potentially teratogenic cells. Muscle-regenerating progenitors can be derived through transient PAX7 expression. To better understand the biology, and to discover potential markers for these cells, here we investigate ...[more]

PMID: 28658631

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Project description:BackgroundPluripotent stem cells present the ability to self-renew and undergo differentiation into any cell type building an organism. Importantly, a lot of evidence on embryonic stem cell (ESC) differentiation comes from in vitro studies. However, ESCs cultured in vitro do not necessarily behave as cells differentiating in vivo. For this reason, we used teratomas to study early and advanced stages of in vivo ESC myogenic differentiation and the role of Pax7 in this process. Pax7 transcription factor plays a crucial role in the formation and differentiation of skeletal muscle precursor cells during embryonic development. It controls the expression of other myogenic regulators and also acts as an anti-apoptotic factor. It is also involved in the formation and maintenance of satellite cell population.MethodsIn vivo approach we used involved generation and analysis of pluripotent stem cell-derived teratomas. Such model allows to analyze early and also terminal stages of tissue differentiation, for example, terminal stages of myogenesis, including the formation of innervated and vascularized mature myofibers.ResultsWe determined how the lack of Pax7 function affects the generation of different myofiber types. In Pax7-/- teratomas, the skeletal muscle tissue occupied significantly smaller area, as compared to Pax7+/+ ones. The proportion of myofibers expressing Myh3 and Myh2b did not differ between Pax7+/+ and Pax7-/- teratomas. However, the area of Myh7 and Myh2a myofibers was significantly lower in Pax7-/- ones. Molecular characteristic of skeletal muscles revealed that the levels of mRNAs coding Myh isoforms were significantly lower in Pax7-/- teratomas. The level of mRNAs encoding Pax3 was significantly higher, while the expression of Nfix, Eno3, Mck, Mef2a, and Itga7 was significantly lower in Pax7-/- teratomas, as compared to Pax7+/+ ones. We proved that the number of satellite cells in Pax7-/- teratomas was significantly reduced. Finally, analysis of neuromuscular junction localization in samples prepared with the iDISCO method confirmed that the organization of neuromuscular junctions in Pax7-/- teratomas was impaired.ConclusionsPax7-/- ESCs differentiate in vivo to embryonic myoblasts more readily than Pax7+/+ cells. In the absence of functional Pax7, initiation of myogenic differentiation is facilitated, and as a result, the expression of mesoderm embryonic myoblast markers is upregulated. However, in the absence of functional Pax7 neuromuscular junctions, formation is abnormal, what results in lower differentiation potential of Pax7-/- ESCs during advanced stages of myogenesis.

Project description:IntroductionPorcine congenital splay leg syndrome (PCS) is a major birth defect in piglets, resulting in lameness and high mortality rates. The multifactorial pathogenesis of PSC is not well understood but includes a polygenic inheritance.MethodsHere, in addition to morphological investigations, we characterized the expression of myogenic genes and functional (proliferation and differentiation) properties of myogenic precursor/satellite cells (SATCs) in 1 day-old PCS piglets, non-affected littermates (LCs), and piglets from PCS-free healthy litters (HCs). In addition, PCS phenotypes were related to the SNP Homer1_rs325197091 within the Homer1 locus, which has been identified as a potential hereditary cause of PCS.Results and discussionSamples from musculus semitendinosus (ST) of PCS piglets had a higher proportion of type II fibers, reflecting myofiber immaturity. In addition, myofiber atrophy, a lower number of myonuclei per fiber (ST), and a higher apoptotic activity (in ST and longissimus dorsi muscle; LD) were found in the PCS group. A higher proportion of cycling committed myoblasts (Pax7+/Ki67+ cells) occurred in samples from PCS-affected piglets, and on the other hand, the mRNA expression of genes involved in differentiation (muscle differentiation 1; MyoD, myogenin; MyoG) was repressed compared with HCs. Cultured SATCs from PCS-affected animals showed a temporal shift in peak expression of Pax7, MyoD, and MyoG toward days 3 and 4 of their 7 days differentiation regime. In vitro experiments with isolated SATCs confirmed the lower differentiation potential and the delayed progression of the myogenic processes in cells from piglets with PCS phenotype. In addition, Pax7 and desmin were differently expressed in Homer1_rs325197091 genotype variants (GG, GA, and AA). Both genes showed the lowest expression in the homozygous AA-variant, which was most frequently found in PCS-affected animals. The homozygous AA-variant was also associated with lower expression of the truncated Homer1-subtype 205. Thus, we hypothesize that in PCS, the balance between Homer1 proteins and its signaling functions is changed in a way detrimental to the myogenic differentiation program. Our results demonstrated direct negative effects of the Homer1 AA genotype on Pax7 expression, but the exact mode of action still needs to be elucidated.

Dataset Information

PAX7 Targets, CD54, Integrin ?9?1, and SDC2, Allow Isolation of Human ESC/iPSC-Derived Myogenic Progenitors.

Publications

PAX7 Targets, CD54, Integrin α9β1, and SDC2, Allow Isolation of Human ESC/iPSC-Derived Myogenic Progenitors.

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