Abstract
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.
Highlights
Pluripotent stem cells present the ability to self-renew and undergo differentiation into any cell type building an organism
In the absence of functional Pax7 neuromuscular junctions, formation is abnormal, what results in lower differentiation potential of Pax7−/− embryonic stem cells (ESCs) during advanced stages of myogenesis
The level of mRNAs encoding Pax3 was significantly higher (Fig. 3b), while the expression of Nuclear factor one X (Nfix), Enolase 3 (Eno3), Muscle creatine kinase (Mck), Myocyte enhancer factor 2A (Mef2a), and Integrin subunit alpha 7 (Itga7) was significantly lower in Pax7−/− teratomas, as compared to Pax7+/+ teratomas (Fig. 3c)
Summary
Pluripotent stem cells present the ability to self-renew and undergo differentiation into any cell type building an organism. Pax 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 acts as an anti-apoptotic factor. The most common in vitro approach to induce myogenic differentiation of ESCs depends either on the formation of embryoid bodies (EBs) and EB outgrowths [2,3,4,5,6,7] or ESC incubation in the presence of demethylating agents [3, 8] These methods are rather inefficient and poorly controllable. Myogenic differentiation could be enhanced by ESC genetic modifications or specific culture conditions involving complicated culture schemes recapitulating environmental changes occurring during embryonic myogenesis ([8,9,10,11], reviewed in [12, 13]). Even under such conditions, differentiation of ESCs is limited to the early stages of myogenesis
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