Myogenic potential of mouse embryonic stem cells lacking functional Pax7 tested in vitro by 5-azacitidine treatment and in vivo in regenerating skeletal muscle

Helinska, Anita; Krupa, M.; Archacka, Karolina; Czerwińska, Areta M.; Stremińska, Władysława; Jańczyk-Ilach, Katarzyna; Ciemerych, Maria A.; Grabowska, Iwona

doi:10.1016/j.ejcb.2016.12.001

Cited by 9 publications

(28 citation statements)

References 73 publications

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“…During cellular differentiation occurring in vivo in developing vertebrate embryo or in vitro in ESC culture the number of expressed miRNAs gradually rises . Such increase was also observed by us in differentiating ESCs, that is, the cells previously shown by us and others to be pluripotent, able to successfully differentiate into ecto‐, endo‐, and mesodermal lineages in vivo as well as in vitro . Many lines of evidence document the differences between various ESC and iPSC lines at the level of transcriptome, epigenome, and their ability to differentiate (e.g., ), but the interplay between ESCs miRNA profiles and their differentiation has not been shown, yet.…”

Section: Discussionsupporting

confidence: 58%

Transient MicroRNA Expression Enhances Myogenic Potential of Mouse Embryonic Stem Cells

et al. 2018

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MicroRNAs (miRNAs) are known regulators of various cellular processes, including pluripotency and differentiation of embryonic stem cells (ESCs). We analyzed differentiation of two ESC lines-D3 and B8, and observed significant differences in the expression of miRNAs and genes involved in pluripotency and differentiation. We also examined if transient miRNA overexpression could serve as a sufficient impulse modulating differentiation of mouse ESCs. ESCs were transfected with miRNA Mimics and differentiated in embryoid bodies and embryoid body outgrowths. miRNAs involved in differentiation of mesodermal lineages, such as miR145 and miR181, as well as miRNAs regulating myogenesis (MyomiRs)-miR1, miR133a, miR133b, and miR206 were tested. Using such approach, we proved that transient overexpression of molecules selected by us modulated differentiation of mouse ESCs. Increase in miR145 levels upregulated Pax3, Pax7, Myod1, Myog, and MyHC2, while miR181 triggered the expression of such crucial myogenic factors as Myf5 and MyHC2. As a result, the ability of ESCs to initiate myogenic differentiation and form myotubes was enhanced. Premature expression of MyomiRs had, however, an adverse effect on myogenic differentiation of ESCs. STEM CELLS 2018;36:655-670 SIGNIFICANCE STATEMENTIn the current study, characterization of microRNomes of mouse embryonic stem cells (ESCs) was performed and the potential use of transient microRNA (miRNA) expression as a tool to induce differentiation of these cells was tested. Analyses of microRNomes of two ESC lines revealed that miRNA expression patterns reflect differentiation abilities of each of the analyzed cell line. Also, it was documented that transient overexpression of selected miRNA is sufficient to affect the phenotype of differentiating ESCs. Using this technique and appropriately chosen miRNAs, we were able to improve myogenic differentiation.

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Section: Discussionsupporting

confidence: 58%

Transient MicroRNA Expression Enhances Myogenic Potential of Mouse Embryonic Stem Cells

et al. 2018

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“…In vitro cultured mouse ESCs are unable to differentiate spontaneously into fully formed, i.e., innervated and vascularized, skeletal muscle tissue. 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]. Unfortunately, these methods are rather inefficient and poorly controllable.…”

Section: Introductionmentioning

confidence: 99%

“…Unfortunately, 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]). However, even under such conditions, differentiation of ESCs is limited to the early stages of myogenesis.…”

Section: Introductionmentioning

confidence: 99%

“…In our previous studies, we analyzed the role of Pax7 in the formation of myoblasts from mouse ESCs differentiating in vitro in embryoid bodies [3] as well as in the presence of methylation reducing agent 5-azacytidine [8]. Using EBs as a differentiation model, we showed that even in the absence of functional Pax7 ESCs could form Pax3+, MyoD+, or Myf5+ myoblasts and multinucleated myotubes [3].…”

Section: Introductionmentioning

confidence: 99%

“…Thus, our in vitro studies confirmed that the absence of functional Pax7 caused positive effect on ESC proliferation and early stages of their myogenic differentiation, as well as on the proliferation of mouse embryonic fibroblasts [4]. On the other hand, in the presence of methylation reducing agent 5-azacytidine, ESCs lacking functional Pax7 (Pax7−/− ESCs) were characterized by higher expression of mesodermal lineagespecific markers, such as platelet-derived growth factor receptor alpha (Pdgfra), and also myogenic cell markers like Pax3, Myf5, and MyoD [8]. We also demonstrated that in vivo Pax7−/− ESCs were able to form teratomas composed of cells and tissue originating from ecto-, endo-, and mesoderm [3].…”

Section: Introductionmentioning

confidence: 99%

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Pax7 as molecular switch regulating early and advanced stages of myogenic mouse ESC differentiation in teratomas

et al. 2020

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Background: Pluripotent 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. Methods: In 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. Results: We 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.

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Stem cell‐based strategies for skeletal muscle tissue engineering

Baldwin

Kim

Sivaraman

et al. 2022

J Tissue Eng Regen Med

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Skeletal muscle tissue engineering has been a key area of focus over the years and has been of interest for developing regenerative strategies for injured or degenerative skeletal muscle tissue. Stem cells have gained increased attention as sources for developing skeletal muscle tissue for subsequent studies or potential treatments. Focus has been placed on understanding the molecular pathways that govern skeletal muscle formation in development to advance differentiation of stem cells towards skeletal muscle fates in vitro. Use of growth factors and transcription factors have long been the method for guiding skeletal muscle differentiation in vitro. However, further research in small molecule induced differentiation offers a xeno-free option that could result from use of animal derived factors.

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Myogenic potential of mouse embryonic stem cells lacking functional Pax7 tested in vitro by 5-azacitidine treatment and in vivo in regenerating skeletal muscle

Cited by 9 publications

References 73 publications

Transient MicroRNA Expression Enhances Myogenic Potential of Mouse Embryonic Stem Cells

Transient MicroRNA Expression Enhances Myogenic Potential of Mouse Embryonic Stem Cells

Pax7 as molecular switch regulating early and advanced stages of myogenic mouse ESC differentiation in teratomas

Stem cell‐based strategies for skeletal muscle tissue engineering

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