Transient MicroRNA Expression Enhances Myogenic Potential of Mouse Embryonic Stem Cells

Abstract

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.

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

“…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.…”

miR-128a Acts as a Regulator in Cardiac Development by Modulating Differentiation of Cardiac Progenitor Cell Populations

“…Different pluripotent stem cell lines do not necessarily behave equally in vitro, e.g., due to epigenomic and transcriptomic differences or variable differentiation abilities [20]. Additionally, miR chromosomal locations and expression profiles can vary between species [21].…”

Regulation of skeletal myogenesis by microRNAs

“…miR‐145a‐5p is gradually upregulated in differentiating C2C12 myoblasts (Du et al, ). Recent findings suggests that miR‐145a‐5p likely acts as a positive regulator of myoblast differentiation (Bem et al, ; Du et al, ). Though the gene targets of miR‐145a‐5p are not uncovered, the genes of endogenous Wnt signaling pathway are increased by miR‐145a‐5p overexpression (Du et al, ), suggesting that miR‐145a‐5p may regulate myogenic differentiation through Wnt signaling pathway.…”

“…MicroRNAs (miRNAs) are a class of ∼22 nucleotide, evolutionally conserved small noncoding RNAs, which negatively regulate gene expression primarily at the posttranscriptional level by destabilizing and degrading their target messenger RNAs (mRNAs) or suppressing their translation through sequence‐specific interactions with 3′‐untranslated regions (3‐UTRs) (Bem et al, ; Mok, Lozano‐Velasco, & Münsterberg, ; Rao, Kumar, Farkhondeh, Baskerville, & Lodish, ). Their biogenesis is summarized in detail in a previous review (Mok et al, ).…”