Pig MYOD1 overexpression vector construction
Myod1/MYOD1 is reportedly identified as a master transcription factor in myogenesis, thereby inducing myogenic transdifferentiation in nonmuscle cells (9, 24–27). Thus, comparative MYOD1 sequence analysis was performed among various species to assess whether pig MYOD1 contains functionally conserved sequences for myogenesis, as in the other species, before vector construction for ectopic pig MYOD1 (pMYOD1) expression. The pig MYOD1 whole protein is composed of 319 amino acids and has a similar size to MYOD1 protein in other species (Fig. 1A). Additionally, in all the analyzed species, including pig, the MYOD1 protein contains acidic domains, histidine and cysteine-rich (H/C) domains, basic-helix1-loop-helix2 (bHLH) domains, and helix3 domains, which are located at 3–56 a.a, 63–99 a.a, 102–167 a.a, and 246–259 a.a (in the case of mouse: 245–258 a.a), respectively, indicating there are identical locations and sizes among the species (28, 29). Subsequently, the MYOD1 sequences from those species were aligned, and the similarity score was assessed at the amino acid level (Fig. 1B and Table 2). In the bHLH domain, Ala114-Thr115 and Arg111 reportedly endow the MYOD1 protein with myogenic activity (30, 31). In fact, these residues (marked in red; Fig. 1B) were confirmed to be conserved in the basic region of all the analyzed species. The MYOD1 whole proteins of pig and ungulates (pig-horse: 94.36%, pig-cow: 95.92%, and pig-sheep: 95.61%) were more similar than those of pig and human (93.12%) or rodent (89.03%). It is noteworthy that the bHLH domain showed a 100.00% similarity between pig and all other species. Altogether, porcine MYOD1, especially the bHLH domain, was identified as an evolutionarily conserved protein, which seems that its role is also conserved in myogenesis across species.
Based on the above analyses, doxycycline (DOX)-inducible pMYOD1 overexpression vectors were generated including the MYOD1 gene isolated from satellite cells in 3-day-old LYD biceps femoris. To verify the function of these constructed vectors, they were introduced into pig embryonic fibroblasts (PEFs) through lentiviral infection for stable transgene expression (4, 27). First, the integration of the exogenous pMYOD1 gene was confirmed by PCR targeting the FUW-tetO-pMYOD1 sequence in gDNA of PEFs infected with a lentivirus carrying the vectors (pMYOD1-PEFs) (Fig. 2A). At day 9, the gene of interest had been inserted stably into the genome of PEFs. Then, MYOD1 expression was confirmed at the protein level using immunostaining in pMYOD1-PEFs (Fig. 2B). The vectors were activated by the addition of DOX, leading to ectopic MYOD1 expression. Finally, the expression pattern of muscle-associated genes (Exo-MYOD1, Endo-MYOD1, PAX7, MYF5, and MYOG) was analyzed in pMYOD1-PEFs using qPCR (Fig. 2C). These genes have been characterized as myogenic lineage-specific markers, such as skeletal muscle progenitor/myoblasts (PAX7, MYF5, and MYOD1) and myocytes (MYOD1 and MYOG) (3). Exo-MYOD1 overexpression by vector activation increased the expression of endogenous muscle-associated genes. During extended cell culture, the expression of these genes was stably maintained. In conclusion, we constructed a DOX-inducible pMYOD1 overexpression vector that triggered the expression of muscle markers in long-term culture, indicating its stable function.
Myogenic transdifferentiation of PEFs by overexpression of MYOD1
The expression of endogenous muscle markers, including Endo-MYOD1, was enhanced via ectopic MYOD1 expression, as shown in Fig. 2C. However, because of the mild changes in each gene, transdifferentiated muscle cells from fibroblasts were not observed. It has been shown that full transdifferentiation is achieved by genetic modulation along with suitable culture conditions for specific cell types (32), suggesting that optimization of culture conditions is required. According to previous studies, a 2-step transdifferentiation protocol was employed with some modification to derive myogenic cells through MYOD1 overexpression (8, 17, 33). In the ‘induction’ step, the myogenic program was activated with the stimulation of transcription factors associated with myogenesis, thereby leading to the commitment into a myogenic lineage. Briefly, pMYOD1-PEFs were treated with various signaling molecules, such as FGF2, SB431542, CHIR99021, and forskolin, which have been shown to be involved in the regulation of myogenesis (8, 14). Then, transdifferentiation was promoted through serum starvation in the ‘differentiation’ step.
To ensure efficient myogenic conversion, we investigated the transition point where exogenous MYOD1 leads to the peak expression of endogenous skeletal muscle-specific genes. In addition, pMYOD1-PEFs were cultured in mitogen-rich media in which myogenic induction was sustained without entering the differentiation process (Fig. 3A). While the FSCHF group had a long cylindrical shape due to elongation of the cytoplasm, a typical fibroblastic and round shape was observed in the control and FGF groups, respectively (Fig. 3B). The alteration of the FSCHF group was maintained by the end of the culture period, resulting in a similar morphology to myoblasts, as previously reported (17, 34). The myogenic genes were upregulated by forced MYOD1 expression (Fig. 3C). Compared to other groups, the relative gene expression of the FSCHF group was higher across all the genes and culture periods. These results suggested that the Exo-MYOD1 effect was enhanced by the combination of four signaling molecules rather than DOX-induced exogenous MYOD1 per se or additional FGF2. While the gene expression of the FGF group was gradually changed, that of the FSCHF group was significantly increased on day 6 and then decreased. The aforementioned expression patterns were observed in all the markers except PAX7, which was upregulated up to day 9 in both groups. Taken together, pMYOD1-PEFs treated with a cocktail of FGF2, SB431542, CHIR99021, and forskolin for 6 days were used for further experiments.
Based on the above observations, we established a myogenic transdifferentiation protocol in which the FSCHF medium for induction into a myogenic lineage was replaced with a low-serum medium for the initiation of differentiation on day 6 (Fig. 4A). The replaced culture condition was classified into two groups distinguished by the addition of DOX (+ DOX and -DOX) to assess whether consistent activation of the myogenic program could enhance transdifferentiation. Notably, multinucleated myotubes via fusion of myoblasts were observed on day 8 in both groups (Fig. 4B). According to the qPCR analyses performed with a sample from day 9, transcripts of the Exo-MYOD1, MYF5, MYOG, and Myosin heavy chain (MHC) genes were upregulated in the +/- DOX groups (Fig. 4C). Unlike that of the other genes, gene expression of Endo-MYOD1 was significantly decreased in the +/- DOX group. Across all of the genes, especially MYF5 and MHC, the + DOX group showed significantly higher expression levels than the -DOX group, demonstrating that continuous MYOD1 overexpression during the differentiation step facilitates myogenic transdifferentiation. The expression of MHC, a marker of late differentiation in myogenesis, was detected in the day 9 sample by immunofluorescence analysis (Fig. 4D). Interestingly, a sarcomere-like structure with a striated pattern was observed, as previously reported (35), indicating that the mature myotube could be assembled. Therefore, the established protocol using ectopic MYOD1 expression and signaling molecules associated with myogenesis, such as FGF2, a TGF-β inhibitor, a WNT activator, and a cAMP activator, enabled fibroblasts to be reprogrammed into skeletal muscle.