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Human first-trimester chorionic villi have a myogenic potential.

Arakawa R, Aoki R, Arakawa M, Saito K - Cell Tissue Res. (2012)

Bottom Line: These cells efficiently differentiated into myotubes after myogenic induction, at which point Nanog and Sox2 were down-regulated, whereas MyoD, myogenin, desmin and dystrophin were up-regulated.To our knowledge, this is the first demonstration that FTCVs can be efficiently directed to differentiate in vitro into skeletal muscle cells that express dystrophin as the last stage marker of myogenic differentiation.The myogenic potential of FTCVs reveals their promise for use in cell therapy for DMD, for which no effective treatment presently exists.

View Article: PubMed Central - PubMed

Affiliation: Affiliated Field of Medical Genetics, Division of Biomedical Engineering and Science, Graduate Course of Medicine, Graduate School of Tokyo Women's Medical University, Tokyo, Japan.

ABSTRACT
First-trimester chorionic-villi-derived cells (FTCVs) are the earliest fetal material that can be obtained for prenatal diagnosis of fetal disorders such as Duchenne muscular dystrophy (DMD). DMD is a devastating X-linked disorder characterized by the absence of dystrophin at the sarcolemma of muscle fibers. Currently, a limited number of treatment options are available for DMD, although cell therapy is a promising treatment strategy for muscle degeneration in DMD patients. A novel candidate source of cells for this approach is FTCVs taken between the 9th and 11th weeks of gestation. FTCVs might have a higher undifferentiated potential than any other tissue-derived cells because they are the earliest fetal material. We examined the expression of mesenchymal stem cell and pluripotent stem cell markers in FTCVs, in addition to their myogenic potential. FTCVs expressed mesenchymal stem cell markers and Nanog and Sox2 transcription factors as pluripotent stem cell markers. These cells efficiently differentiated into myotubes after myogenic induction, at which point Nanog and Sox2 were down-regulated, whereas MyoD, myogenin, desmin and dystrophin were up-regulated. To our knowledge, this is the first demonstration that FTCVs can be efficiently directed to differentiate in vitro into skeletal muscle cells that express dystrophin as the last stage marker of myogenic differentiation. The myogenic potential of FTCVs reveals their promise for use in cell therapy for DMD, for which no effective treatment presently exists.

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qPCR analysis of skeletal muscle markers in the FTCVs. Time course of MyoD (a), myogenin (b) and desmin (c) mRNA expression at the indicated day after treatment with 5-azacytidine. MyoD mRNA was detectable after day 3. Myogenin mRNA was undetectable at day 0. The earliest detectable value was set to 1 in each experiment
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Fig4: qPCR analysis of skeletal muscle markers in the FTCVs. Time course of MyoD (a), myogenin (b) and desmin (c) mRNA expression at the indicated day after treatment with 5-azacytidine. MyoD mRNA was detectable after day 3. Myogenin mRNA was undetectable at day 0. The earliest detectable value was set to 1 in each experiment

Mentions: To evaluate their myogenic potential, the differentiation of the FTCVs into a myogenic lineage was induced by incubation with 5-azacytidine in DMEM with 20% FBS for 24 h, after which they were cultured in DMEM supplemented with 2% horse serum for 21 days. We found that FTCV expression of both Nanog and Sox2 mRNA decreased markedly after 1 day of myogenic induction (Fig. 2g); indeed, the Nanog protein was also down-regulated after myogenic induction (Fig. 2d). During myogenic induction, the morphology of the FTCV population changed to multinucleated myotubes (Fig. 3). We evaluated the expression of the myogenic markers MyoD, myogenin, desmin and dystrophin in the FTCVs by qPCR analysis. These experiments revealed that myogenic differentiation of the FTCVs induced both MyoD and myogenin mRNA expression by day 3, although this expression decreased again by day 7. Moreover, FTCV expression of desmin mRNA was relatively high on day 1 of myogenic differentiation and subsequently decreased to a moderate level (Fig. 4). In addition, RT-PCR analysis clearly showed that dystrophin mRNA expression was increased from day 14 (Fig. 5a), as confirmed by qPCR analysis (Fig. 5b). Immunocytochemistry and Western blot analysis also confirmed that the dystrophin protein was detected in the FTCVs at day 21 (Fig. 6a, d). To evaluate the efficiency of myotube formation, FTCVs and TTCVs were fixed at day 0 and day 21 after plating and stained as described in Materials and methods with an antibody to the myosin heavy chain protein (MF20) in order to determine the fusion index. The results revealed that FTCV-9th cells with a fusion index of 57.3±11.1% had significantly higher myogenicity than FTCV-11th cells (20.9±6.4%) and TTCVs (9.9±1.4%) at day 21 (Fig. 7).Fig. 3


Human first-trimester chorionic villi have a myogenic potential.

Arakawa R, Aoki R, Arakawa M, Saito K - Cell Tissue Res. (2012)

qPCR analysis of skeletal muscle markers in the FTCVs. Time course of MyoD (a), myogenin (b) and desmin (c) mRNA expression at the indicated day after treatment with 5-azacytidine. MyoD mRNA was detectable after day 3. Myogenin mRNA was undetectable at day 0. The earliest detectable value was set to 1 in each experiment
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3316778&req=5

Fig4: qPCR analysis of skeletal muscle markers in the FTCVs. Time course of MyoD (a), myogenin (b) and desmin (c) mRNA expression at the indicated day after treatment with 5-azacytidine. MyoD mRNA was detectable after day 3. Myogenin mRNA was undetectable at day 0. The earliest detectable value was set to 1 in each experiment
Mentions: To evaluate their myogenic potential, the differentiation of the FTCVs into a myogenic lineage was induced by incubation with 5-azacytidine in DMEM with 20% FBS for 24 h, after which they were cultured in DMEM supplemented with 2% horse serum for 21 days. We found that FTCV expression of both Nanog and Sox2 mRNA decreased markedly after 1 day of myogenic induction (Fig. 2g); indeed, the Nanog protein was also down-regulated after myogenic induction (Fig. 2d). During myogenic induction, the morphology of the FTCV population changed to multinucleated myotubes (Fig. 3). We evaluated the expression of the myogenic markers MyoD, myogenin, desmin and dystrophin in the FTCVs by qPCR analysis. These experiments revealed that myogenic differentiation of the FTCVs induced both MyoD and myogenin mRNA expression by day 3, although this expression decreased again by day 7. Moreover, FTCV expression of desmin mRNA was relatively high on day 1 of myogenic differentiation and subsequently decreased to a moderate level (Fig. 4). In addition, RT-PCR analysis clearly showed that dystrophin mRNA expression was increased from day 14 (Fig. 5a), as confirmed by qPCR analysis (Fig. 5b). Immunocytochemistry and Western blot analysis also confirmed that the dystrophin protein was detected in the FTCVs at day 21 (Fig. 6a, d). To evaluate the efficiency of myotube formation, FTCVs and TTCVs were fixed at day 0 and day 21 after plating and stained as described in Materials and methods with an antibody to the myosin heavy chain protein (MF20) in order to determine the fusion index. The results revealed that FTCV-9th cells with a fusion index of 57.3±11.1% had significantly higher myogenicity than FTCV-11th cells (20.9±6.4%) and TTCVs (9.9±1.4%) at day 21 (Fig. 7).Fig. 3

Bottom Line: These cells efficiently differentiated into myotubes after myogenic induction, at which point Nanog and Sox2 were down-regulated, whereas MyoD, myogenin, desmin and dystrophin were up-regulated.To our knowledge, this is the first demonstration that FTCVs can be efficiently directed to differentiate in vitro into skeletal muscle cells that express dystrophin as the last stage marker of myogenic differentiation.The myogenic potential of FTCVs reveals their promise for use in cell therapy for DMD, for which no effective treatment presently exists.

View Article: PubMed Central - PubMed

Affiliation: Affiliated Field of Medical Genetics, Division of Biomedical Engineering and Science, Graduate Course of Medicine, Graduate School of Tokyo Women's Medical University, Tokyo, Japan.

ABSTRACT
First-trimester chorionic-villi-derived cells (FTCVs) are the earliest fetal material that can be obtained for prenatal diagnosis of fetal disorders such as Duchenne muscular dystrophy (DMD). DMD is a devastating X-linked disorder characterized by the absence of dystrophin at the sarcolemma of muscle fibers. Currently, a limited number of treatment options are available for DMD, although cell therapy is a promising treatment strategy for muscle degeneration in DMD patients. A novel candidate source of cells for this approach is FTCVs taken between the 9th and 11th weeks of gestation. FTCVs might have a higher undifferentiated potential than any other tissue-derived cells because they are the earliest fetal material. We examined the expression of mesenchymal stem cell and pluripotent stem cell markers in FTCVs, in addition to their myogenic potential. FTCVs expressed mesenchymal stem cell markers and Nanog and Sox2 transcription factors as pluripotent stem cell markers. These cells efficiently differentiated into myotubes after myogenic induction, at which point Nanog and Sox2 were down-regulated, whereas MyoD, myogenin, desmin and dystrophin were up-regulated. To our knowledge, this is the first demonstration that FTCVs can be efficiently directed to differentiate in vitro into skeletal muscle cells that express dystrophin as the last stage marker of myogenic differentiation. The myogenic potential of FTCVs reveals their promise for use in cell therapy for DMD, for which no effective treatment presently exists.

Show MeSH
Related in: MedlinePlus