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An in vitro culture system that supports robust expansion and maintenance of in vivo engraftment capabilities for myogenic progenitor cells from adult mice.

Wang Z, Cheung D, Zhou Y, Han C, Fennelly C, Criswell T, Soker S - Biores Open Access (2014)

Bottom Line: Long term in vitro expanded mMPC expressed the myogenic stem cell markers Pax3 and Pax7 and formed spontaneously contracting myotubes.Furthermore, expanded mMPC injected into the tibialis anterior muscle of nude mice engrafted and formed myofibers.Collectively, the method developed in this study can be potentially adapted for the expansion of human MPCs to high enough numbers for treatment of muscle injuries in human patients.

View Article: PubMed Central - PubMed

Affiliation: Wake Forest Institute for Regenerative Medicine , Winston-Salem, North Carolina.

ABSTRACT
Muscle cell therapy and tissue engineering require large numbers of functional muscle precursor/progenitor cells (MPCs), making the in vitro expansion of MPCs a critical step for these applications. The cells must maintain their myogenic properties upon robust expansion, especially for cellular therapy applications, in order to achieve efficacious treatment. A major obstacle associated with MPCs expansion is the loss of "stemness," or regenerative capacity, of freshly isolated cells, presumably due to the absence of the native cellular niches. In the current study, we developed an in vitro system that allowed for long-term culture and massive expansion of murine MPCs (mMPCs) with the preservation of myogenic regeneration capabilities. Long term in vitro expanded mMPC expressed the myogenic stem cell markers Pax3 and Pax7 and formed spontaneously contracting myotubes. Furthermore, expanded mMPC injected into the tibialis anterior muscle of nude mice engrafted and formed myofibers. Collectively, the method developed in this study can be potentially adapted for the expansion of human MPCs to high enough numbers for treatment of muscle injuries in human patients.

No MeSH data available.


Related in: MedlinePlus

Myotube formation in expanded mMPCs. (A) In vitro myotube formation in passages 1 (P1), 10 (P10), and 25 (P25) of MPCs. Images were taken at day 7 after cell seeding. Scale bar=100 μm. (B) Desmin expression in the myotubes was assed using anti-desmin antibodies (red) and cell nuclei were stained with DAPI (blue). Right panel shows a representative image of a single myotube showing striations. Scale bar=100 μm. (C) Quantification of myotubes at multiple passages (P1–P11 and P25). Results are presented as the percentage of myotubes with ≥5 nuclei/myotube (mean±SD).
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f3: Myotube formation in expanded mMPCs. (A) In vitro myotube formation in passages 1 (P1), 10 (P10), and 25 (P25) of MPCs. Images were taken at day 7 after cell seeding. Scale bar=100 μm. (B) Desmin expression in the myotubes was assed using anti-desmin antibodies (red) and cell nuclei were stained with DAPI (blue). Right panel shows a representative image of a single myotube showing striations. Scale bar=100 μm. (C) Quantification of myotubes at multiple passages (P1–P11 and P25). Results are presented as the percentage of myotubes with ≥5 nuclei/myotube (mean±SD).

Mentions: Myotube formation, as determined by the presence of multinucleated myotubes, is a measure to determine mMPCs differentiation capacity and can also be used to determine myotube maturity. In vitro expanded mMPCs showed cell fusion and myotube formation for up to passage 25 (Fig. 3A). The myotubes exhibited a striated appearance, suggesting proper sarcomeric organization, and expressed Desmin, a sarcomeric intermediate filament protein (Fig 3B). We subsequently quantified the myotube forming capacities at different passages. About 60%–80% of multinucleated myotubes had five or more nuclei per myotube, consistently from passage 1 to 11, and in passage 25 (Fig 3C), implying the maintenance of in vitro myogenic potential during long-term cell expansion. Furthermore, under the optimized culture conditions (Condition II), we were able to observe spontaneous contraction of the myotubes (Supplementary Movies A–C).


An in vitro culture system that supports robust expansion and maintenance of in vivo engraftment capabilities for myogenic progenitor cells from adult mice.

Wang Z, Cheung D, Zhou Y, Han C, Fennelly C, Criswell T, Soker S - Biores Open Access (2014)

Myotube formation in expanded mMPCs. (A) In vitro myotube formation in passages 1 (P1), 10 (P10), and 25 (P25) of MPCs. Images were taken at day 7 after cell seeding. Scale bar=100 μm. (B) Desmin expression in the myotubes was assed using anti-desmin antibodies (red) and cell nuclei were stained with DAPI (blue). Right panel shows a representative image of a single myotube showing striations. Scale bar=100 μm. (C) Quantification of myotubes at multiple passages (P1–P11 and P25). Results are presented as the percentage of myotubes with ≥5 nuclei/myotube (mean±SD).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4048971&req=5

f3: Myotube formation in expanded mMPCs. (A) In vitro myotube formation in passages 1 (P1), 10 (P10), and 25 (P25) of MPCs. Images were taken at day 7 after cell seeding. Scale bar=100 μm. (B) Desmin expression in the myotubes was assed using anti-desmin antibodies (red) and cell nuclei were stained with DAPI (blue). Right panel shows a representative image of a single myotube showing striations. Scale bar=100 μm. (C) Quantification of myotubes at multiple passages (P1–P11 and P25). Results are presented as the percentage of myotubes with ≥5 nuclei/myotube (mean±SD).
Mentions: Myotube formation, as determined by the presence of multinucleated myotubes, is a measure to determine mMPCs differentiation capacity and can also be used to determine myotube maturity. In vitro expanded mMPCs showed cell fusion and myotube formation for up to passage 25 (Fig. 3A). The myotubes exhibited a striated appearance, suggesting proper sarcomeric organization, and expressed Desmin, a sarcomeric intermediate filament protein (Fig 3B). We subsequently quantified the myotube forming capacities at different passages. About 60%–80% of multinucleated myotubes had five or more nuclei per myotube, consistently from passage 1 to 11, and in passage 25 (Fig 3C), implying the maintenance of in vitro myogenic potential during long-term cell expansion. Furthermore, under the optimized culture conditions (Condition II), we were able to observe spontaneous contraction of the myotubes (Supplementary Movies A–C).

Bottom Line: Long term in vitro expanded mMPC expressed the myogenic stem cell markers Pax3 and Pax7 and formed spontaneously contracting myotubes.Furthermore, expanded mMPC injected into the tibialis anterior muscle of nude mice engrafted and formed myofibers.Collectively, the method developed in this study can be potentially adapted for the expansion of human MPCs to high enough numbers for treatment of muscle injuries in human patients.

View Article: PubMed Central - PubMed

Affiliation: Wake Forest Institute for Regenerative Medicine , Winston-Salem, North Carolina.

ABSTRACT
Muscle cell therapy and tissue engineering require large numbers of functional muscle precursor/progenitor cells (MPCs), making the in vitro expansion of MPCs a critical step for these applications. The cells must maintain their myogenic properties upon robust expansion, especially for cellular therapy applications, in order to achieve efficacious treatment. A major obstacle associated with MPCs expansion is the loss of "stemness," or regenerative capacity, of freshly isolated cells, presumably due to the absence of the native cellular niches. In the current study, we developed an in vitro system that allowed for long-term culture and massive expansion of murine MPCs (mMPCs) with the preservation of myogenic regeneration capabilities. Long term in vitro expanded mMPC expressed the myogenic stem cell markers Pax3 and Pax7 and formed spontaneously contracting myotubes. Furthermore, expanded mMPC injected into the tibialis anterior muscle of nude mice engrafted and formed myofibers. Collectively, the method developed in this study can be potentially adapted for the expansion of human MPCs to high enough numbers for treatment of muscle injuries in human patients.

No MeSH data available.


Related in: MedlinePlus