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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

Engraftment of in vitro expanded mMPC engraft into injured muscle tissue. (A) Nude mice transplanted with 1×105 GFP+ mMPCs at passage 2, 5, and 10 (P2, P5, and P10, respectively), as described in Methods. Tibialis anterior (TA) muscles were harvested 4 weeks after transplantation and processed for fluorescence imaging (epifluorescence). Cross-section of the TA muscles showed large clusters of GFP+ myofibers with centrally localized nuclei (yellow box). Dashed red line (left lower panel) indicates the border between the epifluorescent signal of GFP+ myofibers and the autofluorescence of the adjacent host extensor digitorumlongus (EDL) muscles. White and yellow arrows indicated heterogeneous epifluorescence of GFP+ myofibers. Scale bar=100 μm. (B) Detection of GFP in engrafted muscle. GFP was detected in serial sections of the muscle tissue by epifluorescence (green, a) and staining with anti-GFP antibodies (brown staining, b), showing similar patterns of GFP signal. c. A chimeric myofiber contains host (blue nucleus, black arrow) and donor (brown nucleus, white arrow) nuclei, as determined by staining with anti-GFP antibodies. (C) Quantification of GFP+ myofibers. The number of GFP+ myofibers was determined in 100–200 serial sections of each TA muscle. GFP, green fluorescent protein.
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f4: Engraftment of in vitro expanded mMPC engraft into injured muscle tissue. (A) Nude mice transplanted with 1×105 GFP+ mMPCs at passage 2, 5, and 10 (P2, P5, and P10, respectively), as described in Methods. Tibialis anterior (TA) muscles were harvested 4 weeks after transplantation and processed for fluorescence imaging (epifluorescence). Cross-section of the TA muscles showed large clusters of GFP+ myofibers with centrally localized nuclei (yellow box). Dashed red line (left lower panel) indicates the border between the epifluorescent signal of GFP+ myofibers and the autofluorescence of the adjacent host extensor digitorumlongus (EDL) muscles. White and yellow arrows indicated heterogeneous epifluorescence of GFP+ myofibers. Scale bar=100 μm. (B) Detection of GFP in engrafted muscle. GFP was detected in serial sections of the muscle tissue by epifluorescence (green, a) and staining with anti-GFP antibodies (brown staining, b), showing similar patterns of GFP signal. c. A chimeric myofiber contains host (blue nucleus, black arrow) and donor (brown nucleus, white arrow) nuclei, as determined by staining with anti-GFP antibodies. (C) Quantification of GFP+ myofibers. The number of GFP+ myofibers was determined in 100–200 serial sections of each TA muscle. GFP, green fluorescent protein.

Mentions: To test the capacity of in vitro expanded mMPCs to engraft in an injured muscle, we performed cell transplantation experiments, using GFP-expressing mMPCs in a cardiotoxin muscle injury mouse model. Murine GFP+ MPCs at passages 2, 5, and 10 were injected 24 h after cardiotoxin injection, and the TA muscles were harvested for analyses 28 days later. Large clusters of donor-derived GFP+ myofibers, with centrally localized nuclei were observed (Fig. 4A, B). The GFP+ myofibers exhibited heterogeneity in fluorescence intensity (Fig. 4A, arrows), suggesting mosaic regenerating myofibers (Fig. 4Bc), possibly due to heterogeneous fusion of donor cells with host myofibers, consistent with previous findings.16


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)

Engraftment of in vitro expanded mMPC engraft into injured muscle tissue. (A) Nude mice transplanted with 1×105 GFP+ mMPCs at passage 2, 5, and 10 (P2, P5, and P10, respectively), as described in Methods. Tibialis anterior (TA) muscles were harvested 4 weeks after transplantation and processed for fluorescence imaging (epifluorescence). Cross-section of the TA muscles showed large clusters of GFP+ myofibers with centrally localized nuclei (yellow box). Dashed red line (left lower panel) indicates the border between the epifluorescent signal of GFP+ myofibers and the autofluorescence of the adjacent host extensor digitorumlongus (EDL) muscles. White and yellow arrows indicated heterogeneous epifluorescence of GFP+ myofibers. Scale bar=100 μm. (B) Detection of GFP in engrafted muscle. GFP was detected in serial sections of the muscle tissue by epifluorescence (green, a) and staining with anti-GFP antibodies (brown staining, b), showing similar patterns of GFP signal. c. A chimeric myofiber contains host (blue nucleus, black arrow) and donor (brown nucleus, white arrow) nuclei, as determined by staining with anti-GFP antibodies. (C) Quantification of GFP+ myofibers. The number of GFP+ myofibers was determined in 100–200 serial sections of each TA muscle. GFP, green fluorescent protein.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4048971&req=5

f4: Engraftment of in vitro expanded mMPC engraft into injured muscle tissue. (A) Nude mice transplanted with 1×105 GFP+ mMPCs at passage 2, 5, and 10 (P2, P5, and P10, respectively), as described in Methods. Tibialis anterior (TA) muscles were harvested 4 weeks after transplantation and processed for fluorescence imaging (epifluorescence). Cross-section of the TA muscles showed large clusters of GFP+ myofibers with centrally localized nuclei (yellow box). Dashed red line (left lower panel) indicates the border between the epifluorescent signal of GFP+ myofibers and the autofluorescence of the adjacent host extensor digitorumlongus (EDL) muscles. White and yellow arrows indicated heterogeneous epifluorescence of GFP+ myofibers. Scale bar=100 μm. (B) Detection of GFP in engrafted muscle. GFP was detected in serial sections of the muscle tissue by epifluorescence (green, a) and staining with anti-GFP antibodies (brown staining, b), showing similar patterns of GFP signal. c. A chimeric myofiber contains host (blue nucleus, black arrow) and donor (brown nucleus, white arrow) nuclei, as determined by staining with anti-GFP antibodies. (C) Quantification of GFP+ myofibers. The number of GFP+ myofibers was determined in 100–200 serial sections of each TA muscle. GFP, green fluorescent protein.
Mentions: To test the capacity of in vitro expanded mMPCs to engraft in an injured muscle, we performed cell transplantation experiments, using GFP-expressing mMPCs in a cardiotoxin muscle injury mouse model. Murine GFP+ MPCs at passages 2, 5, and 10 were injected 24 h after cardiotoxin injection, and the TA muscles were harvested for analyses 28 days later. Large clusters of donor-derived GFP+ myofibers, with centrally localized nuclei were observed (Fig. 4A, B). The GFP+ myofibers exhibited heterogeneity in fluorescence intensity (Fig. 4A, arrows), suggesting mosaic regenerating myofibers (Fig. 4Bc), possibly due to heterogeneous fusion of donor cells with host myofibers, consistent with previous findings.16

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