Limits...
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

Long-term in vitro expanded mMPC express muscle stem cell markers. (A) Pax7 and Pax3 expression in expanded MPCs at passage 0 (P0) and 25 (P25). Cells were stained with antibodies against Pax7 and Pax3 (red) and cell nuclei were stained with DAPI (blue). Scale bar=100 μm. (B) Quantification of Pax7 expression (as shown in A) in passages 0–4 (P0–P4) and 20–25 (P20–P25) of MPCs. Results are presented as the frequency of Pax7-positive cells (mean±SD). (C) Myf-5, MyoD, and Myogenin expression in expanded MPCs at P0 and P25. Cells were stained with antibody against Myf5, MyoD, and Myogenin (red) and cell nuclei were stained with DAPI (blue). Scale bar=100 μm. (D) Quantification of Myf-5, MyoD, and Myogenin expression in P0 and P25 of MPCs (as shown in C). Results are presented as the frequency of Myf-5-, MyoD-, and Myogenin-positive cells (mean±SD). DAPI, 4′,6-diamidino-2-phenylindole.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4048971&req=5

f2: Long-term in vitro expanded mMPC express muscle stem cell markers. (A) Pax7 and Pax3 expression in expanded MPCs at passage 0 (P0) and 25 (P25). Cells were stained with antibodies against Pax7 and Pax3 (red) and cell nuclei were stained with DAPI (blue). Scale bar=100 μm. (B) Quantification of Pax7 expression (as shown in A) in passages 0–4 (P0–P4) and 20–25 (P20–P25) of MPCs. Results are presented as the frequency of Pax7-positive cells (mean±SD). (C) Myf-5, MyoD, and Myogenin expression in expanded MPCs at P0 and P25. Cells were stained with antibody against Myf5, MyoD, and Myogenin (red) and cell nuclei were stained with DAPI (blue). Scale bar=100 μm. (D) Quantification of Myf-5, MyoD, and Myogenin expression in P0 and P25 of MPCs (as shown in C). Results are presented as the frequency of Myf-5-, MyoD-, and Myogenin-positive cells (mean±SD). DAPI, 4′,6-diamidino-2-phenylindole.

Mentions: Pax3 and Pax7 are key transcription factors regulating skeletal muscle development26,27 and regeneration.28 To evaluate whether our culture method is capable of maintaining these myogenic markers during extensive in vitro expansion, we measured the expression of Pax7 and Pax3 in passages 0 to 4 (P0–P4) and 20 to 25 (P20–25) of mMPCs. Typical images, as shown in Fig. 2A indicates the presence of Pax7+ and Pax3+ mMPCs at passages 0 and 25. Figure 2B shows the quantification of Pax7+ mMPCs at both low and high passages and determined that 40%–60% of cultured mMPCs were positive for Pax7 expression, indicating that our culture conditions preserved the myogenic properties of mMPCs. In contrast, under the other culture conditions (Conditions III, IV, and V) no more than 10% of cultured mMPCs expressed Pax7 (Supplementary Fig. S2A, B) at passage 0. As a comparison, we also examined Pax7 and Pax3 expression in several cultures of hMPCs that were cultured in growth factor contained medium in collage I–coated tissue culture dishes, as indicated in methods part. Among nine samples tested, only one sample showed Pax3 expression and four showed Pax7 expression. In the hMPC culture in which we observed Pax3 and Pax7 expression, only 3.5% and 4.3% of the cells were positive for Pax3 and Pax7, respectively, suggesting the loss of myogenic “stemness” under nonoptimal culture conditions (Supplementary Fig. S3A, B).


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)

Long-term in vitro expanded mMPC express muscle stem cell markers. (A) Pax7 and Pax3 expression in expanded MPCs at passage 0 (P0) and 25 (P25). Cells were stained with antibodies against Pax7 and Pax3 (red) and cell nuclei were stained with DAPI (blue). Scale bar=100 μm. (B) Quantification of Pax7 expression (as shown in A) in passages 0–4 (P0–P4) and 20–25 (P20–P25) of MPCs. Results are presented as the frequency of Pax7-positive cells (mean±SD). (C) Myf-5, MyoD, and Myogenin expression in expanded MPCs at P0 and P25. Cells were stained with antibody against Myf5, MyoD, and Myogenin (red) and cell nuclei were stained with DAPI (blue). Scale bar=100 μm. (D) Quantification of Myf-5, MyoD, and Myogenin expression in P0 and P25 of MPCs (as shown in C). Results are presented as the frequency of Myf-5-, MyoD-, and Myogenin-positive cells (mean±SD). DAPI, 4′,6-diamidino-2-phenylindole.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4048971&req=5

f2: Long-term in vitro expanded mMPC express muscle stem cell markers. (A) Pax7 and Pax3 expression in expanded MPCs at passage 0 (P0) and 25 (P25). Cells were stained with antibodies against Pax7 and Pax3 (red) and cell nuclei were stained with DAPI (blue). Scale bar=100 μm. (B) Quantification of Pax7 expression (as shown in A) in passages 0–4 (P0–P4) and 20–25 (P20–P25) of MPCs. Results are presented as the frequency of Pax7-positive cells (mean±SD). (C) Myf-5, MyoD, and Myogenin expression in expanded MPCs at P0 and P25. Cells were stained with antibody against Myf5, MyoD, and Myogenin (red) and cell nuclei were stained with DAPI (blue). Scale bar=100 μm. (D) Quantification of Myf-5, MyoD, and Myogenin expression in P0 and P25 of MPCs (as shown in C). Results are presented as the frequency of Myf-5-, MyoD-, and Myogenin-positive cells (mean±SD). DAPI, 4′,6-diamidino-2-phenylindole.
Mentions: Pax3 and Pax7 are key transcription factors regulating skeletal muscle development26,27 and regeneration.28 To evaluate whether our culture method is capable of maintaining these myogenic markers during extensive in vitro expansion, we measured the expression of Pax7 and Pax3 in passages 0 to 4 (P0–P4) and 20 to 25 (P20–25) of mMPCs. Typical images, as shown in Fig. 2A indicates the presence of Pax7+ and Pax3+ mMPCs at passages 0 and 25. Figure 2B shows the quantification of Pax7+ mMPCs at both low and high passages and determined that 40%–60% of cultured mMPCs were positive for Pax7 expression, indicating that our culture conditions preserved the myogenic properties of mMPCs. In contrast, under the other culture conditions (Conditions III, IV, and V) no more than 10% of cultured mMPCs expressed Pax7 (Supplementary Fig. S2A, B) at passage 0. As a comparison, we also examined Pax7 and Pax3 expression in several cultures of hMPCs that were cultured in growth factor contained medium in collage I–coated tissue culture dishes, as indicated in methods part. Among nine samples tested, only one sample showed Pax3 expression and four showed Pax7 expression. In the hMPC culture in which we observed Pax3 and Pax7 expression, only 3.5% and 4.3% of the cells were positive for Pax3 and Pax7, respectively, suggesting the loss of myogenic “stemness” under nonoptimal culture conditions (Supplementary Fig. S3A, B).

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