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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

Comparison of cell culture condition for murine MPC (mMPC) expansion in vitro. (A) Myotube formation by MPCs cultured under five different conditions. (B) Quantification of myotube formation. The data are presented as the number of pixels corresponding to total myotube length per image, as described in Methods (n=4 cultures/condition). (C) Number of MPCs cultured under four different culture conditions, in passages 0–3 (n=4 individual cultures/condition). (D) Number of MPCs, cultured form whole limb muscle tissues of one mouse under condition II, in passages 0–10 (n=4 cultures). (E) Cell cycle analysis using flow cytometry for expanded MPCs at passages 10 (P10) and 25 (P25). (F) Morphology of in vitro expanded MPCs at low (P0) and high passage (P22). Arrows indicate spontaneous myotube formation. Scale bar=200 μm. Data are expressed as mean±SD. *p<0.05. ns, not significant; MPC, muscle precursor/progenitor cell; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum.
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f1: Comparison of cell culture condition for murine MPC (mMPC) expansion in vitro. (A) Myotube formation by MPCs cultured under five different conditions. (B) Quantification of myotube formation. The data are presented as the number of pixels corresponding to total myotube length per image, as described in Methods (n=4 cultures/condition). (C) Number of MPCs cultured under four different culture conditions, in passages 0–3 (n=4 individual cultures/condition). (D) Number of MPCs, cultured form whole limb muscle tissues of one mouse under condition II, in passages 0–10 (n=4 cultures). (E) Cell cycle analysis using flow cytometry for expanded MPCs at passages 10 (P10) and 25 (P25). (F) Morphology of in vitro expanded MPCs at low (P0) and high passage (P22). Arrows indicate spontaneous myotube formation. Scale bar=200 μm. Data are expressed as mean±SD. *p<0.05. ns, not significant; MPC, muscle precursor/progenitor cell; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum.

Mentions: The overarching hypothesis for this study is that specific combination of growth factors and adhesive proteins can preserve the myogenic capacity of MPCs during long-term cell culture expansion. We tested five different culture media and ECM coating (Fig. 1A, B) for supporting mMPC in vitro expansion. The culture conditions being tested were the following: Condition I: Myo medium+nondiluted Matrigel coated dishes; Condition II: Myo medium+1:200 diluted Matrigel coated dishes; Condition III: Myo medium+uncoated tissue culture dishes; Condition IV: DMEM+10% FBS+uncoated dishes; and Condition V: DMEM+10% FBS+1:200 diluted Matrigel-coated dishes. As a first screen, we examined the ability of the cultured mMPCs to fuse and form myotubes in vitro (Fig. 1A, B). All culture conditions supported mMPC (passage 0) fusion into myotubes, but cells grown in Myo medium on Matrigel (Conditions I and II) demonstrated significantly higher total length of myotubes (Fig. 1A, B), suggesting that the combination of Myo medium and Matrigel coating were beneficial for mMPC differentiation.


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)

Comparison of cell culture condition for murine MPC (mMPC) expansion in vitro. (A) Myotube formation by MPCs cultured under five different conditions. (B) Quantification of myotube formation. The data are presented as the number of pixels corresponding to total myotube length per image, as described in Methods (n=4 cultures/condition). (C) Number of MPCs cultured under four different culture conditions, in passages 0–3 (n=4 individual cultures/condition). (D) Number of MPCs, cultured form whole limb muscle tissues of one mouse under condition II, in passages 0–10 (n=4 cultures). (E) Cell cycle analysis using flow cytometry for expanded MPCs at passages 10 (P10) and 25 (P25). (F) Morphology of in vitro expanded MPCs at low (P0) and high passage (P22). Arrows indicate spontaneous myotube formation. Scale bar=200 μm. Data are expressed as mean±SD. *p<0.05. ns, not significant; MPC, muscle precursor/progenitor cell; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum.
© Copyright Policy
Related In: Results  -  Collection

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

f1: Comparison of cell culture condition for murine MPC (mMPC) expansion in vitro. (A) Myotube formation by MPCs cultured under five different conditions. (B) Quantification of myotube formation. The data are presented as the number of pixels corresponding to total myotube length per image, as described in Methods (n=4 cultures/condition). (C) Number of MPCs cultured under four different culture conditions, in passages 0–3 (n=4 individual cultures/condition). (D) Number of MPCs, cultured form whole limb muscle tissues of one mouse under condition II, in passages 0–10 (n=4 cultures). (E) Cell cycle analysis using flow cytometry for expanded MPCs at passages 10 (P10) and 25 (P25). (F) Morphology of in vitro expanded MPCs at low (P0) and high passage (P22). Arrows indicate spontaneous myotube formation. Scale bar=200 μm. Data are expressed as mean±SD. *p<0.05. ns, not significant; MPC, muscle precursor/progenitor cell; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum.
Mentions: The overarching hypothesis for this study is that specific combination of growth factors and adhesive proteins can preserve the myogenic capacity of MPCs during long-term cell culture expansion. We tested five different culture media and ECM coating (Fig. 1A, B) for supporting mMPC in vitro expansion. The culture conditions being tested were the following: Condition I: Myo medium+nondiluted Matrigel coated dishes; Condition II: Myo medium+1:200 diluted Matrigel coated dishes; Condition III: Myo medium+uncoated tissue culture dishes; Condition IV: DMEM+10% FBS+uncoated dishes; and Condition V: DMEM+10% FBS+1:200 diluted Matrigel-coated dishes. As a first screen, we examined the ability of the cultured mMPCs to fuse and form myotubes in vitro (Fig. 1A, B). All culture conditions supported mMPC (passage 0) fusion into myotubes, but cells grown in Myo medium on Matrigel (Conditions I and II) demonstrated significantly higher total length of myotubes (Fig. 1A, B), suggesting that the combination of Myo medium and Matrigel coating were beneficial for mMPC differentiation.

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