A three-dimensional gel bioreactor for assessment of cardiomyocyte induction in skeletal muscle-derived stem cells.
Skeletal muscle-derived stem cells (MDSCs) are able to differentiate into cardiomyocytes (CMs). However, it remains to be investigated whether differentiated CMs contract similar to native CMs. Here, we developed a three-dimensional collagen gel bioreactor (3DGB) that induces a working CM phenotype from MDSCs, and the contractile properties are directly measured as an engineered cardiac tissue. Neonate rat MDSCs were isolated from hind-leg muscles via the preplate technique. Isolated MDSCs were approximately 60% positive to Sca-1 and negative to CD34, CD45, or c-kit antigens. We sorted Sca-1(-) MDSCs and constructed MDSC-3DGBs by mixing MDSCs with acid soluble rat tail collagen type-I and matrix factors. MDSC-3DGB exhibited spontaneous cyclic contraction by culture day 7. MDSC-3DGB expressed cardiac-specific genes and proteins. Histological assessment revealed that cardiac-specific troponin-T and -I expressed in a typical striation pattern and connexin-43 was expressed similar to the native fetal ventricular papillary muscle. beta-Adrenergic stimulation increased MDSC-3DGB spontaneous beat frequency. MDSC-3DGB generated contractile force and intracellular calcium ion transients similar to engineered cardiac tissue from native cardiac cells. Results suggest that MDSC-3DGB induces a working CM phenotype in MDSCs and is a useful 3D culture system to directly assess the contractile properties of differentiated CMs in vitro.
Clause
Kelly C
KC
Children's Hospital of Pittsburgh of UPMC, Pittsburgh, PA, USA.
Tinney
Joseph P
JP
Liu
Li J
LJ
Gharaibeh
Burhan
B
Huard
Johnny
J
Kirk
Jonathan A
JA
Shroff
Sanjeev G
SG
Fujimoto
Kazuro L
KL
Wagner
William R
WR
Ralphe
John C
JC
Keller
Bradley B
BB
Tobita
Kimimasa
K
D000818:Animals; D000831:Animals, Newborn; D001483:Base Sequence; D019149:Bioreactors; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002454:Cell Differentiation; D002478:Cells, Cultured; D003094:Collagen; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D018482:Muscle, Skeletal / Q000166:cytology* / Q000378:metabolism; D009206:Myocardium / Q000166:cytology* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D051381:Rats; D011917:Rats, Inbred Lew; D013234:Stem Cells / Q000166:cytology*
D017931:DNA Primers; D003094:Collagen; D002118:Calcium