TY - JOUR
T1 - Amassive suspensionculturesystemwithmetabolic purification for human pluripotent stem cell-derived cardiomyocytes
AU - Hemmi, Natsuko
AU - Tohyama, Shugo
AU - Nakajima, Kazuaki
AU - Kanazawa, Hideaki
AU - Suzuki, Tomoyuki
AU - Hattori, Fumiyuki
AU - Seki, Tomohisa
AU - Kishino, Yoshikazu
AU - Hirano, Akinori
AU - Okada, Marina
AU - Tabei, Ryota
AU - Ohno, Rei
AU - Fujita, Chihana
AU - Haruna, Tomoko
AU - Yuasa, Shinsuke
AU - Sano, Motoaki
AU - Fujita, Jun
AU - Fukuda, Keiichi
N1 - Publisher Copyright:
©AlphaMed Press 2014.
PY - 2014
Y1 - 2014
N2 - Cardiac regenerative therapy with human pluripotent stem cells (hPSCs), such as human embryonic stem cells and induced pluripotent stem cells, has been hamperedbythe lackofefficient strategies for expanding functional cardiomyocytes (CMs) to clinically relevant numbers. The development of the massive suspension culture system (MSCS) has shed light on this critical issue, although it remains unclear how hPSCs could differentiate into functional CMs using a MSCS. The proliferative rate of differentiating hPSCs in the MSCS was equivalent to that in suspension cultures using nonadherent culture dishes, although the MSCS provided more homogeneous embryoid bodies (EBs), eventually reducing apoptosis. However, pluripotent markers such as Oct3/4 and Tra-1-60 were still expressed in EBs 2 weeks after differentiation, even in the MSCS. The remaining undifferentiated stem cells in such cultures could retain a strong potential for teratoma formation, which is the worst scenario for clinical applications of hPSC-derived CMs. The metabolic purification of CMs in glucose-depleted and lactate-enriched medium successfully eliminated the residual undifferentiated stem cells, resulting in a refined hPSC-derived CM population. In colony formation assays, no Tra-1-60-positive colonies appeared after purification. The nonpurified CMs in the MSCS produced teratomas at a rate of 60%. However,purified CMs never induced teratomas, and enriched CMs showed proper electrophysiological properties and calcium transients. Overall, the combination of a MSCS and metabolic selection is a highly effective and practical approach to purify and enrich massive numbers of functional CMs and provides an essential technique for cardiac regenerative therapy with hPSC-derived CMs.
AB - Cardiac regenerative therapy with human pluripotent stem cells (hPSCs), such as human embryonic stem cells and induced pluripotent stem cells, has been hamperedbythe lackofefficient strategies for expanding functional cardiomyocytes (CMs) to clinically relevant numbers. The development of the massive suspension culture system (MSCS) has shed light on this critical issue, although it remains unclear how hPSCs could differentiate into functional CMs using a MSCS. The proliferative rate of differentiating hPSCs in the MSCS was equivalent to that in suspension cultures using nonadherent culture dishes, although the MSCS provided more homogeneous embryoid bodies (EBs), eventually reducing apoptosis. However, pluripotent markers such as Oct3/4 and Tra-1-60 were still expressed in EBs 2 weeks after differentiation, even in the MSCS. The remaining undifferentiated stem cells in such cultures could retain a strong potential for teratoma formation, which is the worst scenario for clinical applications of hPSC-derived CMs. The metabolic purification of CMs in glucose-depleted and lactate-enriched medium successfully eliminated the residual undifferentiated stem cells, resulting in a refined hPSC-derived CM population. In colony formation assays, no Tra-1-60-positive colonies appeared after purification. The nonpurified CMs in the MSCS produced teratomas at a rate of 60%. However,purified CMs never induced teratomas, and enriched CMs showed proper electrophysiological properties and calcium transients. Overall, the combination of a MSCS and metabolic selection is a highly effective and practical approach to purify and enrich massive numbers of functional CMs and provides an essential technique for cardiac regenerative therapy with hPSC-derived CMs.
KW - Cardiac
KW - Cell culture
KW - Cellular therapy
KW - Differentiation
KW - Embryonic stem cells
KW - Induced pluripotent stem cells
KW - Stem cell transplantation
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U2 - 10.5966/sctm.2014-0072
DO - 10.5966/sctm.2014-0072
M3 - Article
C2 - 25355733
AN - SCOPUS:84913525381
SN - 2157-6564
VL - 3
SP - 1473
EP - 1483
JO - Stem Cells Translational Medicine
JF - Stem Cells Translational Medicine
IS - 12
ER -