Presence of a ROCK inhibitor in extracellular matrix supports more undifferentiated growth of feeder-free human embryonic and induced pluripotent stem cells upon passaging.
Optimization and development of better defined culture methods for human embryonic and induced pluripotent stem cells (hESCs and hiPSCs) will provide an invaluable contribution to the field of regenerative medicine. However, one problem is the vulnerability of hESCs and hiPSCs to apoptosis that causes a low plating efficiency upon passaging. Herein, we have developed a novel hESCs and hiPSCs culture technique that uses ROCK inhibitor (ROCKi) Y-27632 (10 microM) in Matrigel-coated dishes in both serum- and feeder-free culture conditions. This increases plating efficiency during enzymatic and mechanical passaging as compared to its presence solely in culture medium. Under these conditions, hESCs (three lines) and hiPSCs (two lines) retain their typical morphology, a stable karyotype, express pluripotency markers and have the potential to differentiate into derivatives of all three germ layers after long-term culture. Real-time RT-PCR analysis of stemness-related integrins (alphaV, alpha6, and beta1) has demonstrated that their expression increases in the presence of ROCKi. Similar plating efficiencies have been obtained in both hESCs and hiPSCs with a lower concentration of Y-27632 (800 nM) and another ROCKi (HA-1077/Fasudil), thus ruling out the non-specific effects of Y-27632. These results show that addition of ROCKi in the extracellular matrix can increase the plating efficiency of hESCs and hiPSCs during passaging of clusters. This is due not only to an anti-apoptotic effect, but also to an increase in the ECM-cells interaction. Therefore, we believe this method will be useful for both current and future applications of these pluripotent stem cells.
Pakzad
Mohammad
M
Department of Stem Cells and Developmental Biology, Royan Institute for Stem Cell Biology and Technology, ACECR, P.O. Box 19395-4644, Tehran, Iran.
Totonchi
Mehdi
M
Taei
Adeleh
A
Seifinejad
Ali
A
Hassani
Seyedeh Nafiseh
SN
Baharvand
Hossein
H
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives / Q000494:pharmacology; D000577:Amides / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D015415:Biomarkers / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects*; D048430:Cell Shape / Q000187:drug effects; D003094:Collagen / Q000378:metabolism*; D004337:Drug Carriers; D004338:Drug Combinations; D053595:Embryonic Stem Cells / Q000187:drug effects* / Q000201:enzymology; D005109:Extracellular Matrix / Q000378:metabolism*; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000187:drug effects* / Q000201:enzymology; D039503:Integrin alpha6 / Q000235:genetics; D039564:Integrin alphaV / Q000235:genetics; D019012:Integrin beta1 / Q000235:genetics; D007621:Karyotyping; D007797:Laminin / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011509:Proteoglycans / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism
D000577:Amides; D015415:Biomarkers; D004337:Drug Carriers; D004338:Drug Combinations; D039503:Integrin alpha6; D039564:Integrin alphaV; D019012:Integrin beta1; D007797:Laminin; D047428:Protein Kinase Inhibitors; D011509:Proteoglycans; D011725:Pyridines; D012333:RNA, Messenger; C059485:matrigel; C108830:Y 27632; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D003094:Collagen; D054460:rho-Associated Kinases; C049347:fasudil