title: Up-scaling single cell-inoculated suspension culture of human embryonic stem cells.
abstract: We have systematically developed single cell-inoculated suspension cultures of human embryonic stem cells (hESC) in defined media. Cell survival was dependent on hESC re-aggregation. In the presence of the Rho kinase inhibitor Y-27632 (Ri) only approximately 44% of the seeded cells were rescued, but an optimized heat shock treatment combined with Ri significantly increased cell survival to approximately 60%. Mechanistically, our data suggest that E-cadherin plays a role in hESC aggregation and that dissociation and re-aggregation upon passaging functions as a purification step towards a pluripotency markers-enriched population. Mass expansion of hESC was readily achieved by up-scaling 2 ml cultures to serial passaging in 50 ml spinner flasks. A media comparison revealed that mTeSR was superior to KnockOut-SR in supporting cell proliferation and pluripotency. Persistent expression of pluripotency markers was achieved for two lines (hES2, hES3) that were used at higher passages (>86). In contrast, rapid down regulation of Oct4, Tra-1-60, and SSEA4 was observed for ESI049, a clinically compliant line, used at passages 20-36. The up-scaling strategy has significant potential to provide pluripotent cells on a clinical scale. Nevertheless, our data also highlights a significant line-to-line variability and the need for a critical assessment of novel methods with numerous relevant cell lines.
authors.affiliation: Institute of Medical Biology (IMB), Immunos, 138648 Singapore. harmeet.singh@imb.a-star.edu.sg
authors.forename: Harmeet
authors.initials: H
authors.lastname: Singh
authors.forename: Pamela
authors.initials: P
authors.lastname: Mok
authors.forename: Thavamalar
authors.initials: T
authors.lastname: Balakrishnan
authors.forename: Siti Norfiza Binte
authors.initials: SN
authors.lastname: Rahmat
authors.forename: Robert
authors.initials: R
authors.lastname: Zweigerdt
mesh_terms: D000577:Amides / Q000494:pharmacology; D000954:Antigens, Surface / Q000378:metabolism; D015820:Cadherins / Q000494:pharmacology; D018929:Cell Culture Techniques; D049109:Cell Proliferation; D053595:Embryonic Stem Cells / Q000166:cytology*; D006801:Humans; D050814:Octamer Transcription Factor-3 / Q000378:metabolism; D011509:Proteoglycans / Q000378:metabolism; D011725:Pyridines / Q000494:pharmacology; D055746:Stage-Specific Embryonic Antigens / Q000378:metabolism; D013696:Temperature; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D000577:Amides; D000954:Antigens, Surface; D015820:Cadherins; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D011509:Proteoglycans; D011725:Pyridines; D055746:Stage-Specific Embryonic Antigens; C107064:TRA-1-60 antigen, human; C066584:stage-specific embryonic antigen-4; C108830:Y 27632; D054460:rho-Associated Kinases