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Late-Adhering Human Embryonic Stem Cell Clumps During Serial Passage Can Yield Morphologically ‘Normal-Looking’ Colonies

Address correspondence to Dr. Tong Cao, Stem Cell Laboratory, Faculty of Dentistry, National University of Singapore, 5 Lower Kent Ridge Road, Singapore 119074; tel 65 6874 4630, ext 1612; fax 65 6774 5701; e-mail dencaot{at}nus.edu.sg.

During serial passage of human embryonic stem (hES) cells, freshly-seeded hES cell clumps that do not adhere to the newly-plated murine embryonic fibroblast (MEF) feeder layer after culturing overnight, are routinely discarded with the changing of fresh culture media. Preliminary observations revealed that non-adhered hES cell clumps that were still viable possessed a distinct, rounded morphology with ‘sharp,’ clearly-defined edges. Typically >90% of ‘viable’ hES cell clumps with such morphology adhered after a further 24 hr of culture on newly-plated MEF. The remaining non-adhered cell clumps that had ill-defined jagged edges could represent either non-viable hES cells or senescent MEF carried over from the previous sub-culture. Virtually none of these adhered to the newly-plated MEF feeder layer. The overwhelming majority of ‘viable,’ late-adhering hES cell clumps (typically >90%) gave rise to morphologically ‘normal-looking’ hES colonies, which appeared to remain undifferentiated. Additionally, hES cell clumps that were attached together with the majority of the passaged colonies, but were somewhat delayed in the adhesion process, compared to neighbouring colonies, also gave rise to morphologically ‘normal-looking’ undifferentiated colonies that were virtually indistinguishable from neighbouring colonies. Hence, it may be useful to retain late-adhering hES cell clumps during serial passage of hES cells, rather than discarding them.

Keywords: human, embryonic, stem cells, passage, in vitro