Establishment and characteristics of new human embryonic stem cell subline SC6-FF in an allogenic feeder-free culture system

Abstract

The novel human embryonic stem cell (hESC) subline SC6-FF was derived from SC6 cells in an allogenic feeder-free culture system. Key components of the feeder-free culture system were extracellular matrix proteins and conditioned medium from the mesenchymal stem cell line SC5-MSC. These conditions are allogenic for SC6-FF cells. SC6-FF subline underwent more than one hundred cell population doublings and retained a normal diploid karyotype; 46, XX. The average population doubling time was 23.7 ± 0.8 h, similar to that of the parent SC6 line. The presence of undifferentiated hESC markers (alkaline phosphatase activity, Oct-4, SSEA-4, and TRA-1-60) was verified by histochemistry and immunofluorescence. Cells were distinguished from parental cells in size and morphology as a result of spontaneous differentiation. These cells exhibited the ability to differentiate into derivates of three germ layers by expressing common markers of the ectoderm (alpha-fetoprotein), mesoderm (a-actinin) and endoderm (a-fetoprotein) cells. We could conclude that characteristics of the novel feeder-free SC6-FF subline correspond to the status of human embryonic stem cells.