DERIVATION AND CHARACTERISTICS OF THE NEW SUBLINE OF HUMAN EMBRYONIC STEM CELLS SC6-FF IN ALLOGENIC FEEDER-FREE CULTURE SYSTEM.

A new human embryonic stem cell subline SC6-FF has been derived from SC6 cells in allogenic feeder-free culture system. Extracellular matrix proteins and conditioned medium from mesenchymal stem cell line SC6-MSC were the key components of the feeder-free culture system that therefore was allogenic for SC6-FF cells. SC6-FF subline has underwent more than 100 cell population doublings and retained normal diploid human karyotype: 46, XX. The average doubling time of the cell population was 23.7 ± 0.8 h that does not differ from that for the parent SC6 line. The presence of undifferentiated hESCs markers, alkaline phosphatase activity, Oct-4, SSEA-4 and TRA-1-60, has been verified by histochemical and immunofluorescence analysis. Non-directional differentiation of SC6-FF subline has led to development of cells that differ in size and morphology from the cells in the parent population. These cells demonstrate the ability of differentiation in the derivates of three germ layers by expressing the characteristic markers of the ectoderm (alpha-fetoprotein), mesoderm (a-actinin) and endoderm (a-fetoprotein) cells. We can conclude that the obtained characteristics of the new feeder-free SC6-FF sub-line correspond to the status of the human embryonic stem cells.