DEVELOPING OF A NEW FEEDER-FREE SYSTEM AND CHARACTERISATION OF HUMAN EMBRYONIC STEM CELL SUBLINES,
DERIVED IN THIS SYSTEM, UNDER AUTOGENIC AND ALLOGENIC CULTURING
A.M. Koltsova,1 I.V. Voronkina,1 O.F. Gordeeva,2 V.V. Zenin,1 N.V. Lifantseva,2
A.S. Musorina,1 L.V. Smagina,1 T.K. Yakovleva,1 G.G. Poljanskaya 1,*
1 Institute of Cytology, Russian Academy of Sciences, St. Petersburg,
2 Kol'tsov Institute of Developmental Biology, Russian Academy of Sciences, Moscow;
* e-mail: poljansk@mail.cytspb.rssi.ru
A new feeder-free culture system for human embryonic stem cells (hESC) was developed. It consist of extracellular matrix proteins synthesized by feeder cells – mesenchymal
stem cell line SC5-MSC, which was derived from initial hESC line SC5. The major ECM proteins – fibronectin and laminin - that maintain hESC growth in feeder-free system were
identified. An essential component of this system is a SC5-MSC-conditioned medium. Two hESC sublines were derived. The subline SC5-FF was cultured in autogenic and subline
SC7-FF in allogenic system. Sublines SC5-FF and SC7-FF passed through more than 300 and 115 cell population doublings, retained normal diploid karyotype and an ability of
in vitro differentiation into derivates of three germ layers. These sublines express markers of undifferentiated hESC: alkaline phosphatasa, Oct-4, SSEA-4, TRA-1-81 and
multidrug resistance transporter – ABCG2. The RT-PCR analysis revealed that undifferentiated cells SC5-FF subline, like cells of initial feeder-maintained hESC line SC5, expressed
genes OCT4 and NANOG, and germ line specific genes such as DPPA3/STELLA and DAZL. An expression of OCT4, NANOG, DPPA3/STELLA
and DAZL was down-regulated during embryonic bodies differentiation, whereas expression of somatic lineages specific genes like GATA4 and AFP
(extra embryonic and embryonic endoderm), PAX6 (neuroectoderm) and BRY (mesoderm) was up-regulated. The comparative analysis of some typical features
(karyotype structure, the average population doubling time and the number of undifferentiated cells in populations) did not reveal essential differences between initial SC5 and SC7 lines
and their sublines SC5-FF and SC7-FF. This shows that feeder-free culture systems, which are much more stable than any feeder systems, do not break main hESC features during
long cultivation and can be recommended for fundamental, biomedicine and pharmacological investigations, using hESCs.
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