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Human embryonic stem cell (hESС) lines represent a cell population that has unlimited replicative capacity and can differentiate into all somatic cells and germ line. These properties makes hESC lines an excellent tool for human cell and tissue replacement therapy. Traditionally, feeder cells are used to support the hESC growth by producing components of extracellular matrix and growth factors. The use of such culture conditions could lead to contamination of hESC by feeder cells or animal pathogenes and immunogens ( if xenogenic feeder cells are used), Moreover, such conditions are difficult to reproduce because of variations between different batches of feeders, so it is an unsuitable for the production of clinical grade cells. In our work a novel hESC subline SC6-FF was derived in allogenic feeder-free culture conditions from hESC line SC6, which was derived with using of human feeder cells. The used culture system consists of extracellular matrix proteins and conditioned medium, synthesized by feeder cells – mesenchymal stem cell line SC5-MSC, which was previously derived from initial hESC line SC5. The subline SC6-FF passed through more than 150 cell population doublins, retain normal diploid karyotype and ability of in vitro differentiation in the derivates of three germ layers. SC6-FF express the markers of undifferentiated hESC. The comparative analysis of some typical features does not reveal essential differences between initial SC6 line and subline SC6-FF.