REVIEW pre Culture conditions for hESC

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Unformatted text preview: et al. (2005) have demonstrated the role of activin/Nodal/TGF-b pathway in the maintenance of undifferentiated stage of hESC. Very recent results have demonstrate that addition of bFGF, litidium chloride, g-aminobutyric acid (GABA), pipecolic acid, and TGF-b supplements into defined culture medium support undifferentiated growth of hESC (Ludwig et al. 2006), and that neurotrophins may behave as survival factors for hESC (Pyle et al. 2006). Although karyotype analyses have been made after several passages, real long-term studies are missing and the genetic and epigenetic stability of hESC in all these conditions has still to be confirmed. Feeder-free derivation of human embryonic stem cell lines Feeder-free derivation of hESC lines has been successful as described by Klimanskaya et al. (2005) using a mousederived matrix, and more recently by Ludwig et al. (2006) on Matrigel and human laminin. The problem Culture conditions for hESCs 695 with the later feeder-free derivation was that one of the lines had karyotype 47, XXY, which may have come from the embryo. On the contrary, the other line gained an extra chromosome at passage level 40. It has been suggested that feeder-free cultures may be so demanding for the hESC that they become more prone to abnormalities (Draper et al. 2004). The use of extracellular matrix eliminates the risk of transmitting animal pathogens with feeder cells to the hESC and decrease variability in culture conditions. Enzymes (especially trypsin) used in passaging may, on the other hand, push cells to too demanding culture conditions which may induce abnormalities (Draper et al. 2004). The next step towards entirely defined derivation and maintenance of hESC would be the combination of mechanical isolation of ICM, use of human feeder cells derived and cultured in defined medium, and use of defined culture medium in the hESC culture (Fig. 2). In addition to this, even better and more defined culture system with feeder-free conditions (Ludwig et al. 2006) is desirable to minimize the variability in culture conditions. In contrast, deriving and culturing hESC in such demanding conditions would need continuous controls of genetic and epigenetic normality of the cells. Defined culture methods To enable the use of hESC derivates for transplantation in humans, it would be crucial to eliminate the potential contamination by pathogens and animal proteins/substances from the hESC culture methods. Elimination of these variable factors is also important for constant and standardized culture conditions for research purposes. Human ESC for therapeutic as well as for research purposes have to be genetically and epigenetically normal. There is clear indication that hESC cultured in feeder-free conditions may gain chromosomal changes (Draper et al. 2004). Many reviewed studies have not detected any chromosomal abnormalities, but even though karyotypes have been analyzed after several passages, real long-term s...
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