REVIEW pre Culture conditions for hESC

Et al 2000 koivisto et al 2004 stojkovic et al 2004

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Unformatted text preview: et al. 2004, Strelchenko et al. 2004). During optimization of our hESC culture conditions by replacing FBS with serum replacement (SR) in hESC medium, we made the interesting observation that hESC were proliferating faster in SR-containing medium than they did in FBS-containing medium (Koivisto et al. 2004). To further study this, we Culture medium composition for human embryonic stem cells Traditionally, hESC culture medium contained FBS (Thomson et al. 1998, Reubinoff et al. 2000). It has, by most of the teams, been replaced by alternatives. The use of human serum instead of FBS in hESC culture medium has been reported (Richards et al. 2002). The problem is that FBS as well as human serum are complex mixtures containing unknown compounds, and serum batches vary in capability of maintaining hESC at an undifferentiated stage. Serum may also contain factors inducing hESC differentiation. www.reproduction-online.org Figure 1 A colony of human embryonic stem cell (hESC) line HS420 (passage 1) derived by mechanical isolation of inner cell mass. The hESC line was derived and grown on human foreskin fibroblasts in SR-containing medium. This line has been fully characterized. Magnification !100. Reproduction (2006) 132 691–698 694 H Skottman and O Hovatta made gene expression profiles using DNA microarrays to compare whole genome gene expression changes between hESC cultured in FBS- and SR-containing media (Skottman et al. 2005). Although the stem cells characteristics of these cells (the expression of many known hESC markers) and their differentiation capacity in embryoid bodies were similar, surprisingly, over 100 genes were found to be significantly differentially expressed when hESC cultured in serum-containing medium were compared with those cultured in SR medium. As a conclusion, we suggested that such changes may have fundamental importance for hESC. The gene expression changes should be monitored as a part of cell culture optimization aiming at establishment of robust and costeffective culture system of hESC. We have also established SR-containing derivation conditions for our recent hESC lines (Inzunza et al. 2005), but even though the use of SR in derivation and maintenance of hESC is a step forward it is not an entirely defined procedure. Feeder-free culture methods for human embryonic stem cells The contribution of feeder cells in hESC cultures is not entirely understood, but it has been suggested that feeder cells provide both a suitable attachment substrate and important soluble factors for the maintenance of undifferentiated hESC. However, such factors remain the most poorly defined component of hESC cultures. Feeder-free cultures would be free of many difficulties of the standardization and optimization procedures, and they would make analyses of the hESC properties easier. Substrate Feeder-free propagation of hESC has been feasible on Matrigel-coated plates and SR-containing conditioned medium (medium conditioned overnight incubation with mouse...
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This document was uploaded on 09/21/2013.

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