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  • Our xeno free culture conditions

    2018-11-12

    Our xeno-free culture conditions made single cell passaging possible, and we could also generate EBs from single cell suspensions. Although both hESC lines retained a normal karyotype, we did observe an accumulation of a chromosomal abnormality in one of our cultures. After 13 passages under xeno-free culture conditions 65% of the gli1 were normal, whereas 35% showed a trisomy of chromosome 12, which is known as a recurrent aberration in hESC associated with high proliferation rates (Draper et al., 2004; Spits et al., 2008). This clone also showed a low number of EBs formed which might be associated with the changed karyotype. As the parallel cultures exhibited a normal karoytype, this observation is probably a randomly appearing phenotype, which is not associated with the culture system itself. However, single cell passaging could contribute to promotion of chromosomal abnormalities, especially when leading to high proliferation rates. It has been shown before that CA1 and CA2 are able to form teratomas when injected into immune-deficient mice (Adewumi et al., 2007). This capacity is maintained after long-term culture under xeno-free culture conditions. In addition, in the present study we quantified the change in marker gene expression for ecto-, endo-, and mesodermal lineages in embryoid bodies by qPCR, to detect differences in differentiation capacities after long-term culture under xeno-free conditions. Despite the random fluctuation of spontaneous differentiation levels, lineage differentiation was similar in EBs derived from xeno-free hESC cultures compared to MEF/SR cultures. In particular, endo- and mesodermal markers were found to be very consistently expressed. The ectodermal markers were expressed similarly in hESC and EBs. Only Sox1 was absent in hESC and induced in EBs. We also formed neurospheres from single cell hESC in serum-free medium and could detect a strong up-regulation of Sox1 in both CA1 and CA2 with both XHEF sources proving the capacity of the cells to form neural progenitors (data not shown). We attempted to culture CA1 and CA2 in a feeder-free system gli1 using CELLstart from Invitrogen as a matrix and XHEF-conditioned HEScGRO medium, but cultures showed increasing levels of differentiation and poor proliferation and could not be maintained for more than 3 passages (data not shown). Recently, Meng et al. (2010) published a feeder-free system using matrix isolated from human foreskin fibroblasts and HEScGRO medium, where cells needed 10 passages of adaptation to the new conditions. This indicates that a combination of culture media, matrixes, and passaging methods is critical for hESC growth in xeno-free culture systems. Interestingly, CA1 and CA2 could be transferred to XHEF feeders without an adaption phase of mechanical passaging and differentiation was absent from these xeno-free cultures from the first passage. The lack of an adaption phase makes these fibroblasts valuable for derivation of hESC or under xeno-free conditions. Recently, one of these embryonic fibroblast cells (XHEFb) was used to derive iPS cells using piggyBac transposition, a safe method to generate patient-specific pluripotent stem cell lines (Woltjen et al., 2009; Kaji et al., 2009). Our xeno-free culture system will also allow the derivation of iPS cells under “safe” culture conditions, which reduce the risk of transmitting pathogens.
    Material and methods
    Acknowledgments
    Introduction Since human embryonic stem (hES) cells were first established by Thomson et al. in 1998, promising results have been obtained with these cells (Thomson et al., 1998). However, owing to ethical problems and concerns about clinical safety, hES cells have not yet been used in clinical studies. Nonhuman primates and their ES and induced pluripotent stem (iPS) cells are expected to be effective preclinical models given their close genetic relationships to humans, as compared with rodents (Hearn, 2001; Nakatsuji and Suemori, 2002). Sasaki and co-workers recently established common marmoset ES (cmES) and iPS cell lines, and green fluorescent protein (GFP)-transgenic marmosets (Sasaki et al., 2005; Sasaki et al., 2009; Tomioka et al., 2010), and described the efficient differentiation of neural cells from cmES cells (Sasaki et al., 2005). Chen et al. also reported successful differentiation of cardiomyocytes from cmES cells and described their characterization (Chen et al., 2008).