In vitro culture conditions favoring selection of chromosomal abnormalities in human ES cells

Abstract

Previous studies in several laboratories have demonstrated inadvertent chromosomal abnormalities in long-term cultured human embryonic stem cells (HESC). Here, using a two-step selection process we report a functional adaptation of a HESC line, HS181, towards a decreased dependence of extra cellular matrix (ECM) for in vitro survival, that is for growth directly onto a plastic surface. Successful adaptation was paralleled with a karyotype change in 100% of the cells to 47,XX,del(7)(q11.2),+i(12)(p10). The resulting adapted population showed increased survival and growth on plastic and also maintained expression of HESC markers, but showed a decreased pluripotency, as demonstrated by results from embryoid body (EB) formation in vitro. The finding of reduced pluripotency may not be totally unexpected since the variant cells were selected for self-renewal and proliferation, not differentiation during the adaptation to growth on plastic. In the light of recent models of a germ cell origin of HESC it is of particular interest that similar to many of the reported spontaneous HESC mutants, one of the identified specific chromosome abnormalities, i(12p), has also been strongly implicated for human germ cell cancer. However, the mutated HESC variant carrying this mutation failed to grow as a xeno-graft in a mouse model in vivo. This is surprising and needs a further mechanistic analysis for its explanation. Increased knowledge of genetic integrity of HESC may have significance on the understanding of mechanisms for tumor progression and thus strategy for treatments, particularly for tumors occurring in early life.

Human Embryonic Stem Cells Are Capable of Executing G1/S Checkpoint Activation

“…Embryonic stem cells and other cells with stem cell features, such as induced pluripotent cells, must be extensively propagated in culture, which increases the risk of accumulating changes to their genomes [2,[18][19][20]. It is currently thought that this genomic instability is at least in part linked to ineffective G1 checkpoint pathways that normally guard somatic cells from such damage.…”

Concise Review: Induced Pluripotent Stem Cells Versus Embryonic Stem Cells: Close Enough or Yet Too Far Apart?

“…Mutations could bring certain advantages for the change of cell fate, thus representing a strong mutagenic factor. Subsequent proliferation and adaptation to the in vitro culture conditions is another important cause of mutations, although common for other cell lines too, including the ESC where gross mutations have already been noted [73][74][75].…”

“…Overall, even though iPSC obviously need to pass through one additional selection process, reprogramming, iPSC and ESC are not drastically different considering the mutational load. In addition, there is some correlation between mutations found in late passage human ESC, iPSC, and cancers cells [75][76][77]. Thus, pluripotent cells cultured in vitro are by definition proliferating and can acquire specific aberrations that support growth advantage, similar to tumor progression, that eventually take over the population.…”

Genome Stability in Embryonic Stem Cells

“…The reason why these chromosomes are more frequently involved is not clear, although it has been proposed that their alterations confer a selective and/or proliferative advantage to cells carring the mutations. The gain of part or of the entire chromosome 12 has been found in many hESC lines (i.e., BG01, BG02, BG03, H1, H7, H9, H14 and HS181) and observed in many indipendent laboratories (Brimble et al, 2001;Mitalipova et al, 2005;Ludwig et al, 2006;Imreh et al, 2006). The presence of an additional copy of chromosome 17 is another frequent abnormality found in hESCs (Brimble et al, 2001;Mitalipova et al, 2005).…”

“…Generally, ESCs are derived and maintained in vitro with a co-culture protocol on a feeder layer of mitotically inactivated fibroblast cells (mouse embryonic or immortalized fibroblasts) or on defined supportive matrixes (i.e., gelatin, fibronectin or matrigel TM ). Whether using the former or the latter, genetic alterations were observed both in mESC and hESC lines (Cowan et al, 2004;Rosler et al, 2004;Mitalipova et al, 2005;Maitra et al, 2005;Guo et al, 2005;Longo et al, 2005;Imreh et al, 2006;Sugawara et al, 2006;Rebuzzini et al, 2008a;Rebuzzini et al, 2008b), suggesting that the presence or absence of a supporting cellular feeder layer can not exclude the onset of aberrations in the ESCs genome. A fundamental component of the ESC medium is the serum of animal (calf or bovine) or artificial (knockout serum replacement of defined composition) origin.…”