Genomic instability in human embryonic stem cells is associated to culture density and medium acidification.

Human embryonic stem cells (hESC) show great promise for clinical and research applications, but genomic instability hampers the development of their full potential. Here we demonstrate that increased culture density causes medium acidification due to lactic acid accumulation. hESC are able to cope with this, but acquire increased DNA damage associated to DNA replication stress. Also, single-cell genomics analysis reveals a strong correlation of the occurrence of de novo CNVs and culture density after only five days of culture. However, by an improved control over the accumulation of metabolites through more frequent medium changes, we were able to counter the effect of culture density on DNA damage and CNVs. These data underline the importance of optimal culture conditions, even at short-term, in the light of bringing hESC to their full potential. We isolated 29 single hESC from the lowest density group (A) and 30 of the highest-density group (D) and compared CNV occurence at a resolution of 7 consecutive clones.