Sunday, March 15, 2015

Stem Cells: Unstable in Culture

Stem Cells (SC) can differentiate into many different cell types, offering the potential of replacing failing cells, tissues, or even entire organs with new ones generated from the patient’s own or related donor SC (National Academy of Sciences Workshop summary).  To be useful for therapy in the clinic, it would be necessary to grow and expand SCs in culture.

The authors explored the proliferation of human embryonic SC (HESC), which are prepared from disrupted embryos, and the less-controversial human inducible pluripotent stem cells (hiPSCs), which can be prepared from several adult tissues, including blood, skin, and fat.  They obtained 1 HESC line, WA09, from the WiCell Research Institute and they generated 3 hiPSC lines from fetal dermal fibroblasts by over-expressing the ‘standard reprogramming factors’ (pluripotency-conferring genes, transduced OCT4/POU5F1, SOX2, KLF4, and MYC).

They compared four standard SC culture conditions: with or without a feeder cell layer and enzymatic or “mechanical” (dissection) disruption, with 6 replicate cultures per condition, for over 100 “passages” (transfers to fresh cultures). Previous studies cited here revealed genomic changes (small duplications) that are not detectable by karyotyping, particularly on chromosome 12, where the pluripotency-related gene NANOG is encoded, and chromosome 20, where the survival gene Bcl-xL is encoded.  In addition to measuring proliferation, telomere length, pluripotency by teratoma formation, they also analyzed over a million reference SNPs around the genome and used those SNPs to assess copy number variation (CNV). 

Not surprisingly, genomic changes increased with time in culture, both in aberration number (A) and total length (B) (Figure 2, shown, WA09 HESC: left duplications and right deletions).  The number of aberrations was lowest in “EcmMech” condition, i.e. cultures without feeder cells (only extracellular matrix, ECM), and disrupted mechanically (blue line).  The number and length of aberrations was worst with MefEnz (green line), cultured with feeder cells (mouse embryo fibroblasts, Mef) and disrupted enzymatically.  They conclude that there is a “need for careful assessment of the effects of culture conditions on cells intended for clinical therapies”.  

Increased Risk of Genetic and Epigenetic Instability in Human Embryonic Stem Cells Associated with Specific Culture Conditions  Garitaonandia et al. PLoS One 10(2), February 25, 2015

1 comment:

blue native sds page said...

great job in the stem cells culture researches. Indeed SC culture may be affected by many factors, such as endogenous regulation and exogenous regulation. And what you did is really helpful for my stem cells culture project.