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Reprogramming: The Prize for the Best Pluripotent Stem Cell Goes To………

Review of “Abnormalities in human pluripotent cells due to reprogramming mechanisms” from Nature by Stuart P. Atkinson

Induced pluripotent stem cell (iPSC) technology aims to provide patient-specific stem cells to be used in the replacement of cells and tissues in human diseases and disorders. However, high levels of genetic and epigenetic abnormalities have been observed in iPSCs, and it is not fully understood if these errors arise during the reprogramming process or if they represent an incomplete reprogramming stage. Now in a gargantuan effort, researchers from the laboratories of Joseph R. Ecker, Louise C. Laurent, and Shoukhrat Mitalipov have subjected genetically matched sets of human in vitro fertilization-derived embryonic stem cells (IVF ESCs), iPSCs and nuclear transfer ESCs (NT ESCs) to genome-wide analyses and report these findings in Nature [1].

The group investigated 4 different types of pluripotent cells:

  • four NT ESC lines derived from fetal human dermal fibroblasts (HDFs) - NT1–NT4 [2]
  • two iPSC lines from the same HDFs using retroviral vectors - iPS-R1 and iPS-R2 [3]
  • five iPSC lines from the same HDFs using Sendai-virus-based vectors - iPS-S1-5 [4]
  • two IVF ESC lines (human ES Oregon (hESO)-7 and hESO-8) derived using IVF of oocytes from the same egg donor used for SCNT [2]

For each cell type, an in depth genome wide analysis of the cells to investigate the scale of differences in the NT ESCs and iPSCs as compared to the “gold standard” IVF ESCs. Their findings, in brief, were:

  • Copy Number Variations (CNVs) – using high-throughput single nucleotide polymorphism (SNP) analysis
    • No statistically significant differences between the three cell types (although limited by small sample number)
  • Global DNA Methylation - using Infinium HumanMethylation450 BeadChip
    • Methylation of NT ESCs are more similar to IVF ESCs, than to iPSCs
    • iPSCs retain higher levels of residual somatic epigenetic memory
  • DNA methylation at imprinted and X-chromosome inactivation (XCI) regions
    • NT ESCs are more similar to IVF ESCs, than to iPSCs 
    • XCI loci methylation levels displayed variation across the lines
  • Autosomal non-imprinted loci
    • iPSCs generally displayed higher methylation levels than NT ESCs as compared to IVF ESCs
  • High-coverage base-resolution methylation – using Methyl C-sequencing
    • iPSCs displayed higher levels of differentially methylated regions (DMRs) than NT ESCs as compared to IVF ESCs
  • Non-CG Methylation in NT ESCs
    • Analysis suggests that NT ESCs are reprogrammed to a state nearing that of IVF ESCs, compared to iPSCs
  • Global gene expression – using strand specific RNA-sequencing
    • iPSCs aberrantly expressed more genes than NT ESCs as compared to IVF ESCs

Overall, their findings suggest that transcription factor-mediated iPSC-generation suffers from incomplete epigenetic reprogramming, while NT-ESCs are highly similar to IVF ESC controls. Specifically, iPSCs suffer the persistence of somatic patterns of CpG methylation [5], higher aberrations in non-CpG methylation, and so an affected gene expression profile. The authors therefore suggest that the study of oocyte-based reprogramming may allow us to elucidate reprogramming conditions which more faithfully recapitulate the conditions of the ooplasm. The next questions is whether this research will shift the focus, both in funding and research, away from iPSC-based technologies and towards NT-based ESC production.

References

  1. Ma H, Morey R, O'Neil RC, et al. Abnormalities in human pluripotent cells due to reprogramming mechanisms. Nature 2014;511:177-183.
  2. Tachibana M, Amato P, Sparman M, et al. Human embryonic stem cells derived by somatic cell nuclear transfer. Cell 2013;153:1228-1238.
  3. Lowry WE, Richter L, Yachechko R, et al. Generation of human induced pluripotent stem cells from dermal fibroblasts. Proc Natl Acad Sci U S A 2008;105:2883-2888.
  4. Fusaki N, Ban H, Nishiyama A, et al. Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome. Proc Jpn Acad Ser B Phys Biol Sci 2009;85:348-362.
  5. Kim K, Doi A, Wen B, et al. Epigenetic memory in induced pluripotent stem cells. Nature 2010;467:285-290.