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iPSC Production – Select Your Factors Wisely

Review of “Developmental Potential of iPSCs Is Greatly Influenced by Reprogramming Factor Selection” from Cell by Stuart P. Atkinson

Current methodologies for induced pluripotent stem cell (iPSC) production from somatic cells are thought to negatively affect reprograming efficiency, the quality of the cells produced, and their differentiation capacity, as compared to embryonic stem cells (ESCs) produced by somatic cell nuclear transfer (SCNT). However, the specific contributions of any given parameter to these deficiencies are relatively unknown. Now in a study published in Cell Stem Cell led by Rudolf Jaenisch, researchers have demonstrated that these deficiencies are linked to the specific reprogramming factors utilized [1].

The group initially investigated the transcription factors Sall4, Nanog, Esrrb, and Lin28 (SNEL) as reprogramming factors, based on them being key master regulators of Oct4 and Sox2, Dnmt3b and Nanog. Lentiviral transduction of the individual factors generated fully pluripotent iPSC colonies, but at a low efficiency (SNEL-iPSCs). Chimera formation through injecting SNEL-iPSCs into host blastocysts generated high-grade chimeras with germline transmission. Interestingly, comparisons of SNEL-iPSCs and iPSCs generated using the more classic combination of Oct4, Sox2, Klf4 and Myc (OSKM-iPSCs) in 4n complementation assays found that SNEL-iPSC (“high quality” iPSCs) injection led to the generation of five times as many pups as for OSKM-iPSCs (“poor quality” iPSCs). Researchers consider the 4n, or tetraploid, complementation assay the most stringent test for pluripotency, and involves the injection of pluripotent cells into a tetraploid embryo (either at the morula or blastocyst stage) where they contribute to the developing fetus and the 4n cells contribute to the extra-embryonic tissues. Experiments using iPSCs cultured under 2i medium conditions (LIF, GSK3b, and Mek 1/2 inhibitor-containing medium), found similar results.

The authors next assessed the possible negative contribution of Myc through the generation of OSK-iPSCs and, indeed, a larger proportion of colonies passed the 4n complementation test, although still lower than for SNEL-iPSCs, suggesting that factors other than Myc negatively affect reprogramming in OSK-iPSCs. Indeed, leaving out the two potent oncogenes Myc and Lin28, and using Oct4, Sox2, Sall4, Nanog, and Esrrb (OSSNE) to generate iPSCs yielded the highest number of these “poor quality” iPSCs.

Transcriptional comparisons between poor- and high-quality iPSCs revealed that a 1,765 gene signature was highly correlated with 4n competency. While gene ontology and pathway analysis gave categories associated with the control of cellular growth and division, amongst other specific developmental pathways and phenotypes, standard motif enrichment analysis on the 1,765 gene promoters demonstrated enrichment for transcription factors necessary for early embryonic development and ESC self-renewal. Further assessments found that DNA methylation differences, variations in the transcript levels of ESC key master regulators, and differences in the establishment of ESC-specific super enhancers (associated with genes that determine cell identity [2]) were not able to distinguish between poor- and high-quality iPSCs. Some genomic aberrations were observed in OSKM-iPSCs, with higher levels of the DNA damage associated -H2A.X phosphorylation, although assessment of sister chromatid exchange (sensitive indicators of genomic stress and instability) at the single cell level demonstrated no differences between OSKM-iPSCs (poor quality) and SNEL-iPSCs (high quality). However, assessment of chromosomal aberrations occurring due to long transient genomic instability during reprogramming did find that around 20% of OSK- and OSKM-iPSC lines displayed trisomy of chromosome 8, whereas no SNEL-iPSCs contained this aberration. The final check by the group concerned defective H2A.X deposition, known to occur in OSKM-iPSCs that do not support ‘‘all-iPS’’ mice development in 4n complementation experiments [3], and which may be linked to g-H2A.X phosphorylation. Interestingly, while SNEL-iPSC lines displayed patterns similar to parental ESCs, poor-quality lines displayed different deposition patterns, suggesting that the recapitulation of a faithful H2A.X pattern may be the key to the generation of high-quality iPSCs.

The message seems clear: iPSC quality depends on the choice of transcription factors used. However, it is important to note that the authors generated high quality iPSCs at a lower efficiency. Efficiency of reprogramming is often used as a surrogate marker for “quality” of iPSCs, under the supposition that more is better. This suggests that the expression of regulators of the main pluripotency network factors leads to a more endogenous-like expression level for said factors and, while this leads to a longer, less efficient process, the final product is of higher quality. Does this hold for human cells? Unfortunately, the authors note that the SNEL factors were not sufficient to generate human iPSCs, which may reflect the inherent differences in mouse pluripotent cells and human pluripotent cells.

References

  1. Buganim Y, Markoulaki S, van Wietmarschen N, et al. The Developmental Potential of iPSCs Is Greatly Influenced by Reprogramming Factor Selection. Cell Stem Cell 2014;15:295-309.
  2. Hnisz D, Abraham BJ, Lee TI, et al. Super-enhancers in the control of cell identity and disease. Cell 2013;155:934-947.
  3. Wu T, Liu Y, Wen D, et al. Histone Variant H2A.X Deposition Pattern Serves as a Functional Epigenetic Mark for Distinguishing the Developmental Potentials of iPSCs. Cell Stem Cell 2014;15:281-294.