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A New Strategy to Efficiently Identify Cells Undergoing Reprogramming

Review of Reprogramming progressive cells display low CAG promoter activityfrom STEM CELLS by Stuart P. Atkinson

Researchers led by Shangqin Guo (Yale University, New Haven, CT, USA) recently linked the lack of activity of the MKL1 (Megakaryoblast Leukemia 1)/SRF (serum response factor) transcription factor pathway, which controls the expression of genes that antagonize pluripotency, with the attainment of the pluripotent state during somatic cell reprogramming [1]. The synthetic CAG promoter, which derives part of its sequence from the chicken actin promoter/enhancer [2, 3], can act as a readout for endogenous MLK1/SRF activity, and now, Hu et al. describe the selection of cells with high inherent reprogramming potential through the evaluation of CAG promoter activity as a possible means to significantly improve reprogramming efficiency.

As described in their new STEM CELLS article [4], the authors evaluated the reprogramming of mouse embryonic fibroblasts with an inducible OCT4, SOX2, KLF4, and MYC (OSKM) polycistronic cassette and confirmed a link between reprogramming propensity and the decreased activity of the MKL1/SRF transcription factor pathway. Additionally, the study linked the increased likelihood of reprogramming with smaller cell size and a more rapid cell cycle, which had been linked to cells correctly undergoing reprogramming [5].

Given that SRF mediates the expression of target genes through binding to a well-characterized promoter transcription factor-binding site known as the CArG site (CCW6GG) and that the CAG promoter contains two well‐conserved CArG sites [2, 3], the authors evaluated the activity of fluorescent reporters driven by the CAG promoter during early reprogramming. Encouragingly, the study found a tight correlation between the decreased activity of this promoter and the increased likelihood of reprogramming to pluripotency and demonstrated that cells with low CAG promoter activity arise and expand during cell cycle acceleration during the early reprogramming of both mouse and human fibroblasts.

While these findings may lead to the development of a convenient and widely applicable means to enrich those few cells undergoing efficient reprogramming, this data may also help to reveal the molecular processes controlling the passage of somatic cells into a pluripotent state.

For more novel strategies that aim to increase reprogramming efficiency and take iPSC-derived cells closer to therapeutic applications, stay tuned to the Stem Cells Portal!

References

  1. Hu X, Liu ZZ, Chen X, et al., MKL1-actin pathway restricts chromatin accessibility and prevents mature pluripotency activation. Nature Communications 2019;10:1695.
  2. Stoflet ES, Schmidt LJ, Elder PK, et al., Activation of a muscle-specific actin gene promoter in serum-stimulated fibroblasts. Molecular Biology of the Cell 1992;3:1073-1083.
  3. Ng S-Y, Gunning P, Liu S-H, et al., Regulation of the human β-actin promoter by upstream and intron domains. Nucleic Acids Research 1989;17:601-615.
  4. Hu X, Wu Q, Zhang J, et al., Reprogramming progressive cells display low CAG promoter activity. STEM CELLS 2021;39:43-54.
  5. Guo S, Zi X, Schulz Vincent P, et al., Nonstochastic Reprogramming from a Privileged Somatic Cell State. Cell 2014;156:649-662.