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Two miRNA clusters reveal alternative paths in late-stage reprogramming.

Ronald J Parchem1, Julia Ye1, Robert L Judson1

  • 1The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, Center for Reproductive Sciences, University of California, San Francisco, San Francisco, California, 94143, USA; Department of Urology, University of California, San Francisco, San Francisco, California, 94143, USA.

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Reprogramming somatic cells into induced pluripotent stem cells (iPSCs) can follow multiple paths. Adding Sall4 to Oct4, Sox2, and Klf4 (OSK) factors promotes a specific miRNA activation sequence, enhancing iPSC generation.

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Area of Science:

  • Cellular reprogramming
  • Stem cell biology
  • Epigenetics

Background:

  • Ectopic expression of Oct4, Sox2, and Klf4 (OSK) factors can induce pluripotency in somatic cells.
  • Understanding the precise cellular pathways during reprogramming is crucial for efficient iPSC generation.

Purpose of the Study:

  • To investigate the transcriptional activation patterns of pluripotent miRNA clusters (mir-290 and mir-302) during OSK-induced reprogramming.
  • To determine if the order of miRNA activation influences reprogramming efficiency and cell trajectory.

Main Methods:

  • Utilized knockin reporter systems to track individual cell transcriptional activation of mir-290 and mir-302 loci.
  • Compared miRNA activation sequences during embryonic development, ESC differentiation, and OSK-induced reprogramming.
  • Assessed the impact of adding Sall4 to the OSK cocktail on reprogramming efficiency and miRNA locus activation order.

Main Results:

  • During normal development and differentiation, mir-290 and mir-302 were activated sequentially.
  • OSK-induced reprogramming showed stochastic, non-ordered activation of these miRNA loci.
  • Addition of Sall4 to OSK resulted in a reversed, ordered activation (mir-302 then mir-290) and improved reprogramming efficiency.

Conclusions:

  • Cells can adopt diverse trajectories during the late stages of induced pluripotent stem cell reprogramming.
  • The combination of reprogramming factors significantly influences the sequence of miRNA activation and overall reprogramming efficiency.