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The cells of the blastocyst inner cell mass only remain pluripotent for a short time. This state of pluripotency and self-renewal can be maintained in embryonic stem (ES) cell culture by adding specific chemicals or growth factors to ensure the cells can continue dividing and later differentiate into different cell types. In some cases, the cells are grown on a feeder layer of differentiated cells, which provides the growth factors and extracellular matrix components necessary for stem cell...
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Resistance to naive and formative pluripotency conversion in RSeT human embryonic stem cells.

Kevin G Chen1, Kory R Johnson2, Kyeyoon Park1

  • 1NIH Stem Cell Unit, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, United States.

Stem Cells (Dayton, Ohio)
|August 22, 2025
PubMed
Summary

RSeT human embryonic stem cells (hESCs) represent a distinct pluripotent state, differing from naive and primed states. These cells show unique growth properties and lack key naive pluripotency markers, offering new insights into stem cell state transitions.

Keywords:
RSeT mediumcell proliferationhuman embryonic stem cellsnaive pluripotencytranscriptome

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Area of Science:

  • Stem Cell Biology
  • Developmental Biology
  • Genomics

Background:

  • Human embryonic stem cells (hESCs) exhibit naive and primed pluripotent states.
  • Previous work identified heterogeneous naive pluripotent states from various protocols.
  • Understanding pluripotent state heterogeneity is crucial for stem cell applications.

Purpose of the Study:

  • To characterize a commercial RSeT-based pluripotent state.
  • To investigate RSeT hESC behavior under different growth conditions.
  • To define the transcriptomic and biochemical properties of RSeT hESCs in relation to other pluripotent states.

Main Methods:

  • Cell culture under normoxia and hypoxia.
  • Single-cell plating efficiency assays.
  • Integrative transcriptome analysis.
  • Analysis of surface marker expression (SUSD2, CD75).
  • Biochemical assays for signaling pathway dependence (FGF2, JAK, TGFβ).

Main Results:

  • RSeT hESCs can grow without hypoxia but show variable growth and plating efficiency.
  • RSeT hPSCs lack transcriptomic hallmarks of naive and formative pluripotency.
  • RSeT hESCs resemble early post-implantation embryos, similar to primary hESCs.
  • Naive surface markers SUSD2 and CD75 are not significantly expressed.
  • RSeT hESCs exhibit cell line-specific dependence on FGF2 and co-independency on JAK/TGFβ signaling.

Conclusions:

  • RSeT hESCs represent a novel pluripotent state downstream of naive pluripotency.
  • RSeT medium may restrict naive pluripotent potential by sustaining FGF2 activity.
  • This study enhances understanding of in vitro pluripotent state transitions.