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Derivation of Human Embryonic Stem Cells by Immunosurgery
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Deriving rabbit embryonic stem cells by small molecule inhibitors.

Jiao Liu1, Xiumei Zhu1, Jinshan Li1

  • 1Jiangsu Key Laboratory for Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University Nanjing 210046, Jiangsu, P. R. China.

American Journal of Translational Research
|September 10, 2019
PubMed
Summary

Researchers developed domed pluripotent rabbit embryonic stem cells (rbES) using specific inhibitors for MEK, GSK3, and PKC pathways. Basic fibroblast growth factor and leukemia inhibitory factor are essential for rbES derivation and maintenance.

Keywords:
Rabbitembryonic stem cellssignal transductionsmall molecules

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Area of Science:

  • Stem Cell Biology
  • Developmental Biology
  • Mammalian Reproduction

Background:

  • Previous development of pluripotent rabbit embryonic stem cells (rbES) utilized a culture system with basic fibroblast growth factor (bFGF), leukemia inhibitory factor (LIF), noggin, and Y-27632 (iFLY).
  • Enhancing the derivation efficiency of domed pluripotent rbES requires exploring novel culture conditions and signaling pathway modulation.

Purpose of the Study:

  • To investigate the efficacy of inhibiting MEK, GSK3, and PKC signaling pathways for deriving domed pluripotent rbES.
  • To identify the essential components and optimal concentrations of inhibitors required for efficient rbES derivation.

Main Methods:

  • Culturing rbES in a defined medium supplemented with knockout serum replacement (KOSR), LIF, bFGF, and a combination of three inhibitors (3i): PD0325901 (MEK inhibitor), CHIR99021 (GSK3 inhibitor), and Gö6983 (PKC inhibitor).
  • Assessing pluripotency markers (Rex1, ERAS, Oct4, Klf4, Sox2, c-myc) via RT-PCR and immunofluorescence.
  • Evaluating the necessity of each component in the 3i medium and bFGF by targeted deletion experiments.

Main Results:

  • Domed pluripotent rbES were successfully derived using the 3i medium (PD0325901, CHIR99021, Gö6983) in combination with bFGF and LIF.
  • bFGF and LIF were confirmed as indispensable for rbES derivation and maintenance.
  • The 3i medium was crucial for achieving the domed morphology characteristic of pluripotent rbES.
  • Domed rbES expressed key naïve ES cell markers and showed positive staining for pluripotency factors.
  • Optimal concentrations for the 3i components were determined to be 0.75 µM PD0325901, 2.25 µM CHIR99021, and 4.5 µM Gö6983.

Conclusions:

  • The combination of MEK, GSK3, and PKC pathway inhibition is essential for deriving domed pluripotent rbES.
  • Signaling pathway networks play a critical role in the self-renewal, propagation, and maintenance of ES cells.
  • This study provides insights into deriving authentic rabbit ES cells, an important understudied model species.