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Updated: Jan 19, 2026
Derivation of Human Embryonic Stem Cells by Immunosurgery
Published on: December 13, 2007
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.
Abstract:
We previously developed pluripotent rabbit embryonic stem cells (rbES) using a culture system supplemented with basic fibroblast growth factor (bFGF) and leukemia inhibitory factor (LIF), noggin and Y-27632 (referred to as iFLY). In present work, we explored multiple approaches to enhance the chance of deriving domed pluripotent rbES cells by inhibition of MEK, GSK, and PKC signaling pathways. Domed stated rbES were derived in defined medium supplemented with 15% KOSR, 103 IU/mL mouse LIF, 10 ng/mL bFGF and three inhibitors to the MEK (PD0325901, 1 µM), GSK3 (CHIR99021, 3 µM) and PKC (Gö6983, 5 µM) (3i). Domed rbES were passaged every 3-4 days till passage 3-4 for the designated experiments. We showed that bFGF and LIF are indispensable for the derivation and maintenance of rbES; whereas the 3i medium containing inhibitors to the MEK (PD0325901), GSK3 (CHIR99021) and PKC (Gö6983) were necessary for deriving domed rbES. Domed rbES possessed naïve ES markers as Rex1 and ERAS in addition to Oct4, Klf4, Sox 2 and c-myc by RT-PCR. Domed rbES showed positive staining for Rex1, Fgf4, Klf4, Nanog and Oct4 by immunofluorescence chemistry. Further deleting either one factor in 3i medium as CHIR99021, PD0325901, Gö6983 or bFGF resulted in disappearing of domed rbES colonies. The optimal concentrations of 3i contained 0.75 µM PD0325901, 2.25 µM CHIR99021, and 4.5 µM Gö6983. Our work, in combination of different inhibitors for deriving rabbit ES, supports that the network of signal pathways plays an important role in ES self-renew, propagation and maintenance, and sheds light on deriving authentic properties of rbES in an important yet understudied model animal species.
Insights
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.
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.
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