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Stable generation of serum- and feeder-free embryonic stem cell-derived mice with full germline-competency by using a GSK3 specific inhibitor.

ABSTRACT: C57BL/6 (B6)-derived embryonic stem (ES) cells are not widely used to generate knockout mice despite the advantage of a well-defined genetic background because of poor developmental potential. We newly established serum- and feeder-free B6 ES cells with full developmental potential by using leukemia inhibitory factor (LIF) and 6-bromoindirubin-3'-oxime (BIO), a glycogen synthase kinase-3 (GSK3) inhibitor. BIO treatment significantly increased the expression levels of 364 genes including pluripotency markers such as Nanog and Klf family. Unexpectedly, by aggregating or microinjecting those ES cells to each eight-cell-stage diploid embryo, we stably generated germline-competent ES-derived mice. Furthermore, founder mice completely derived from female XO, heterozygous, or homozygous mutant B6 ES cells were directly available for intercross breeding and phenotypic analysis. We hereby propose that serum- and feeder-free B6 ES cells stimulated with LIF plus GSK3 inhibitor are valuable for generating mouse models on B6 background.

SUBMITTER: Sato H

PROVIDER: S-EPMC2726955 | biostudies-literature | 2009 Jun

REPOSITORIES: biostudies-literature

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Stable generation of serum- and feeder-free embryonic stem cell-derived mice with full germline-competency by using a GSK3 specific inhibitor.

Sato Hiromu H Amagai Keiko K Shimizukawa Rie R Tamai Yoshitaka Y

Genesis (New York, N.Y. : 2000) 20090601 6

C57BL/6 (B6)-derived embryonic stem (ES) cells are not widely used to generate knockout mice despite the advantage of a well-defined genetic background because of poor developmental potential. We newly established serum- and feeder-free B6 ES cells with full developmental potential by using leukemia inhibitory factor (LIF) and 6-bromoindirubin-3'-oxime (BIO), a glycogen synthase kinase-3 (GSK3) inhibitor. BIO treatment significantly increased the expression levels of 364 genes including pluripot ...[more]

PMID: 19391115

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Project description:BackgroundThe establishment of stable porcine embryonic stem cells (pESCs) can contribute to basic and biomedical research, including comparative developmental biology, as well as assessing the safety of stem cell-based therapies. Despite these advantages, most pESCs obtained from in vitro blastocysts require complex media and feeder layers, making routine use, genetic modification, and differentiation into specific cell types difficult. We aimed to establish pESCs with a single cell-passage ability, high proliferative potency, and stable in long-term culture from in vitro-derived blastocysts using a simplified serum-free medium.MethodsWe evaluated the establishment efficiency of pESCs from in vitro blastocysts using various basal media (DMEM/F10 (1:1), DMEM/F12, and a-MEM) and factors (FGF2, IWR-1, CHIR99021, and WH-4-023). The pluripotency and self-renewal capacity of the established pESCs were analyzed under feeder or feeder-free conditions. Ultimately, we developed a simplified culture medium (FIW) composed of FGF2, IWR-1, and WH-4-023 under serum-free conditions.ResultsThe pESC-FIW lines were capable of single-cell passaging with short cell doubling times and expressed the pluripotency markers POU5F1, SOX2, and NANOG, as well as cell surface markers SSEA1, SSEA4, and TRA-1-60. pESC-FIW showed a stable proliferation rate and normal karyotype, even after 50 passages. Transcriptome analysis revealed that pESC-FIW were similar to reported pESC maintained in complex media and showed gastrulating epiblast cell characteristics. pESC-FIW were maintained for multiple passages under feeder-free conditions on fibronectin-coated plates using mTeSR™, a commercial medium used for feeder-free culture, exhibiting characteristics similar to those observed under feeder conditions.ConclusionsThese results indicated that inhibition of WNT and SRC was sufficient to establish pESCs capable of single-cell passaging and feeder-free expansion under serum-free conditions. The easy maintenance of pESCs facilitates their application in gene editing technology for agriculture and biomedicine, as well as lineage commitment studies.

Dataset Information

Stable generation of serum- and feeder-free embryonic stem cell-derived mice with full germline-competency by using a GSK3 specific inhibitor.

Publications

Stable generation of serum- and feeder-free embryonic stem cell-derived mice with full germline-competency by using a GSK3 specific inhibitor.

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