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A stable chimeric fibroblast growth factor (FGF) can successfully replace basic FGF in human pluripotent stem cell culture.

ABSTRACT: Fibroblast growth factors (FGFs) are essential for maintaining self-renewal in human embryonic stem cells and induced pluripotent stem cells. Recombinant basic FGF (bFGF or FGF2) is conventionally used to culture pluripotent stem cells; however, because of the instability of bFGF, repeated addition of fresh bFGF into the culture medium is required in order to maintain its concentration. In this study, we demonstrate that a heat-stable chimeric variant of FGF, termed FGFC, can be successfully used for maintaining human pluripotent stem cells. FGFC is a chimeric protein composed of human FGF1 and FGF2 domains that exhibits higher thermal stability and protease resistance than do both FGF1 and FGF2. Both human embryonic stem cells and induced pluripotent stem cells were maintained in ordinary culture medium containing FGFC instead of FGF2. Comparison of cells grown in FGFC with those grown in conventional FGF2 media showed no significant differences in terms of the expression of pluripotency markers, global gene expression, karyotype, or differentiation potential in the three germ lineages. We therefore propose that FGFC may be an effective alternative to FGF2, for maintenance of human pluripotent stem cells.

SUBMITTER: Onuma Y

PROVIDER: S-EPMC4388338 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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A stable chimeric fibroblast growth factor (FGF) can successfully replace basic FGF in human pluripotent stem cell culture.

Onuma Yasuko Y Higuchi Kumiko K Aiki Yasuhiko Y Shu Yujing Y Asada Masahiro M Asashima Makoto M Suzuki Masashi M Imamura Toru T Ito Yuzuru Y

PloS one 20150407 4

Fibroblast growth factors (FGFs) are essential for maintaining self-renewal in human embryonic stem cells and induced pluripotent stem cells. Recombinant basic FGF (bFGF or FGF2) is conventionally used to culture pluripotent stem cells; however, because of the instability of bFGF, repeated addition of fresh bFGF into the culture medium is required in order to maintain its concentration. In this study, we demonstrate that a heat-stable chimeric variant of FGF, termed FGFC, can be successfully use ...[more]

PMID: 25850016

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Project description:BACKGROUND: Cryopreserved ovarian cortical tissue acts as a source of primordial follicles (PF) which can either be auto-transplanted or cultured in vitro to obtain mature oocytes. This offers a good opportunity to attain biological parenthood to individuals with gonadal insufficiency including cancer survivors. However, role of various intra- and extra-ovarian factors during PF growth initiation still remain poorly understood. Ovarian biology has assumed a different dimension due to emerging data on presence of pluripotent very small embryonic-like stem cells (VSELs) and ovarian germ stem cells (OGSCs) in ovary surface epithelium (OSE) and the concept of postnatal oogenesis. The present study was undertaken to decipher effect of follicle stimulating hormone (FSH) and basic fibroblast growth factor (bFGF) on the growth initiation of PF during organ culture with a focus on ovarian stem cells. METHODS: Serum-free cultures of marmoset (n=3) and human (young and peri-menopausal) ovarian cortical tissue pieces were established. Cortical tissue pieces stimulated with FSH (0.5 IU/ml) or bFGF (100 ng/ml) were collected on Day 3 for histological and molecular studies. Gene transcripts specific for pluripotency (Oct-4A, Nanog), early germ cells (Oct-4, c-Kit, Vasa) and to reflect PF growth initiation (oocyte-specific Gdf-9 and Lhx8, and granulosa cells specific Amh) were studied by q-RTPCR. RESULTS: A prominent proliferation of OSE (which harbors stem cells) and transition of PF to primary follicles was observed after FSH and bFGF treatment. Ovarian stem cells were found to be released on the culture inserts and retained the potential to spontaneously differentiate into oocyte-like structures in extended cultures. q-RTPCR analysis revealed an increased expression of gene transcripts specific for VSELs, OGSCs and early germ cells suggestive of follicular transition. CONCLUSION: The present study shows that both FSH and bFGF stimulate stem cells present in OSE and also lead to PF growth initiation. Thus besides being a source of PF, cryopreserved ovarian cortical tissue could also be a source of stem cells which retain the ability to spontaneously differentiate into oocyte-like structures in vitro. Results provide a paradigm shift in the basic understanding of FSH action and also offer a new perspective to the field of oncofertility research.

Dataset Information

A stable chimeric fibroblast growth factor (FGF) can successfully replace basic FGF in human pluripotent stem cell culture.

Publications

A stable chimeric fibroblast growth factor (FGF) can successfully replace basic FGF in human pluripotent stem cell culture.

Similar Datasets

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