Various types of feeder cells have been adopted for the culture of human embryonic stem cells (hESCs) to improve their attachment and provide them with stemness-supporting factors. However, feeder cells differ in their capacity to support the growth of undifferentiated hESCs. Here, we compared the expression and secretion of four well-established regulators of hESC pluripotency and/or differentiation among five lines of human foreskin fibroblasts and primary mouse embryonic fibroblasts throughout a standard hESC culture procedure. We found that human and mouse feeder cells secreted comparable levels of TGF beta 1. However, mouse feeder cells secreted larger quantities of activin A than human feeder cells. Conversely, FGF-2, which was produced by human feeder cells, could not be detected in culture media from mouse feeder cells. The quantity of BMP-4 was at about the level of detectability in media from all feeder cell types, although BMP-4 dimers were present in all feeder cells. Production of TGF beta 1, activin A, and FGF-2 varied considerably among the human-derived feeder cell lines. Low- and high-producing human feeder cells as well as mouse feeder cells were evaluated for their ability to support the undifferentiated growth of hESCs. We found that a significantly lower proportion of hESCs maintained on human feeder cell types expressed SSEA3, an undifferentiated cell marker. Moreover, SSEA3 expression and thus the pluripotent hESC compartment could be partially rescued by addition of activin A. Cumulatively, these results suggest that the ability of a feeder layer to promote the undifferentiated growth of hESCs is attributable to its characteristic growth factor production.
- MeSH
- aktiviny biosyntéza genetika MeSH
- antigeny sacharidové asociované s nádorem genetika MeSH
- biologické markery metabolismus MeSH
- buněčná diferenciace MeSH
- DNA primery genetika MeSH
- druhová specificita MeSH
- embryonální antigeny specifické pro určité stadium vývoje MeSH
- embryonální kmenové buňky cytologie metabolismus MeSH
- exprese genu MeSH
- fibroblastový růstový faktor 2 biosyntéza genetika MeSH
- glykosfingolipidy genetika MeSH
- kokultivační techniky metody MeSH
- kostní morfogenetické proteiny analýza genetika MeSH
- kostní morfogenetický protein 4 MeSH
- kultivační média speciální analýza MeSH
- lidé MeSH
- messenger RNA genetika metabolismus MeSH
- myši MeSH
- pluripotentní kmenové buňky cytologie metabolismus MeSH
- proliferace buněk MeSH
- sekvence nukleotidů MeSH
- transformující růstový faktor beta1 biosyntéza genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
- Názvy látek
- activin A MeSH Prohlížeč
- aktiviny MeSH
- antigeny sacharidové asociované s nádorem MeSH
- biologické markery MeSH
- BMP4 protein, human MeSH Prohlížeč
- Bmp4 protein, mouse MeSH Prohlížeč
- DNA primery MeSH
- embryonální antigeny specifické pro určité stadium vývoje MeSH
- fibroblastový růstový faktor 2 MeSH
- glykosfingolipidy MeSH
- kostní morfogenetické proteiny MeSH
- kostní morfogenetický protein 4 MeSH
- kultivační média speciální MeSH
- messenger RNA MeSH
- stage-specific embryonic antigen-3 MeSH Prohlížeč
- transformující růstový faktor beta1 MeSH
BACKGROUND: Aberrant expression of either growth factors or growth factor-receptors by stromal cells can be an important factor promoting the growth of solid tumours. It may also affect differentiation of malignant cells and support tumour spread. The aim of the present study was to investigate the hypothesis that basic-fibroblast growth factor (bFGF) and platelet-derived growth factor (PDEGF) may be involved in tumour-stromal microenvironment interactions in primary malignant melanomas. MATERIALS AND METHODS: PDEGF and bFGF expression in malignant cells and surrounding stromal elements was assessed using indirect immunohistochemistry. RESULTS: It was confirmed that PDEGF can be involved in the reciprocal interactions between tumour cells and stroma, including aberrant angiogenesis. Interestingly, bFGF was present both in malignant melanoma lesions and benign nevi accompanied by different intracellular localisation of the protein, suggesting its implication in regulation of nevus cell proliferation and maturation. CONCLUSION: The present results suggest that bFGF and PDEGF participate in malignant melanoma progression.
- MeSH
- buňky stromatu metabolismus patologie MeSH
- destičkový růstový faktor biosyntéza MeSH
- fibroblastový růstový faktor 2 biosyntéza MeSH
- lidé MeSH
- melanom metabolismus patologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- destičkový růstový faktor MeSH
- fibroblastový růstový faktor 2 MeSH
Although the detection of several components of the fibroblast growth factor (FGF) signaling pathway in human embryonic stem cells (hESCs) has been reported, the functionality of that pathway and effects on cell fate decisions are yet to be established. In this study we characterized expression of FGF-2, the prototypic member of the FGF family, and its receptors (FGFRs) in undifferentiated and differentiating hESCs; subsequently, we analyzed the effects of FGF-2 on hESCs, acting as both exogenous and endogenous factors. We have determined that undifferentiated hESCs are abundant in several molecular-mass isoforms of FGF-2 and that expression pattern of these isoforms remains unchanged under conditions that induce hESC differentiation. Significantly, FGF-2 is released by hESCs into the medium, suggesting an autocrine activity. Expression of FGFRs in undifferentiated hESCs follows a specific pattern, with FGFR1 being the most abundant species and other receptors showing lower expression in the following order: FGFR1 --> FGFR3 --> FGFR4 --> FGFR2. Initiation of differentiation is accompanied by profound changes in FGFR expression, particularly the upregulation of FGFR1. When hESCs are exposed to exogenous FGF-2, extracellular signal-regulated kinases are phosphorylated and thereby activated. However, the presence or absence of exogenous FGF-2 does not significantly affect the proliferation of hESCs. Instead, increased concentration of exogenous FGF-2 leads to reduced outgrowth of hESC colonies with time in culture. Finally, the inhibitor of FGFRs, SU5402, was used to ascertain whether FGF-2 that is released by hESCs exerts its activities via autocrine pathways. Strikingly, the resultant inhibition of FGFR suppresses activation of downstream protein kinases and causes rapid cell differentiation, suggesting an involvement of autocrine FGF signals in the maintenance of proliferating hESCs in the undifferentiated state. In conclusion from our data, we propose that this endogenous FGF signaling pathway can be implicated in self-renewal or differentiation of hESCs.
- MeSH
- buněčná diferenciace MeSH
- embryo savčí cytologie MeSH
- fibroblastový růstový faktor 2 biosyntéza fyziologie MeSH
- fosforylace MeSH
- kmenové buňky cytologie účinky léků metabolismus MeSH
- lidé MeSH
- mitogenem aktivované proteinkinasy metabolismus MeSH
- protein - isoformy MeSH
- pyrroly farmakologie MeSH
- receptor fibroblastových růstových faktorů, typ 2 biosyntéza fyziologie MeSH
- receptory fibroblastových růstových faktorů biosyntéza fyziologie MeSH
- signální transdukce fyziologie MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- FGFR2 protein, human MeSH Prohlížeč
- fibroblastový růstový faktor 2 MeSH
- mitogenem aktivované proteinkinasy MeSH
- protein - isoformy MeSH
- pyrroly MeSH
- receptor fibroblastových růstových faktorů, typ 2 MeSH
- receptory fibroblastových růstových faktorů MeSH
- SU 5402 MeSH Prohlížeč
