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Combined influence of basal media and fibroblast growth factor on the expansion and differentiation capabilities of adipose-derived stem cells.

Ahearne M, Lysaght J, Lynch AP - Cell Regen (Lond) (2014)

Bottom Line: FGF has a negative effective on passage 4 ASC adipogenesis and high glucose media plus FGF-enhanced osteogenic capacity of passage 4 ASCs.High glucose media plus FGF appeared to be the most beneficial for priming ASCs to induce a keratocyte phenotype.These findings demonstrate the reciprocal effect FGF and basal media have on ASCs.

View Article: PubMed Central - PubMed

Affiliation: Trinity Centre for Bioengineering, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin, Ireland ; Department of Mechanical and Manufacturing Engineering, School of Engineering, Trinity College Dublin, Dublin, Ireland.

ABSTRACT

Background: Interest in adipose-derived stem cells (ASCs) has increased in recent years due to their multi-linage differentiation capabilities. While much work has been done to optimize the differentiation media, few studies have focused on examining the influence of different expansion media on cell behavior. In this study, three basal media (low glucose Dulbecco's modified Eagle's medium (DMEM), high glucose DMEM and DMEM-F12) supplemented with or without fibroblast growth factor 2 (FGF) were examined to assess their suitability for expanding ASCs.

Findings: Flow cytometry, colony-forming unit assays (CFU-Fs) and differentiation assays were utilized to study cell behavior. High glucose media CFU-Fs produced fewest colonies while the addition of FGF increased colony size. By passage 2, the majority of cells were positive for CD44, 45, 73, 90 and 105 and negative for CD14, 31 and 45, indicating a mesenchymal phenotype. A sub-population of CD34 positive cells was present among passage 2 cells; however, by passage 4 the cells were negative for CD34. FGF has a negative effective on passage 4 ASC adipogenesis and high glucose media plus FGF-enhanced osteogenic capacity of passage 4 ASCs. FGF supplemented basal media were most suitable for chondrogenesis. High glucose media plus FGF appeared to be the most beneficial for priming ASCs to induce a keratocyte phenotype.

Conclusions: These findings demonstrate the reciprocal effect FGF and basal media have on ASCs. This research has implications for those interested regenerating bone, cartilage, cornea or adipose tissues.

No MeSH data available.


Related in: MedlinePlus

P2 and P4 ASCs cultured from two different donors in chemically defined differentiation media after 21 days. (A) Adipogenic assay stained using oil red O and quantified by measuring the percentage of surface area stained; (B) osteogenic assay stained using alizarin red and quantified by measuring the light absorbance at 405 nm; (C) chondrogenic pellet assay stained using alcian blue and nuclear fast red and quantified using biochemical assays for DNA and GAG. n = 3, ● represents a significant difference between FGF and the equivalent non-FGF-supplemented media; o represents a significant difference between P2 and P4 cells; *significant difference between donors, p < 0.05; scalebar = 20 μm for A and B and 100 μm for C, representative images display the results from P2 donor 1.
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Fig3: P2 and P4 ASCs cultured from two different donors in chemically defined differentiation media after 21 days. (A) Adipogenic assay stained using oil red O and quantified by measuring the percentage of surface area stained; (B) osteogenic assay stained using alizarin red and quantified by measuring the light absorbance at 405 nm; (C) chondrogenic pellet assay stained using alcian blue and nuclear fast red and quantified using biochemical assays for DNA and GAG. n = 3, ● represents a significant difference between FGF and the equivalent non-FGF-supplemented media; o represents a significant difference between P2 and P4 cells; *significant difference between donors, p < 0.05; scalebar = 20 μm for A and B and 100 μm for C, representative images display the results from P2 donor 1.

Mentions: The findings of this study would suggest that choosing the optimal media for expanding ASCs is dependent on the final application. The addition of FGF was found to cause a significant increase in adipogenesis (Figure 3A) at P2 when added to F12 media (p < 0.05). By P4, cells expanded in FGF-supplemented media showed a decrease in adipogenesis regardless of the basal media. The expansion media had no significant effect on the osteogenic potential of P2 cells although HG + FGF-cultured cells were significantly better at undergoing osteogenesis than cells expanded in the other media at P4 (Figure 3B). Cells expanded in FGF-supplemented media displayed a higher chondrogenic capacity when compared to cells expanded in media without FGF regardless of the basal media used (Figure 3C). The center of pellets formed from cells that has not been pre-cultured in FGF appeared to contain substantially less sGAG than FGF-expanded cells. These findings would correlate with several other studies which have found that FGF primes cells, thus enabling them to become more chondrogenic when moved to a chemically defined differentiation media [16, 17].Figure 3


Combined influence of basal media and fibroblast growth factor on the expansion and differentiation capabilities of adipose-derived stem cells.

Ahearne M, Lysaght J, Lynch AP - Cell Regen (Lond) (2014)

P2 and P4 ASCs cultured from two different donors in chemically defined differentiation media after 21 days. (A) Adipogenic assay stained using oil red O and quantified by measuring the percentage of surface area stained; (B) osteogenic assay stained using alizarin red and quantified by measuring the light absorbance at 405 nm; (C) chondrogenic pellet assay stained using alcian blue and nuclear fast red and quantified using biochemical assays for DNA and GAG. n = 3, ● represents a significant difference between FGF and the equivalent non-FGF-supplemented media; o represents a significant difference between P2 and P4 cells; *significant difference between donors, p < 0.05; scalebar = 20 μm for A and B and 100 μm for C, representative images display the results from P2 donor 1.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig3: P2 and P4 ASCs cultured from two different donors in chemically defined differentiation media after 21 days. (A) Adipogenic assay stained using oil red O and quantified by measuring the percentage of surface area stained; (B) osteogenic assay stained using alizarin red and quantified by measuring the light absorbance at 405 nm; (C) chondrogenic pellet assay stained using alcian blue and nuclear fast red and quantified using biochemical assays for DNA and GAG. n = 3, ● represents a significant difference between FGF and the equivalent non-FGF-supplemented media; o represents a significant difference between P2 and P4 cells; *significant difference between donors, p < 0.05; scalebar = 20 μm for A and B and 100 μm for C, representative images display the results from P2 donor 1.
Mentions: The findings of this study would suggest that choosing the optimal media for expanding ASCs is dependent on the final application. The addition of FGF was found to cause a significant increase in adipogenesis (Figure 3A) at P2 when added to F12 media (p < 0.05). By P4, cells expanded in FGF-supplemented media showed a decrease in adipogenesis regardless of the basal media. The expansion media had no significant effect on the osteogenic potential of P2 cells although HG + FGF-cultured cells were significantly better at undergoing osteogenesis than cells expanded in the other media at P4 (Figure 3B). Cells expanded in FGF-supplemented media displayed a higher chondrogenic capacity when compared to cells expanded in media without FGF regardless of the basal media used (Figure 3C). The center of pellets formed from cells that has not been pre-cultured in FGF appeared to contain substantially less sGAG than FGF-expanded cells. These findings would correlate with several other studies which have found that FGF primes cells, thus enabling them to become more chondrogenic when moved to a chemically defined differentiation media [16, 17].Figure 3

Bottom Line: FGF has a negative effective on passage 4 ASC adipogenesis and high glucose media plus FGF-enhanced osteogenic capacity of passage 4 ASCs.High glucose media plus FGF appeared to be the most beneficial for priming ASCs to induce a keratocyte phenotype.These findings demonstrate the reciprocal effect FGF and basal media have on ASCs.

View Article: PubMed Central - PubMed

Affiliation: Trinity Centre for Bioengineering, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin, Ireland ; Department of Mechanical and Manufacturing Engineering, School of Engineering, Trinity College Dublin, Dublin, Ireland.

ABSTRACT

Background: Interest in adipose-derived stem cells (ASCs) has increased in recent years due to their multi-linage differentiation capabilities. While much work has been done to optimize the differentiation media, few studies have focused on examining the influence of different expansion media on cell behavior. In this study, three basal media (low glucose Dulbecco's modified Eagle's medium (DMEM), high glucose DMEM and DMEM-F12) supplemented with or without fibroblast growth factor 2 (FGF) were examined to assess their suitability for expanding ASCs.

Findings: Flow cytometry, colony-forming unit assays (CFU-Fs) and differentiation assays were utilized to study cell behavior. High glucose media CFU-Fs produced fewest colonies while the addition of FGF increased colony size. By passage 2, the majority of cells were positive for CD44, 45, 73, 90 and 105 and negative for CD14, 31 and 45, indicating a mesenchymal phenotype. A sub-population of CD34 positive cells was present among passage 2 cells; however, by passage 4 the cells were negative for CD34. FGF has a negative effective on passage 4 ASC adipogenesis and high glucose media plus FGF-enhanced osteogenic capacity of passage 4 ASCs. FGF supplemented basal media were most suitable for chondrogenesis. High glucose media plus FGF appeared to be the most beneficial for priming ASCs to induce a keratocyte phenotype.

Conclusions: These findings demonstrate the reciprocal effect FGF and basal media have on ASCs. This research has implications for those interested regenerating bone, cartilage, cornea or adipose tissues.

No MeSH data available.


Related in: MedlinePlus