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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

Percentage of P2 and P4 ASCs isolated from two different donors that were positive for CD14, CD19, CD31, CD34, CD44, CD45, CD73, CD90, CD105and CD146.
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Fig1: Percentage of P2 and P4 ASCs isolated from two different donors that were positive for CD14, CD19, CD31, CD34, CD44, CD45, CD73, CD90, CD105and CD146.

Mentions: All the ASCs were positive and negative for appropriate markers normally associated with mesenchymal stem cells apart from CD34 where there was a separate population of cells at P2 expressing this marker, the size of which varied depending on the donor and the type of culture media used (FigureĀ 1). By passage 4, the majority of cells were CD34 negative. CD34 is most commonly used as a hematopoietic progenitor and endothelial cell marker [12, 13]; although given the almost complete lack of CD45 and CD31 positive cells, these effectively eliminate the presence of CD34 positive hematopoietic and endothelial cells among the cell population. While it has generally been believed that expanded mesenchymal stem cells are negative for CD34 [1], its presence has been found among freshly isolated mesenchymal stem cell populations [14]. Among adipose derived cells, the percentage of CD34 positive cells has been shown to decrease with culture time and may indicate an immature phenotype prior to differentiation towards a specific linage [15] although from our findings there was no obvious correlation between the size of the population of CD34 positive cells and their differential capabilities. In this study, the cells at P2 had undergone 4 to 5 population doublings while those cells at P4 had undergone 10 to 12 population doublings although this assumes that all cells initially seeded are ASCs and all cells proliferate at the same rate.


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)

Percentage of P2 and P4 ASCs isolated from two different donors that were positive for CD14, CD19, CD31, CD34, CD44, CD45, CD73, CD90, CD105and CD146.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4325938&req=5

Fig1: Percentage of P2 and P4 ASCs isolated from two different donors that were positive for CD14, CD19, CD31, CD34, CD44, CD45, CD73, CD90, CD105and CD146.
Mentions: All the ASCs were positive and negative for appropriate markers normally associated with mesenchymal stem cells apart from CD34 where there was a separate population of cells at P2 expressing this marker, the size of which varied depending on the donor and the type of culture media used (FigureĀ 1). By passage 4, the majority of cells were CD34 negative. CD34 is most commonly used as a hematopoietic progenitor and endothelial cell marker [12, 13]; although given the almost complete lack of CD45 and CD31 positive cells, these effectively eliminate the presence of CD34 positive hematopoietic and endothelial cells among the cell population. While it has generally been believed that expanded mesenchymal stem cells are negative for CD34 [1], its presence has been found among freshly isolated mesenchymal stem cell populations [14]. Among adipose derived cells, the percentage of CD34 positive cells has been shown to decrease with culture time and may indicate an immature phenotype prior to differentiation towards a specific linage [15] although from our findings there was no obvious correlation between the size of the population of CD34 positive cells and their differential capabilities. In this study, the cells at P2 had undergone 4 to 5 population doublings while those cells at P4 had undergone 10 to 12 population doublings although this assumes that all cells initially seeded are ASCs and all cells proliferate at the same rate.

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