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Isolation of adipose and bone marrow mesenchymal stem cells using CD29 and CD90 modifies their capacity for osteogenic and adipogenic differentiation.

Davies OG, Cooper PR, Shelton RM, Smith AJ, Scheven BA - J Tissue Eng (2015)

Bottom Line: Maintaining cells in fluorescence-activated cell sorting buffer did not affect adipose-derived cell viability, but a significant (p < 0.05) reduction was found in bone marrow-derived cell viability.Additionally, CD29(+)/CD90(+) selection was associated with a significant decrease in the expression of Lin28, Sox2, Nanog and CD73 in adipose-derived cell cultures, whereas differences in stem cell-associated gene expression were not observed in sorted bone marrow-derived cell cultures.In summary, this study demonstrated that fluorescence-activated cell sorting had differential effects on adipose-derived cells and bone marrow-derived cells, and both CD29(+)/CD90(+) cells displayed a significantly reduced capacity for osteogenic/adipogenic differentiation.

View Article: PubMed Central - PubMed

Affiliation: School of Dentistry, University of Birmingham, Birmingham, UK ; Wolfson School of Mechanical and Manufacturing Engineering, Loughborough University, Loughborough, UK.

ABSTRACT
Mesenchymal stem cells isolated from rats are frequently used for tissue engineering research. However, considerable differences have been identified between rat mesenchymal stem cells and those derived from humans, and no defined panel of markers currently exists for the isolation of these cells. The aim of this study was to examine the effects of cell sorting for CD29(+)/CD90(+) cells from rat adipose and bone marrow tissues on their differentiation and expression of stem cell-associated genes. Flow cytometry showed 66% and 78% CD29(+)/CD90(+) positivity within passage 1 of adipose and bone marrow cultures, respectively. CD29(+)/CD90(+) cells showed a reduction in both osteogenic and adipogenic differentiation when compared with unsorted cells, as determined by alizarin red and Oil Red-O staining, respectively. These findings could not entirely be explained by fluorescence-activated cell sorting-induced cell injury as sort recovery was only modestly affected in adipose-derived cells. Maintaining cells in fluorescence-activated cell sorting buffer did not affect adipose-derived cell viability, but a significant (p < 0.05) reduction was found in bone marrow-derived cell viability. Additionally, CD29(+)/CD90(+) selection was associated with a significant decrease in the expression of Lin28, Sox2, Nanog and CD73 in adipose-derived cell cultures, whereas differences in stem cell-associated gene expression were not observed in sorted bone marrow-derived cell cultures. In summary, this study demonstrated that fluorescence-activated cell sorting had differential effects on adipose-derived cells and bone marrow-derived cells, and both CD29(+)/CD90(+) cells displayed a significantly reduced capacity for osteogenic/adipogenic differentiation. In conclusion, we identify that maintaining heterogeneity within the mesenchymal stem cell population may be important for optimal differentiation.

No MeSH data available.


Related in: MedlinePlus

Osteogenic and adipogenic differentiation analysed by (1) alizarin red (AR) and (2) Oil Red-O (ORO) staining, respectively. AR staining and quantification were used to identify calcium deposition. ORO staining and quantification were used to identify lipid accumulation. For both assays, CD29+/CD90+, unlabelled and unsorted (a) ADC and (b) BMDC cultures were compared. Both analyses were performed following a 21-day culture in osteogenic or adipogenic medium.*p < 0.05, n = 3.
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fig3-2041731415592356: Osteogenic and adipogenic differentiation analysed by (1) alizarin red (AR) and (2) Oil Red-O (ORO) staining, respectively. AR staining and quantification were used to identify calcium deposition. ORO staining and quantification were used to identify lipid accumulation. For both assays, CD29+/CD90+, unlabelled and unsorted (a) ADC and (b) BMDC cultures were compared. Both analyses were performed following a 21-day culture in osteogenic or adipogenic medium.*p < 0.05, n = 3.

Mentions: Osteogenic differentiation was analysed using AR staining and quantification. AR staining indicated that calcium deposition was significantly (p < 0.05) reduced in CD29+/CD90+ cultures when compared with unsorted and unlabelled controls (Figure 3(1)). Significant (p < 0.05) differences in AR staining were observed between the CD29+/CD90+ and the unlabelled controls (i.e. cells processed through the FACS instrument but lacking primary antibody) in both ADC and BMDC cultures. A significant (p < 0.05) reduction in AR staining was also observed when unlabelled cells were compared with unsorted cells.


Isolation of adipose and bone marrow mesenchymal stem cells using CD29 and CD90 modifies their capacity for osteogenic and adipogenic differentiation.

Davies OG, Cooper PR, Shelton RM, Smith AJ, Scheven BA - J Tissue Eng (2015)

Osteogenic and adipogenic differentiation analysed by (1) alizarin red (AR) and (2) Oil Red-O (ORO) staining, respectively. AR staining and quantification were used to identify calcium deposition. ORO staining and quantification were used to identify lipid accumulation. For both assays, CD29+/CD90+, unlabelled and unsorted (a) ADC and (b) BMDC cultures were compared. Both analyses were performed following a 21-day culture in osteogenic or adipogenic medium.*p < 0.05, n = 3.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2 - License 3
Show All Figures
getmorefigures.php?uid=PMC4555348&req=5

fig3-2041731415592356: Osteogenic and adipogenic differentiation analysed by (1) alizarin red (AR) and (2) Oil Red-O (ORO) staining, respectively. AR staining and quantification were used to identify calcium deposition. ORO staining and quantification were used to identify lipid accumulation. For both assays, CD29+/CD90+, unlabelled and unsorted (a) ADC and (b) BMDC cultures were compared. Both analyses were performed following a 21-day culture in osteogenic or adipogenic medium.*p < 0.05, n = 3.
Mentions: Osteogenic differentiation was analysed using AR staining and quantification. AR staining indicated that calcium deposition was significantly (p < 0.05) reduced in CD29+/CD90+ cultures when compared with unsorted and unlabelled controls (Figure 3(1)). Significant (p < 0.05) differences in AR staining were observed between the CD29+/CD90+ and the unlabelled controls (i.e. cells processed through the FACS instrument but lacking primary antibody) in both ADC and BMDC cultures. A significant (p < 0.05) reduction in AR staining was also observed when unlabelled cells were compared with unsorted cells.

Bottom Line: Maintaining cells in fluorescence-activated cell sorting buffer did not affect adipose-derived cell viability, but a significant (p < 0.05) reduction was found in bone marrow-derived cell viability.Additionally, CD29(+)/CD90(+) selection was associated with a significant decrease in the expression of Lin28, Sox2, Nanog and CD73 in adipose-derived cell cultures, whereas differences in stem cell-associated gene expression were not observed in sorted bone marrow-derived cell cultures.In summary, this study demonstrated that fluorescence-activated cell sorting had differential effects on adipose-derived cells and bone marrow-derived cells, and both CD29(+)/CD90(+) cells displayed a significantly reduced capacity for osteogenic/adipogenic differentiation.

View Article: PubMed Central - PubMed

Affiliation: School of Dentistry, University of Birmingham, Birmingham, UK ; Wolfson School of Mechanical and Manufacturing Engineering, Loughborough University, Loughborough, UK.

ABSTRACT
Mesenchymal stem cells isolated from rats are frequently used for tissue engineering research. However, considerable differences have been identified between rat mesenchymal stem cells and those derived from humans, and no defined panel of markers currently exists for the isolation of these cells. The aim of this study was to examine the effects of cell sorting for CD29(+)/CD90(+) cells from rat adipose and bone marrow tissues on their differentiation and expression of stem cell-associated genes. Flow cytometry showed 66% and 78% CD29(+)/CD90(+) positivity within passage 1 of adipose and bone marrow cultures, respectively. CD29(+)/CD90(+) cells showed a reduction in both osteogenic and adipogenic differentiation when compared with unsorted cells, as determined by alizarin red and Oil Red-O staining, respectively. These findings could not entirely be explained by fluorescence-activated cell sorting-induced cell injury as sort recovery was only modestly affected in adipose-derived cells. Maintaining cells in fluorescence-activated cell sorting buffer did not affect adipose-derived cell viability, but a significant (p < 0.05) reduction was found in bone marrow-derived cell viability. Additionally, CD29(+)/CD90(+) selection was associated with a significant decrease in the expression of Lin28, Sox2, Nanog and CD73 in adipose-derived cell cultures, whereas differences in stem cell-associated gene expression were not observed in sorted bone marrow-derived cell cultures. In summary, this study demonstrated that fluorescence-activated cell sorting had differential effects on adipose-derived cells and bone marrow-derived cells, and both CD29(+)/CD90(+) cells displayed a significantly reduced capacity for osteogenic/adipogenic differentiation. In conclusion, we identify that maintaining heterogeneity within the mesenchymal stem cell population may be important for optimal differentiation.

No MeSH data available.


Related in: MedlinePlus