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Effects of different serum conditions on osteogenic differentiation of human adipose stem cells in vitro

INTRODUCTION: Currently, human adipose stem cells (hASCs) are differentiated towards osteogenic lineages using culture medium supplemented with L-ascorbic acid 2-phosphate (AsA2-P), dexamethasone (Dex) and beta-glycerophosphate (β-GP). Because this osteogenic medium (OM1) was initially generated for...

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Autores principales: Kyllönen, Laura, Haimi, Suvi, Mannerström, Bettina, Huhtala, Heini, Rajala, Kristiina M, Skottman, Heli, Sándor, George K, Miettinen, Susanna
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3706769/
https://www.ncbi.nlm.nih.gov/pubmed/23415114
http://dx.doi.org/10.1186/scrt165
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author Kyllönen, Laura
Haimi, Suvi
Mannerström, Bettina
Huhtala, Heini
Rajala, Kristiina M
Skottman, Heli
Sándor, George K
Miettinen, Susanna
author_facet Kyllönen, Laura
Haimi, Suvi
Mannerström, Bettina
Huhtala, Heini
Rajala, Kristiina M
Skottman, Heli
Sándor, George K
Miettinen, Susanna
author_sort Kyllönen, Laura
collection PubMed
description INTRODUCTION: Currently, human adipose stem cells (hASCs) are differentiated towards osteogenic lineages using culture medium supplemented with L-ascorbic acid 2-phosphate (AsA2-P), dexamethasone (Dex) and beta-glycerophosphate (β-GP). Because this osteogenic medium (OM1) was initially generated for the differentiation of bone marrow-derived mesenchymal stem cells, the component concentrations may not be optimal for the differentiation of hASCs. After preliminary screening, two efficient osteogenic media (OM2 and OM3) were chosen to be compared with the commonly used osteogenic medium (OM1). To further develop the culture conditions towards clinical usage, the osteo-inductive efficiencies of OM1, OM2 and OM3 were compared using human serum (HS)-based medium and a defined, xeno-free medium (RegES), with fetal bovine serum (FBS)-based medium serving as a control. METHODS: To compare the osteo-inductive efficiency of OM1, OM2 and OM3 in FBS-, HS- and RegES-based medium, the osteogenic differentiation was assessed by alkaline phosphatase (ALP) activity, mineralization, and expression of osteogenic marker genes (runx2A, DLX5, collagen type I, osteocalcin, and ALP). RESULTS: In HS-based medium, the ALP activity increased significantly by OM3, and mineralization was enhanced by both OM2 and OM3, which have high AsA2-P and low Dex concentrations. ALP activity and mineralization of hASCs was the weakest in FBS-based medium, with no significant differences between the OM compositions due to donor variation. However, the qRT-PCR data demonstrated significant upregulation of runx2A mRNA under osteogenic differentiation in FBS- and HS-based medium, particularly by OM3 under FBS conditions. Further, the expression of DLX5 was greatly stimulated by OM1 to 3 on day 7 when compared to control. The regulation of collagen type I, ALP, and osteocalcin mRNA was modest under induction by OM1 to 3. The RegES medium was found to support the proliferation and osteogenic differentiation of hASCs, but the composition of the RegES medium hindered the comparison of OM1, OM2 and OM3. CONCLUSIONS: Serum conditions affect hASC proliferation and differentiation significantly. The ALP activity and mineralization was the weakest in FBS-based medium, although osteogenic markers were upregulated on mRNA level. When comparing the OM composition, the commonly used OM1 was least effective. Accordingly, higher concentration of AsA2-P and lower concentration of Dex, as in OM2 and OM3, should be used for the osteogenic differentiation of hASCs in vitro.
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spelling pubmed-37067692013-07-15 Effects of different serum conditions on osteogenic differentiation of human adipose stem cells in vitro Kyllönen, Laura Haimi, Suvi Mannerström, Bettina Huhtala, Heini Rajala, Kristiina M Skottman, Heli Sándor, George K Miettinen, Susanna Stem Cell Res Ther Research INTRODUCTION: Currently, human adipose stem cells (hASCs) are differentiated towards osteogenic lineages using culture medium supplemented with L-ascorbic acid 2-phosphate (AsA2-P), dexamethasone (Dex) and beta-glycerophosphate (β-GP). Because this osteogenic medium (OM1) was initially generated for the differentiation of bone marrow-derived mesenchymal stem cells, the component concentrations may not be optimal for the differentiation of hASCs. After preliminary screening, two efficient osteogenic media (OM2 and OM3) were chosen to be compared with the commonly used osteogenic medium (OM1). To further develop the culture conditions towards clinical usage, the osteo-inductive efficiencies of OM1, OM2 and OM3 were compared using human serum (HS)-based medium and a defined, xeno-free medium (RegES), with fetal bovine serum (FBS)-based medium serving as a control. METHODS: To compare the osteo-inductive efficiency of OM1, OM2 and OM3 in FBS-, HS- and RegES-based medium, the osteogenic differentiation was assessed by alkaline phosphatase (ALP) activity, mineralization, and expression of osteogenic marker genes (runx2A, DLX5, collagen type I, osteocalcin, and ALP). RESULTS: In HS-based medium, the ALP activity increased significantly by OM3, and mineralization was enhanced by both OM2 and OM3, which have high AsA2-P and low Dex concentrations. ALP activity and mineralization of hASCs was the weakest in FBS-based medium, with no significant differences between the OM compositions due to donor variation. However, the qRT-PCR data demonstrated significant upregulation of runx2A mRNA under osteogenic differentiation in FBS- and HS-based medium, particularly by OM3 under FBS conditions. Further, the expression of DLX5 was greatly stimulated by OM1 to 3 on day 7 when compared to control. The regulation of collagen type I, ALP, and osteocalcin mRNA was modest under induction by OM1 to 3. The RegES medium was found to support the proliferation and osteogenic differentiation of hASCs, but the composition of the RegES medium hindered the comparison of OM1, OM2 and OM3. CONCLUSIONS: Serum conditions affect hASC proliferation and differentiation significantly. The ALP activity and mineralization was the weakest in FBS-based medium, although osteogenic markers were upregulated on mRNA level. When comparing the OM composition, the commonly used OM1 was least effective. Accordingly, higher concentration of AsA2-P and lower concentration of Dex, as in OM2 and OM3, should be used for the osteogenic differentiation of hASCs in vitro. BioMed Central 2013-02-15 /pmc/articles/PMC3706769/ /pubmed/23415114 http://dx.doi.org/10.1186/scrt165 Text en Copyright © 2013 Kyllönen et al.; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research
Kyllönen, Laura
Haimi, Suvi
Mannerström, Bettina
Huhtala, Heini
Rajala, Kristiina M
Skottman, Heli
Sándor, George K
Miettinen, Susanna
Effects of different serum conditions on osteogenic differentiation of human adipose stem cells in vitro
title Effects of different serum conditions on osteogenic differentiation of human adipose stem cells in vitro
title_full Effects of different serum conditions on osteogenic differentiation of human adipose stem cells in vitro
title_fullStr Effects of different serum conditions on osteogenic differentiation of human adipose stem cells in vitro
title_full_unstemmed Effects of different serum conditions on osteogenic differentiation of human adipose stem cells in vitro
title_short Effects of different serum conditions on osteogenic differentiation of human adipose stem cells in vitro
title_sort effects of different serum conditions on osteogenic differentiation of human adipose stem cells in vitro
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3706769/
https://www.ncbi.nlm.nih.gov/pubmed/23415114
http://dx.doi.org/10.1186/scrt165
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