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Enhanced differentiation of mesenchymal stromal cells by three-dimensional culture and azacitidine

View Article: PubMed Central - PubMed

ABSTRACT

Background: Mesenchymal stromal cells (MSCs) are useful for cell therapy because of their potential for multilineage differentiation. However, MSCs that are expanded in traditional two-dimensional (2D) culture systems eventually lose their differentiation abilities. Therefore, we investigated whether azacitidine (AZA) supplementation and three-dimensional culture (3D) could improve the differentiation properties of MSCs.

Methods: 2D- or 3D-cultured MSCs which were prepared according to the conventional or hanging-drop culture method respectively, were treated with or without AZA (1 µM for 72 h), and their osteogenic and adipogenic differentiation potential were determined and compared.

Results: AZA treatment did not affect the cell apoptosis or viability in both 2D- and 3D-cultured MSCs. However, compared to conventionally cultured 2D-MSCs, AZA-treated 2D-MSCs showed marginally increased differentiation abilities. In contrast, 3D-MSCs showed significantly increased osteogenic and adipogenic differentiation ability. When 3D culture was performed in the presence of AZA, the osteogenic differentiation ability was further increased, whereas adipogenic differentiation was not affected.

Conclusion: 3D culture efficiently promoted the multilineage differentiation of MSCs, and in combination with AZA, it could help MSCs to acquire greater osteogenic differentiation ability. This optimized culture method can enhance the therapeutic potential of MSCs.

No MeSH data available.


MSC osteogenesis. (A) ALP staining was performed after the osteogenic differentiation of MSCs treated with or without AZA. (B) The transcript levels of Runx-2 were analyzed in the various MSC populations after inducing osteogenesis with osteogenic medium for 72 h (N=4, a)P <0.05).
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Figure 2: MSC osteogenesis. (A) ALP staining was performed after the osteogenic differentiation of MSCs treated with or without AZA. (B) The transcript levels of Runx-2 were analyzed in the various MSC populations after inducing osteogenesis with osteogenic medium for 72 h (N=4, a)P <0.05).

Mentions: First, ALP staining was performed to identify the osteogenic potential of the different groups of cells. Osteogenesis was highest in AZA-treated 3D-MSCs, followed by that in 3D-MSCs, AZA-treated 2D-MSCs, and 2D-MSCs (Fig. 2A). Real-time PCR analysis revealed increased expression levels of the osteogenesis-related gene Runx-2 in 3D-MSCs than in 2D-MSCs, although the difference was not statistically significant (Fig. 2B). Furthermore, the Runx-2 expression levels were significantly increased in AZA-treated 3D-MSCs than in 2D-MSCs and AZA-treated 2D-MSC, but were not significantly different from those observed in 3D-MSCs. These results indicate that both AZA treatment and 3D culture could improve MSC osteogenesis (N=4, a)P<0.05).


Enhanced differentiation of mesenchymal stromal cells by three-dimensional culture and azacitidine
MSC osteogenesis. (A) ALP staining was performed after the osteogenic differentiation of MSCs treated with or without AZA. (B) The transcript levels of Runx-2 were analyzed in the various MSC populations after inducing osteogenesis with osteogenic medium for 72 h (N=4, a)P <0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5383582&req=5

Figure 2: MSC osteogenesis. (A) ALP staining was performed after the osteogenic differentiation of MSCs treated with or without AZA. (B) The transcript levels of Runx-2 were analyzed in the various MSC populations after inducing osteogenesis with osteogenic medium for 72 h (N=4, a)P <0.05).
Mentions: First, ALP staining was performed to identify the osteogenic potential of the different groups of cells. Osteogenesis was highest in AZA-treated 3D-MSCs, followed by that in 3D-MSCs, AZA-treated 2D-MSCs, and 2D-MSCs (Fig. 2A). Real-time PCR analysis revealed increased expression levels of the osteogenesis-related gene Runx-2 in 3D-MSCs than in 2D-MSCs, although the difference was not statistically significant (Fig. 2B). Furthermore, the Runx-2 expression levels were significantly increased in AZA-treated 3D-MSCs than in 2D-MSCs and AZA-treated 2D-MSC, but were not significantly different from those observed in 3D-MSCs. These results indicate that both AZA treatment and 3D culture could improve MSC osteogenesis (N=4, a)P<0.05).

View Article: PubMed Central - PubMed

ABSTRACT

Background: Mesenchymal stromal cells (MSCs) are useful for cell therapy because of their potential for multilineage differentiation. However, MSCs that are expanded in traditional two-dimensional (2D) culture systems eventually lose their differentiation abilities. Therefore, we investigated whether azacitidine (AZA) supplementation and three-dimensional culture (3D) could improve the differentiation properties of MSCs.

Methods: 2D- or 3D-cultured MSCs which were prepared according to the conventional or hanging-drop culture method respectively, were treated with or without AZA (1 &micro;M for 72 h), and their osteogenic and adipogenic differentiation potential were determined and compared.

Results: AZA treatment did not affect the cell apoptosis or viability in both 2D- and 3D-cultured MSCs. However, compared to conventionally cultured 2D-MSCs, AZA-treated 2D-MSCs showed marginally increased differentiation abilities. In contrast, 3D-MSCs showed significantly increased osteogenic and adipogenic differentiation ability. When 3D culture was performed in the presence of AZA, the osteogenic differentiation ability was further increased, whereas adipogenic differentiation was not affected.

Conclusion: 3D culture efficiently promoted the multilineage differentiation of MSCs, and in combination with AZA, it could help MSCs to acquire greater osteogenic differentiation ability. This optimized culture method can enhance the therapeutic potential of MSCs.

No MeSH data available.