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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.


Related in: MedlinePlus

(A) Immunophenotyping results of mouse MSCs. After harvesting, 2D-MSCs, AZA-treated 2D-MSCs, 3D-MSCs, and AZA-treated 3D-MSCs were stained with antibodies and analyzed by flow cytometry. The cells were strongly positive for MSC-specific markers such as CD29, CD44, and SCA-1, and negative for the CD31, CD34, c-Kit, and FLK-1 markers. (B) Effects of AZA on MSC viability. Relative proliferation rates were determined by the MTS assay. (C) MSC apoptosis was determined by flow cytometry based on PI uptake and Annexin V-FITC labeling (N=4, a)P < 0.05, b)P <0.01).
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Figure 1: (A) Immunophenotyping results of mouse MSCs. After harvesting, 2D-MSCs, AZA-treated 2D-MSCs, 3D-MSCs, and AZA-treated 3D-MSCs were stained with antibodies and analyzed by flow cytometry. The cells were strongly positive for MSC-specific markers such as CD29, CD44, and SCA-1, and negative for the CD31, CD34, c-Kit, and FLK-1 markers. (B) Effects of AZA on MSC viability. Relative proliferation rates were determined by the MTS assay. (C) MSC apoptosis was determined by flow cytometry based on PI uptake and Annexin V-FITC labeling (N=4, a)P < 0.05, b)P <0.01).

Mentions: Phenotypic changes between 2D-MSCs, AZA-treated 2D-MSCs, 3D-MSCs, and AZA-treated 3D-MSCs were evaluated by flow cytometry. No changes in the MSC-negative markers CD31, CD34, c-Kit, and FLK-1 were detected across all cell groups. However, the expression of the MSC-positive markers CD44 and SCA-1 were significantly decreased in 3D-MSCs and AZA-treated 3D-MSCs than in the 2D-MSC groups (with or without AZA treatment). These results indicate that the 3D culture treatment induced a change in the MSC phenotype (Fig. 1A).


Enhanced differentiation of mesenchymal stromal cells by three-dimensional culture and azacitidine
(A) Immunophenotyping results of mouse MSCs. After harvesting, 2D-MSCs, AZA-treated 2D-MSCs, 3D-MSCs, and AZA-treated 3D-MSCs were stained with antibodies and analyzed by flow cytometry. The cells were strongly positive for MSC-specific markers such as CD29, CD44, and SCA-1, and negative for the CD31, CD34, c-Kit, and FLK-1 markers. (B) Effects of AZA on MSC viability. Relative proliferation rates were determined by the MTS assay. (C) MSC apoptosis was determined by flow cytometry based on PI uptake and Annexin V-FITC labeling (N=4, a)P < 0.05, b)P <0.01).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: (A) Immunophenotyping results of mouse MSCs. After harvesting, 2D-MSCs, AZA-treated 2D-MSCs, 3D-MSCs, and AZA-treated 3D-MSCs were stained with antibodies and analyzed by flow cytometry. The cells were strongly positive for MSC-specific markers such as CD29, CD44, and SCA-1, and negative for the CD31, CD34, c-Kit, and FLK-1 markers. (B) Effects of AZA on MSC viability. Relative proliferation rates were determined by the MTS assay. (C) MSC apoptosis was determined by flow cytometry based on PI uptake and Annexin V-FITC labeling (N=4, a)P < 0.05, b)P <0.01).
Mentions: Phenotypic changes between 2D-MSCs, AZA-treated 2D-MSCs, 3D-MSCs, and AZA-treated 3D-MSCs were evaluated by flow cytometry. No changes in the MSC-negative markers CD31, CD34, c-Kit, and FLK-1 were detected across all cell groups. However, the expression of the MSC-positive markers CD44 and SCA-1 were significantly decreased in 3D-MSCs and AZA-treated 3D-MSCs than in the 2D-MSC groups (with or without AZA treatment). These results indicate that the 3D culture treatment induced a change in the MSC phenotype (Fig. 1A).

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.


Related in: MedlinePlus