Blood Res.  2017 Mar;52(1):18-24. 10.5045/br.2017.52.1.18.

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

• Bae YJ
• Kwon YR
• Kim HJ
• Lee S
• Kim YJ

• KMID: 2375199
• DOI: http://doi.org/10.5045/br.2017.52.1.18

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.

Keyword

Three-dimensional culture; 5-azacytidine; Mesenchymal stromal cells; Osteogenesis; Adipogenesis

MeSH Terms

Adipogenesis
Apoptosis
Azacitidine*
Cell- and Tissue-Based Therapy
Mesenchymal Stromal Cells*
Methods
Osteogenesis
Azacitidine

Figure

• 

  Fig. 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).

• 

  Fig. 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).

• 

  Fig. 3 MSC adipogenesis. (A) Oil red O staining was performed after adipogenic differentiation of MSCs treated with or without AZA treatment. (B) The transcript levels of Ppar-γ were analyzed in the various MSC populations after adipogenesis induction with adipogenic medium for 72 h. β-2 microglobulin was used as an internal control (N=4).

• 

  Fig. 4 CFU-F assay and expression of stemness markers. MSCs were plated at a density of 500 cells/plate and incubated for 14 days for the CFU-F assay. (A) The number of colonies (diameter≥2 mm) was counted. The expression levels of Oct4 (B), Nanog (C), and Cxcr4 (D) in control and AZA-treated MSCs were assessed by real time PCR analysis (N=4).

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  Fig. 5 Representative photographs showing senescence-associated β-galactosidase (blue) staining of MSCs before and after exposure to AZA. The scale bar represents 100 µm.

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