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

Yoo-Jin Bae; Yong-Rim Kwon; Hye Joung Kim; Seok Lee; Yoo-Jin Kim

doi:10.5045/br.2017.52.1.18

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Original Article

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Blood Res 2017; 52(1):

Published online March 27, 2017

https://doi.org/10.5045/br.2017.52.1.18

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

Yoo-Jin Bae¹, Yong-Rim Kwon¹, Hye Joung Kim¹, Seok Lee^2,3, and Yoo-Jin Kim^1,2,3*

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¹Laboratory of Hematological Disease and Immunology, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea.

²Leukemia Research Institute, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea.

³Department of Hematology, Catholic Blood and Marrow Transplantation Center, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea.

Correspondence to : Yoo-Jin Kim, M.D., Ph.D. Department of Hematology, Catholic Blood and Marrow Transplantation Center, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, 222 Banpo-daero, Seocho-gu, Seoul 06591, Korea. yoojink@catholic.ac.kr

Received: November 10, 2016; Revised: December 16, 2016; Accepted: January 6, 2017

Abstract

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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 ?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.

Keywords Three-dimensional culture, 5-azacytidine, Mesenchymal stromal cells, Osteogenesis, Adipogenesis

Article

Original Article

Blood Res 2017; 52(1): 18-24

Published online March 27, 2017 https://doi.org/10.5045/br.2017.52.1.18

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

Yoo-Jin Bae¹, Yong-Rim Kwon¹, Hye Joung Kim¹, Seok Lee^2,3, and Yoo-Jin Kim^1,2,3*

¹Laboratory of Hematological Disease and Immunology, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea.

²Leukemia Research Institute, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea.

³Department of Hematology, Catholic Blood and Marrow Transplantation Center, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea.

Correspondence to: Yoo-Jin Kim, M.D., Ph.D. Department of Hematology, Catholic Blood and Marrow Transplantation Center, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, 222 Banpo-daero, Seocho-gu, Seoul 06591, Korea. yoojink@catholic.ac.kr

Received: November 10, 2016; Revised: December 16, 2016; Accepted: January 6, 2017

Abstract

Background

Methods

Results

Conclusion

Keywords: Three-dimensional culture, 5-azacytidine, Mesenchymal stromal cells, Osteogenesis, Adipogenesis

Abstract

Fig 1.

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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).

Blood Research 2017; 52: 18-24https://doi.org/10.5045/br.2017.52.1.18

Fig 2.

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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).

Blood Research 2017; 52: 18-24https://doi.org/10.5045/br.2017.52.1.18

Fig 3.

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Figure 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).

Blood Research 2017; 52: 18-24https://doi.org/10.5045/br.2017.52.1.18

Fig 4.

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Figure 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).

Blood Research 2017; 52: 18-24https://doi.org/10.5045/br.2017.52.1.18

Fig 5.

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Figure 5.

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

Blood Research 2017; 52: 18-24https://doi.org/10.5045/br.2017.52.1.18

Table 1 . Primer sequences used for the real-time RT-PCR analysis..