3D culturing of human adipose-derived stem cells enhances their pluripotency and differentiation abilities

doi:10.1016/j.jmst.2020.05.003

Abstract

Abstract:

Human mesenchymal stem cells, such as human adipose-derived stem cells (hASCs), are typically cultured on a two-dimensional (2D) monolayer material surface, on which 2D culturing methods are easily performed and time-saving. However, hASCs usually suffer from decreased pluripotency and differentiation ability when cultured with a 2D monolayer culturing method compared to hASCs cultured with a three-dimensional (3D) culturing method, such as suspension culture. In this study, we evaluated whether the pluripotency and differentiation ability of hASCs can be reversibly changed during sequential cultivation with 2D and 3D culturing processes. The hASCs cultivated with a 3D culturing process after 2D culture showed at least 2-fold enhanced pluripotency (Sox2, Nanog, and OCT4) compared with that of hASCs cultured with the 2D culture process alone. Furthermore, hASCs obtained from the 3D culture process expressed increased levels of differentiation markers of chondrocytes and osteoblasts compared with hASCs obtained from the 2D culture process when hASCs were induced to differentiate. However, their pluripotency and differentiation ability were extensively reduced when hASCs were shifted from 3D culture to 2D culture and vice versa, which indicates that hASCs show reversibility in terms of their pluripotency and differentiation ability depending on their environment in 2D and 3D culture. The reversibility of pluripotency and differentiation ability were found to last for at least 5 passages in culture during the alternative and sequential culture of cells with 2D and 3D culturing processes. Our study revealed the importance of the culture microenvironment in maintaining the pluripotency and differentiation ability of hASCs, which may reduce the effects of the aging process in hASCs. We discuss whether the environment of stem cell culture (i.e., 2D or 3D cultivation) can affect stem cell fate in terms of pluripotency and differentiation reversibility.

Key words: 2D culture, 3D culture, Stem cell, Differentiation, Osteoblast

Tzu-Cheng Sung, Chao-Wen Heish, Henry Hsin-Chung Lee, Jhe-Yu Hsu, Chun-Ko Wang, Jia-Hua Wang, Yu-Ru Zhu, Shih-Hsi Jen, Shih-Tien Hsu, Abdurahman H. Hirad, Abdullah A. Alarfaj, Akon Higuchi. 3D culturing of human adipose-derived stem cells enhances their pluripotency and differentiation abilities[J]. J. Mater. Sci. Technol., 2021, 63: 9-17.

Figures/Tables 5

Fig. 1. Human ASCs cultivated in 2D or grown in sequential cultivation by using 2D and 3D culturing processes. (A) Procedure used for hASCs cultivated in 2D culturing conditions (upper case) or 2D and 3D sequential culturing conditions (lower case). (B) Morphologies and live/dead staining of hASCs in 2D culturing conditions. The scale bars indicate 100 μm. (C) Morphologies and live/dead staining of hASCs in 2D and 3D sequential culturing conditions. The scale bars indicate 100 μm.

Fig. 2. Flow cytometry analysis of hASCs cultivated in 2D culturing conditions or 2D and 3D sequential culturing conditions. (A) CD34, CD44, CD90 and CD105 expression of hASCs cultivated in 2D culturing conditions at passage 4. (B) CD34, CD44, CD90 and CD105 expression of hASCs cultivated in sequential 2D and 3D culturing conditions at passage 4. (C) The levels of CD markers analyzed by flow cytometry on hASCs grown in 2D and 3D sequential culturing conditions at passage 4. *p < 0.05.

Fig. 3. Pluripotency protein expression of hASCs in 2D culturing conditions or sequential 2D and 3D culturing conditions. (A) Sox2 (a, red), Oct3/4 (d, red) and SSEA4 (e, green) expression of hASCs cultivated in 2D culturing conditions at passage 5. The cells were evaluated by dual immunostaining with Hoechst 33342 (b, f) to stain nuclei (blue). The merged fluorescence image (c) of Sox2 (a) and Hoechst (b) staining and the merged fluorescence image (g) of Oct3/4 (d), SSEA4 (e) and Hoechst (f) staining are also shown. (B) Sox2 (a, red), Oct3/4 (d, red) and SSEA4 (e, green) expression of hASCs cultivated in sequential 2D and 3D culturing conditions at passage 6. The cells were evaluated by dual immunostaining with Hoechst 33342 (b, f) to stain nuclei (blue). The merged fluorescence image (c) of Sox2 (a) and Hoechst (b) staining and the merged fluorescence image (g) of Oct3/4 (d), SSEA4 (e) and Hoechst (f) staining are also shown. (C) Relative gene expression levels of Oct3/4 (black closed circle), Sox2 (red closed square), and Nanog (green closed square) on hASCs cultivated in 2D culturing conditions (a) and in sequential 2D and 3D culturing conditions (b), as analyzed by qRT-PCR. *p < 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4. Osteogenic and chondrogenic differentiation of hASCs cultivated in sequential 2D and 3D culturing conditions. (A) Micrograph images of cells analyzed by Alizarin Red S staining (a, b) and von Kossa staining (c, d) after osteogenic differentiation of hASCs for 28 days in sequential 2D and 3D culturing conditions; passage 5 (a, c) in 2D culture conditions and passage 6 (b, d) in 3D culture conditions are shown. The scale bar represents 100 μm. (B) The level of the osteogenic differentiation of cells analyzed by Alizarin Red S and von Kossa staining using ImageJ software (http://rsb.info.nih.gov/ij/) after chondrogenic differentiation for 28 days in sequential 2D and 3D culturing conditions at passage 5 (2D) and passage 6 (3D). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5. Neural cell differentiation of hASCs cultivated in sequential 2D and 3D culturing conditions. (A) The morphologies of cells after the neural cell differentiation of hASCs for 1 day (a) and 21 days (b) in sequential 2D and 3D culturing conditions at passage 5 (2D). The scale bars indicate 100 μm. (c-f) Expression of the neuronal marker protein β the III-tubulin (c, green) and astrocyte marker protein GFAP (d, red) on cells after neural cell differentiation for 21 days in sequential 2D and 3D culturing conditions at passage 5 (2D). The cells were evaluated by dual immunostaining with Hoechst 33342 (e) to stain nuclei (blue). The merged fluorescence image (f) of βIII-tubulin (c), GFAP (d) and Hoechst 33342 (e) staining is also shown. The scale bars indicate 200 μm. (B) The morphologies of cells after the neural cell differentiation of hASCs for 1 day (a) and 21 days (b) in sequential 2D and 3D culturing conditions at passage 6 (3D). The scale bars indicate 100 μm. (c-f) Expression of the neuronal marker protein βIII-tubulin (c, green) and the astrocyte marker protein GFAP (d, red) on cells after neural cell differentiation for 21 days in sequential 2D and 3D culturing conditions at passage 6 (3D). The cells were evaluated by dual immunostaining with Hoechst 33342 (e) to stain nuclei (blue). The merged fluorescence image (f) of βIII-tubulin (c), GFAP (d) and Hoechst 33342 (e) staining is also shown. The scale bars indicate 200 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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