Int J Stem Cells.  2020 Jul;13(2):246-256. 10.15283/ijsc20019.

Proliferation, Differentiation and Immunoregulatory Capacities of Brown and White Adipose-Derived Stem Cells from Young and Aged Mice

• Yang YM
• Dong XH
• Ma WC
• Guan LH
• Wang YH
• Huang XH
• Chen JF
• Zhao X
• Yang SM
• Jiang XX
• Wen N

• KMID: 2504337
• DOI: http://doi.org/10.15283/ijsc20019

Abstract

Background and Objectives
Adipose tissue is a source of mesenchymal stem cells, which have the potential to differentiate into various types of cells. Adipose-derived stem cells (ADSCs) are now recognized as an accessible, abundant, and reliable stem cells suitable for tissue engineering and regenerative medicine applications. However, few literatures gave a comprehensive report on the capacities of ADSCs harvested from different sites. Especially, the capacities of ADSCs from aged mice remained unclear. In this study, we investigated several main capacities of brown adipose derived stem cells (B-ADSCs) and white adipose derived stem cells (W-ADSCs) from both young and aged mice.
Methods and Results
When isolated from young mice, B-ADSCs showed a stronger proliferation rate and higher osteogenic, adipogenic and myocardial differentiation ability than W-ADSCs. Carboxy fluorescein diacetate succinimidyl ester (CFSE) labeling test suggested no significant difference in immunosuppression capacity between B-ADSCs and W-ADSCs. Similarly, no difference between these two were found in several immune related molecules, such as programmed death-ligand 1 (PD-L1), intercellular cell adhesion molecule (ICAM-1), vascular cell adhesion molecule (VCAM-1), inducible nitric oxide synthase (iNOS), tumour necrosis factor-α (TNF-α), interleukin 10 (IL10), and suppressor of cytokine signaling 1 (socs1). When isolated from aged mice, B-ADSCs also showed a stronger proliferation rate and higher osteogenic, adipogenic and myocardial differentiation ability than W-ADSCs; however, it demonstrated an attenuated immunosuppression capacity compared to W-ADSCs.
Conclusions
In summary, our data showed that ADSCs’ characteristics were tissue source dependent and changed with age. It provided evidence for choosing the right tissue-specific ADSCs for clinical application and fundamental research.

Keyword

Brown adipose derived stem cells; White adipose derived stem cells; Differentiation; Immunoregulation; Aged mice

Figure

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  Fig. 1 Morphological and phenotypic characteristics of B-ADSC and W-ADSC. (A) Morphology of cultured B-ADSC and W-ADSC of P1, P3, P5 of 3-to-4-week C57BL/6 mice were analyzed by microscopy; each experiment was observed three fields. Cell surface markers of 3-to-4-week C57BL/6 mice (B) and of 7-to-8-month C57BL/6 mice (C) were analyzed by flow cytometry. Scale bars represent 500 μm in the first and the third columns, and 125 μm in the second and the fourth columns.

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  Fig. 2 Proliferation and cell-cycle phase of B-ADSC and W-ADSC. (A) The cell proliferation curve was made by cell counting every two days for 8 days. PI staining of 3-to-4-week C57BL/6 mice (B) and 7-to-8-month C57BL/6 mice (C) were analyzed by flow cytometry. The first red line, the second oblique lines, and the third red-filled peak indicate the G0/G1, S, and G2/M phases, respectively.

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  Fig. 3 Osteogenic and adipogenic differentiation capacity of B-ADSC and W-ADSC. ADSCs were cultured in osteogenic (A) and adipogenic (C) differentiation for different time points, and were stained with ALP (bar=250 μm) or Oil Red O (bar=125 μm). They were analyzed for Alp, Runx2, Ocn, Osterix (B), Ppar-γ, Cebp-α, and Fabp4 (D) by qRT-PCR. (E) ADSCs of 7-to-8-month C57BL/6 mice were cultured in osteogenic or adipogenic medium for the indicated times, and were stained with ALP (bar=250 μm) or Oil Red O (bar=125 μm). They were analyzed for Alp, Runx2, Ocn, Osterix, Ppar-γ, Cebp-α, and Fabp4 by qRT-PCR (F). (Three experiments were performed, and each experiment was observed three fields.) *p<0.05, **p<0.01.

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  Fig. 4 Myocardial differentiation capacity of B-ADSCs and W-ADSCs. Cells from 3-to-4-week (A) and 7-to-8-month (B) C57BL/6 mice were cultured on carbon nanotube (CNT) for 3 days; cTnT, GATA4, and Nkx2.5 expression were examined by qRT-PCR. *p<0.05, **p<0.01.

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  Fig. 5 B-ADSCs and W-ADSCs showed no significant effect on PD-L1, ICAM-1, and VCAM-1 expression under stimulation (representative of 3 experiments). B-ADSCs and W-ADSCs were cultured with stimulation of TNF-α and IFN-γ at different levels - 0 ng/ml, 2 ng/ml, 5 ng/ml, and 10 ng/ml for 12 hours. The expression of PD-L1 (A), and ICAM-1 and VCAM-1 (B) were analyzed by flow cytometry. (C) B-ADSCs and W-ADSCs were cultured with stimulation of TNF-α and IFN-γ at 0 ng/ml and 2 ng/ml for 12 hours. The expression of iNOS, IL10, TNFα and socs1 were examined by qRT-PCR.

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  Fig. 6 Immunosuppressive capacity of B-ADSC and W-ADSC in vitro (representative of 3 experiments). CFSE-labeled CD3+ T cells (A) and CD4+ T cells (B) were cultured alone or co-cultured with different numbers of ADSCs from 3-to-4-week C57BL/6 mice for 48 hours in RPMI 1640 complete medium, supplemented with PMA (50 ng/ml) and ionomycin (1 μg/ml). (C) CFSE-labeled CD3+ T cells were cultured alone or co-cultured with different numbers of ADSCs from 7-to-8-month C57BL/6 mice. Cells were subjected to flow cytometry for T cell proliferation as detected by the CFSE signal.

Reference

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