Comparative Analysis of the Therapeutic Effects of Amniotic Membrane and Umbilical Cord Derived Mesenchymal Stem Cells for the Treatment of Type 2 Diabetes

Abstract

Type 2 diabetes mellitus (T2DM), one of the most common carbohydrate metabolism disorders, is characterized by chronic hyperglycemia and insulin resistance (IR), and has become an urgent global health challenge. Mesenchymal stem cells (MSCs) originating from perinatal tissues such as umbilical cord (UC) and amniotic membrane (AM) serve as ideal candidates for the treatment of T2DM due to their great advantages in terms of abundant source, proliferation capacity, immunomodulation and plasticity for insulin-producing cell differentiation. However, the optimally perinatal MSC source to treat T2DM remains elusive. This study aims to compare the therapeutic efficacy of MSCs derived from AM and UC (AMMSCs and UCMSCs) of the same donor in the alleviation of T2DM symptoms and explore the underlying mechanisms. Our results showed that AMMSCs and UCMSCs displayed indistinguishable immunophenotype and multi-lineage differentiation potential, but UCMSCs had a much higher expansion capacity than AMMSCs. Moreover, we uncovered that single-dose intravenous injection of either AMMSCs or UCMSCs could comparably reduce hyperglycemia and improve IR in T2DM db/db mice. Mechanistic investigations revealed that either AMMSC or UCMSC infusion could greatly improve glycolipid metabolism in the liver of db/db mice, which was evidenced by decreased liver to body weight ratio, reduced lipid accumulation, upregulated glycogen synthesis, and increased Akt phosphorylation. Taken together, these data indicate that the same donor-derived AMMSCs and UCMSCs possessed comparable effects and shared a similar hepatoprotective mechanism on the alleviation of T2DM symptoms.

Keywords: Amniotic membrane; Hyperglycemia; Liver; Mesenchymal stem cells; Type 2 diabetes; Umbilical cord.

Fig. 1

Biological characterization of AMMSCs and UCMSCs originating from the same donor. (A) Representative pictures of the morphology of AMMSCs and UCMSCs. The pictures were taken with a light microscope at 4× (top) and 10 × (bottom) magnification. Scale bar = 100 μm. (B) Representative histograms of immunophenotypic characterization of AMMSCs and UCMSCs by flow cytometry analysis. Blue areas indicate isotype control staining, and red areas represent specific marker expression. (C) Representative images of adipocytes (top), chondrocytes (middle) and osteocytes (bottom) differentiated from AMMSCs or UCMSCs. The images were captured at 40× (top), 20× (middle) and 40 × (bottom) magnification, respectively. Scale bar = 50 μm.

Fig. 2

The growth curves and gene expression patterns of AMMSCs and UCMSCs. (A) Growth curve analyses of AMMSCs and UCMSCs. (B-I) mRNA expression levels of HGF (B), IGF1 (C), FGF2 (D), VEGF (E), IL-6 (F), IL-11 (G), IDO1 (H) and TGF-β (I) in AMMSCs and UCMSCs with or without IFN-γ stimulation. The results are shown as mean ± SEM (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 3

Metabolic phenotypes of db/db mice after AMMSC or UCMSC administration. Random blood glucose concentrations (A) and body weights (B) of different group mice were determined at indicated time points. Changes of blood glucose concentrations (C) and area under the curve (D) from the IPITT in different group mice at two weeks after MSC or saline infusion. Changes of blood glucose levels (E) and area under the curve (F) from the IPGTT in mice of different groups at two weeks after MSC or saline infusion. Data are displayed as mean ± SEM. Normal group: n = 6 mice; Saline group: n = 7 mice; UCMSCs group: n = 7 mice; AMMSCs group: n = 8 mice. AUC: area under the curve. *P or ^#P < 0.05, **P or ^##P < 0.01, ***P < 0.001. In (A) and (C), *: UCMSCs group vs. saline group; #: AMMSCs group vs. saline group.

Fig. 4

MSC administration decreased lipid accumulation and enhanced glycogen storage in the liver of db/db mice. The ratios of liver to body weight (A) and ratios of EWAT to body weight (B) were calculated in different group mice at five weeks after MSC or saline infusion. (C) Representative pictures of PAS staining in the liver sections of different group mice. Scale bar = 200 μm. (D) Quantification of PAS-positive areas in the livers of different group mice. (E) Representative pictures of H&E staining in mouse liver sections. Scale bar = 200 μm. (F) Quantification of lipid droplet areas in the livers of different group mice. Quantitative data are shown as mean ± SEM (n = 5-7 mice per group). *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 5

MSC administration improved Akt phosphorylation and glycolipid metabolism dysfunction and reduced inflammation in the liver. (A) Western blotting analysis of Akt and p-Akt levels in the livers of different group mice. (B) Quantification of the ratio of p-Akt to Akt protein level in mouse livers. n = 4 mice per group. (C-I) Gene expression patterns of G6PC (C), FASN (D), SREBP-1 C (E), PPAR-α (F), F4/80 (G), IFN-γ (H) and TNF-α (I) in the livers of different group mice. n = 6-7 mice per group. Quantitative data are displayed as mean ± SEM. *P < 0.05, ***P < 0.001.

Fig. 6

Working model of perinatal MSCs in alleviating T2DM symptoms. Single-dose intravenous infusion of either AMMSCs or UCMSCs from the same donor comparably alleviates T2DM symptoms in db/db mice through improving glycolipid metabolism, enhancing insulin sensitivity, and decreasing inflammation in the liver.