Therapeutic Effects of Adipose Stem Cells from Diabetic Mice for the Treatment of Type 2 Diabetes

Abstract

To assess the potential therapeutic effects of adipose tissue-derived mesenchymal stem cells (ASCs) for the treatment of type 2 diabetes (T2D), we compared the phenotype and functionality of ASCs isolated from high-fat diet and streptozotocin (STZ)-induced T2D and the leptin receptor-deficient (db/db) mice with cells from healthy C57BL/6 mice. ASCs from T2D or db/db mice showed similar expression patterns of cellular markers and abilities to differentiate into adipocytes, osteoblasts, and chondrocytes. However, the rate of proliferation was reduced. ASCs from db/db mice secreted less hepatocyte growth factor (HGF). T2D mice receiving a single intravenous injection of T2D or db/db ASCs showed increased insulin sensitivity, reduced inflammation and fat content in adipose tissue and the liver and increased pancreatic β cell mass through 5 weeks post-infusion. Our data show that, although ASCs from T2D or db/db mice had inferior proliferative capacity compared to cells from healthy controls, improved insulin sensitivity and less β cell death was seen in T2D mice receiving mesenchymal stem cell (MSC) therapy. This study offers evidence that ASCs from diabetic donors have the potential to be used for cell therapy in the treatment of insulin resistance and T2D.

Keywords: adipose stem cells; cell therapy; db/db mice; insulin resistance; type 2 diabetes; type 2 diabetes mouse model; β cell mass.

Graphical abstract

Figure 1

Generation of T2D Mouse Model by HFD Feeding and STZ Injection (A) Changes in body weights in C57BL/6 mice fed with normal chow (chow, n = 15) or high-fat diet with STZ (T2D, n = 39). (B) Random (non-fasting) blood glucose levels in C57BL/6 mice fed with normal chow or high-fat diet and STZ injection. (C and D) Blood glucose levels (C) and area under the curve (D) in chow and T2D mice during the IPGTT. (E and F) Blood glucose levels (E) and area above the curve (F) during the ITT in chow and T2D mice before cell infusion (5–10 mice were included in each group). *p < 0.05 and **p < 0.01, ANOVA test. Error bars represent SD.

Figure 2

Characterization of ASCs Harvested from Different Donors (A) Representative micrographs of ASCs from C57BL/6, T2D, and db/db mice observed under light microscopy. (B) Representative micrographs of ASC-derived adipocytes identified by oil red O staining, osteocytes by alizarin red staining, and chondrocytes by toluidine blue staining. Scale bars, 100 μm. (C) Expression of CD105, CD29, CD45, and CD34 in ASCs analyzed by flow cytometry. Red lines represent cells stained with corresponding isotype control antibodies, and blue lines represent cells stained with individual antibodies. (D) Growth curves of C57BL/6, T2D, and db/db ASCs. (E–G) Amounts of HGF (E), TGF-β (F), and VEGF (G) secreted by different ASCs. Cells at passage three were used for the experiments. Data are from at least three individual experiments. **p < 0.01, ANOVA test. Error bars represent SD.

Figure 3

The Effects of ASC Infusion on Mouse Body Weights and Blood Glucose Levels (A and B) Changes in body weights (A) and percentages of body weight changes (B) after ASC injection in chow-fed mice receiving PBS; T2D mice receiving PBS; and T2D mice receiving C57BL/6, T2D, or db/db ASCs. (C and D) Non-fasting blood glucose levels (C) and percentages of changes of blood glucoses (D) in chow-fed mice and T2D mice receiving C57BL/6, T2D, or db/db ASCs. Each group contains 6–8 mice. Error bars represent SD.

Figure 4

Metabolic Phenotypes of Mice at 2 and 5 Weeks after Treatment (A and B) Blood glucose levels (A) and areas under the curve (B) during the IPGTT in chow-fed mice and T2D mice receiving PBS or C57BL/6, T2D, or db/db ASCs at 2 weeks after treatment. (C and D) Blood glucose levels (C) and areas under the curve (D) in mice at 2 weeks after cell infusion. (E and F) Blood glucose levels (E) and areas under the curve (F) during the IPGTT in chow-fed mice or T2D mice receiving C57BL/6, T2D, or db/db ASCs at 5 weeks after ASC or PBS infusion. (G and H) Blood glucose levels (G) and areas above the curve (H) during the ITT at 5 weeks after cell infusion. n = 4–6 per group; *p < 0.05 and **p < 0.01, Student’s t test. Error bars represent SD.

Figure 5

ASC Infusion Increases Pancreatic β Cell Mass (A) Representative immunofluorescence staining of pancreases harvested from chow-fed mice and T2D mice receiving PBS or C57BL/6, T2D, or db/db ASCs. Red represents insulin⁺ cells. Blue stains for nuclei. Scale bars, 100 μm. (B) Statistics of insulin⁺ area in pancreases from each treatment group. (C) Plasma insulin levels in chow-fed mice and T2D mice receiving PBS or C57BL/6, T2D, or db/db ASCs (n = 6 in each group). (D–F) Relative mRNA expressions of TNF-α (D), NOD2 (E), and F4/80 (F) in pancreases of chow-fed mice or T2D mice receiving PBS or C57BL/6, T2D, or db/db ASCs at 5 weeks after cell infusion, measured by RT-PCR analysis. At least three mice were included in each group. *p < 0.05 and **p < 0.01, ANOVA test. Error bars represent SD.

Figure 6

ASC Infusion Reduces Adiposity in Adipose Tissue (A) Epididymal fat weights of chow-fed mice and T2D mice receiving PBS or C57BL/6, T2D, or db/db ASCs. (B) Representative micrographs of H&E staining of adipose tissue sections from mice from each treatment group. Scale bar, 100 μm. (C) Mean fat bubble diameters (n = 200) of epididymal adipocytes of mice from each group. Tissues from at least 3 mice in each group were analyzed. p < 0.05 and **p < 0.01, Student’s t test. Error bars represent SD.

Figure 7

ASC Infusion Reduces Liver Steatosis and Inflammation and Increases Insulin Receptor Expression in Liver (A) Liver weights in chow-fed mice and T2D mice receiving PBS or C57BL/6, T2D, or db/db ASCs at 5 weeks after cell infusion. (B) Representative micrographs of H&E staining of liver tissue sections. Scale bar, 100 μm. (C) Mean vacuole diameters in livers. (D–G) Relative mRNA expressions of IL-6 (D), TNF-α (E), F4/80 (F), and InsR (G) in livers. Samples from 5–6 individual mice were analyzed. *p < 0.05 and **p < 0.01, one-way ANOVA. Error bars represent SD.