Transplantation of betatrophin-expressing adipose-derived mesenchymal stem cells induces β-cell proliferation in diabetic mice

Figures

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  Figure 1. Biological characteristics of adipose-derived mesenchymal stem cells (ADMSCs) overexpressing betatrophin. (A) RT-qPCR of betatrophin mRNA expression following transduction of ADMSCs with a control lentivirus (MSC) and betatrophin-expressing lentivirus (MSC-BET). Data represent the mean ± standard deviation; n=3 for each bar. (B) Western blot analysis for betatrophin expression following transduction of ADMSCs with a control lentivirus (ADMSCs) and betatrophin-expressing lentivirus (ADMSCs-BET). (C) Phase contrast images of adherent fibroblast-like AdMSCs and AdMSCs-BET. (D) Histogram of ADMSCs stained with PI. The G0/G1 and G2/M peaks are separated by the S phase distribution.

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  Figure 2. Surface phenotype and differentiation ability of adipose-derived mesenchymal stem cells (ADMSCs) overexpressing betatrophin. (A) ADMSCs and ADMSCs-BET were incubated with specific surface marker antibodies or isotype control antibodies and subjected to flow cytometry analysis. (B) Representative images of ADMSCs and ADMSCs-BET differentiation. Images are shown at ×100 magnification.

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  Figure 3. Betatrophin-expressing adipose-derived mesenchymal stem cells (ADMSCs) promote the proliferation of human islets in a co-culture system. (A) Staining for the proliferation marker, Ki67 (red), and nuclei (DAPI, blue) in human islets cultured alone (islet) or with ADMSCs (ADMSCs + islets) or ADMSCs-BET (ADMSCs-BET + islets). (B) Proportion of Ki67-positive cells among islet, ADMSCs + islets, and ADMSCs-BET + islet groups. Data are represented as the means ± SD (n=3 per condition). *,†P<0.0167, significant difference compared with *Islets only, and †ADMSCs + islets.

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  Figure 4. Betatrophin-expressing adipose-derived mesenchymal stem cells (ADMSCs) promote the function of human islets in a co-culture system. (A) Insulin and glucagon mRNA levels in islets cultured alone (islets) or with ADMSCs (ADMSCs + islets) or ADMSCs-BET (ADMSCs-BET + islet). (B) mRNA levels of transcription factors in islets cultured alone (islet) or with ADMSCs (ADMSCs + islet) or ADMSCs-BET (ADMSCs-BET + islets). (C) Insulin levels from the four indicated groups are shown upon stimulation with Arg and KCl at 20 and 50 min. (D) Glucose-induced insulin secretion in islets cultured alone (islets) or with ADMSCs (ADMSCs + islets) or ADMSC-BET (ADMSCs-BET + islets) after stimulation with 60 and 300 mg/dl of glucose. (E) The ADP/ATP ratio of the 3 different groups was detected.

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  Figure 5. Enhanced anti-inflammatory and anti-apoptotic effects of adipose-derived mesenchymal stem cell (ADMSCs)-BET co-cultured with islets. (A) The protein levels of inflammatory- and apoptosis-related proteins in the supernatant of the human islets cultured alone (islet) or with ADMSCs (MSC + islet) or ADMSCs-BET (ADMSCs-BET + islets). Data are presented as the mean ± standard deviation; n=3 for each bar. (B) Western blot analysis of active caspase-3 protein levels in islets cultured alone (islets) or with ADMSCs (ADMSCs + islets) or ADMSCs-BET (ADMSCs-BET + islets). (C) Single cell suspensions derived from different co-culture settings were stained with Annexin V and PI and followed by flow cytometric analysis.

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  Figure 6. Reversion of the hyperglycemic condition and body weight in mice with streptozotocin (STZ)-induced diabetes by adipose-derived mesenchymal stem cells (ADMSCs). Mice with STZ-induced diabetes were transplanted with 1×106 ADMSCs (ADMSCs) or ADMSCs-BET (ADMSCs-BET) via a tail vein injection. Mice in control group received PBS injected via the tail vein. Blood glucose levels (A) and body weight (B) were determined every 7 days throughout the 28-day study period (n=8 for each line). (C) The ratio of β-cells in the islets (n=3 for each bar). Data are presented as the means ± standard deviation. All measurements were all performed on the same day for each group; however, the values for each measurement were plotted to avoid overlapping of SD bars from each group. *†‡Significant difference as compared with the *control, †ADMSCs and ‡healthy BALB/c groups.

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  Figure 7. Administration of adipose-derived mesenchymal stem cells (ADMSCs)-BET enhances the proliferation of islets in vivo. Mice with streptozotocin (STZ)-induced diabetes were transplanted with 1×106 ADMSCs or ADMSCs-BET (ADMSCs-BET) via a tail vein injection. Mice in control group received PBS injected via the tail vein. The islets were harvested at 7 days following the ADMSC injection, and sections were stained with anti-vimentin (control), anti-insulin, anti-Ki67 antibodies.

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  Figure 8. Adipose-derived mesenchymal stem cell (ADMSCs)-BET induce betatrophin overexpression and increased C-peptide and insulin levels in streptozotocin (STZ)-induced diabetic mice. The betatrophin expression levels in (A) liver and (B) blood plasma as well as the levels of plasma (C) C-peptide and (D) insulin were determined on day 28 following injection of ADMSCs (ADMSCs) or those overexpressing betatrophin (MSC-BET). Data are presented as the means ± standard deviation; n=3 for each bar.