Sertoli Cells Engineered to Express Insulin to Lower Blood Glucose in Diabetic Mice

Abstract

Long-term survival of allo- and xenotransplanted immune-privileged Sertoli cells (SCs) is well documented suggesting that SCs can be used to deliver foreign proteins for cell-based gene therapy. The aim of this study was to use a lentivirus carrying proinsulin cDNA to achieve stable expression and lowering of blood glucose levels (BGLs). A SC line transduced with the lentivirus (MSC-LV-mI) maintained stable insulin expression in vitro. These MSC-LV-mI cells were transplanted and grafts were analyzed for cell survival, continued proinsulin mRNA, and insulin protein expression. All grafts contained MSC-LV-mI cells that expressed proinsulin mRNA and insulin protein. Transplantation of MSC-LV-mI cells into diabetic mice significantly lowered BGLs for 4 days after transplantation. Interestingly, in three transplanted SCID mice and one transplanted BALB/c mouse, the BGLs again significantly lowered by day 50 and 70, respectively. This is the first time SC transduced with a lentiviral vector was able to stably express insulin and lower BGLs. In conclusion, a SC line can be modified to stably express therapeutic proteins (e.g., insulin), thus taking us one step further in the use of SCs as an immune-privileged vehicle for cell-based gene therapy.

Keywords: Sertoli cells; cell-based gene therapy; insulin; lentivirus; transplantation.

FIG. 1.

Mouse Sertoli cells transduced with LV-mI express insulin mRNA and protein. (A) LV-mI, contains a cPPT; CMV promoter; furin-modified mouse proinsulin 2 cDNA (mIns); IRES; eGFP cDNA; WPRE and the Bsd resistance gene. (B) RT-PCR was performed to detect mouse proinsulin mRNA. β-actin was used as a control. RT negative (−ve) controls containing RNA instead of cDNA was used to rule out genomic DNA contamination. (C) Cell supernatant from MSC-LV-mI and MSC-EhI-Zs cells was collected and ELISA was performed to detect insulin secretion. Data shown are the mean ± SEM. The asterisk denotes a significant difference in insulin secretion by MSC-LV-mI (blue bar) compared with MSC-EhI-Zs (green bar) cells as determined by unpaired Student's t-test (p ≤ 0.05). (D) MSC-1 cells transduced with a lentiviral vector containing furin-modified mouse proinsulin cDNA (MSC-LV-mI) were immunostained for insulin (red, D). (E) MSC-LV-mI cells immunostained with secondary antibody were used as negative controls. Cell nuclei were immunostained with DAPI (blue, D and E). Bsd, Blasticidin; CMV, cytomegalovirus; cPPT, central polypurine tract; eGFP, enhanced green fluorescent protein; IRES, internal ribosome entry site; SEM, standard error of the mean; WPRE, Woodchuck Posttranscriptional Regulatory Element.

FIG. 2.

Six million MSC-LV-mI cells transplanted to BALB/c mice. Six million MSC-LV-mI were transplanted into diabetic BALB/c mice (n = 8). (A and C) The graft-bearing kidneys were collected at day 20 posttransplantation and immunostained for large T antigen (MSC-1 cell marker, brown, A, n = 5) and insulin (brown, C, n = 5). (E) Cells from the MSC-LV-mI grafts were collected at day 20, cultured for 2 days, and immunostained for insulin (red, E, n = 3). (G) Average BGLs of animals transplanted with MSC-LV-mI, MSC-EhI-Zs, or nontransduced MSC-1 cells. Bars represent mean ± SD. Statistical significance of difference versus day 0 was calculated by one-way ANOVA followed by Tukey's post hoc test, ^#represents p = 0.056. (H) RT-qPCR was performed for mouse proinsulin using mRNA isolated from MSC-LV-mI (white bar, n = 4) or nontransduced MSC-1 (black bar, n = 4) cell grafts collected from mice 20 days posttransplantation. Data shown are the mean ± SEM. The asterisk denotes a significant difference in MSC-LV-mI insulin mRNA expression compared with nontransduced MSC-1 cells as determined by unpaired Student's t-test (p ≤ 0.05). Cell nuclei were counterstained with Hematoxylin (blue, A–D) or DAPI (blue, E and F). Insets are the high magnification images of (A) and (C). Arrows in the inset (C) indicate insulin-positive MSC-LV-mI cells. (B, D, and F) are negative controls for (A, C, and E), respectively. In (A, B, and D), the dotted line separates the graft (above) from the kidney (below). BGL, blood glucose level; SD, standard deviation; ANOVA, analysis of variance.

FIG. 3.

Twenty million MSC-LV-mI cells transplanted to SCID mice. (A) Twenty million MSC-LV-mI (n = 11) or nontransduced MSC-1 (n = 10) cells were transplanted underneath the kidney capsule of diabetic SCID mice and BGLs were measured. (B) Data represent the average BGLs of three mice, transplanted with MSC-LV-mI cells, which had decreased BGLs at the end of the study (below 20 mM). Bars represent mean ± SD. Statistical significance of difference versus day 0 was calculated by one-way ANOVA followed by Tukey's post hoc test, * = p ≤ 0.05. (C and I) The MSC-LV-mI (C, n = 6) or nontransduced MSC-1 (I, n = 10) cell grafts were collected at day 50 posttransplantation and immunostained for large T antigen (brown). (E) The MSC-LV-mI grafts were immunostained for insulin (brown, n = 6). (G) Cells from the MSC-LV-mI grafts (n = 4) were collected, cultured for 2 days, and immunostained for insulin (red). (J) RT-qPCR was performed for mouse proinsulin using mRNA isolated from MSC-LV-mI (white bar, n = 6) or nontransduced MSC-1 (black bar, n = 6) grafts collected at 50 days posttransplantation. Data shown are the mean ± SEM. The asterisk denotes a significant difference in MSC-LV-mI insulin mRNA expression compared with nontransduced MSC-1 cells as determined by unpaired Student's t-test (p ≤ 0.05). (D, F, and H) are negative controls for (C, E, and G), respectively. Cell nuclei were counterstained with Hematoxylin (blue, C–F) or DAPI (blue, G and H). Insets are the high magnification images of (C, E, and I). Arrows in the inset (E) indicate insulin-positive MSC-LV-mI cells. In (C, D, and I), the dotted line separates the graft (above) from the kidney (below).

FIG. 4.

Twenty million MSC-LV-mI cells transplanted to BALB/c mice. (A) Twenty million MSC-LV-mI (n = 10) or nontransduced MSC-1 cells (n = 3) were transplanted underneath the kidney capsule of diabetic BALB/c mice and BGLs were measured. (B) Data represent the individual BGLs of three mice, transplanted with MSC-LV-mI cells that had achieved euglycemia at day 1 posttransplantation. Bars represent mean ± SD. Statistical significance of difference versus day 0 was calculated by one-way ANOVA followed by Tukey's post hoc test. (C and I) The MSC-LV-mI (C, n = 7) and nontransduced MSC-1 cell (I, n = 3) grafts were collected at day 50 and 70 posttransplantation, respectively, and immunostained for large T antigen (brown). (E) The MSC-LV-mI grafts were immunostained for insulin (brown, E, n = 7). (G) Cells were isolated from MSC-LV-mI grafts (n = 3) at day 50, cultured for 2 days, and immunostained for insulin (red). (J) RT-qPCR was performed for mouse proinsulin using mRNA isolated from MSC-LV-mI (white bar, n = 7) or nontransduced MSC-1 (black bar, n = 3) grafts collected from mice at 50 and 70 days posttransplantation, respectively. Data shown are the mean ± SEM. The asterisk denotes a significant difference in MSC-LV-mI insulin mRNA expression compared with nontransduced MSC-1 cells as determined by unpaired Student's t-test (p ≤ 0.05). (D, F, and H) are negative controls for (C, E, and G), respectively. Cell nuclei were counterstained with Hematoxylin (blue, C–F) or DAPI (blue, G and H). Arrows in the inset (E) indicate insulin-positive MSC-LV-mI cells. Insets are the high magnification images of (C, E, and I). In (C, D, and I), the dotted line separates the graft (above) from the kidney (below).