Genetically engineered immune privileged Sertoli cells: A new road to cell based gene therapy

Abstract

Sertoli cells are immune privileged cells, important for controlling the immune response to male germ cells as well as maintaining the tolerogenic environment in the testis. Additionally, ectopic Sertoli cells have been shown to survive and protect co-grafted cells when transplanted across immunological barriers. The survival of ectopic Sertoli cells has led to the idea that they could be used in cell based gene therapy. In this review, we provide a brief overview of testis immune privilege and Sertoli cell transplantation, factors contributing to Sertoli cell immune privilege, the challenges faced by viral vector gene therapy, the use of immune privileged cells in cell based gene therapy and describe several recent studies on the use of genetically engineered Sertoli cells to provide continuous delivery of therapeutic proteins.

Figure 1. SC isolated from transgenic mice (TgN (GFPU) 5Nagy) survive and express GFP when transplanted as allografts in BALB/c mice. Thirteen million SC were transplanted into the renal subcapsular space of BALB/c mice. The graft bearing kidney was collected at day 60 post-transplantation (A and B) and double immunostained for GATA-4 (SC marker, A) and GFP (B).

Figure 2. Production of insulin protein by NPSC transduced with adenoviral vector carrying furin modified human insulin cDNA. (A and B) NPSC were cultured overnight as a monolayer on chamber slides, transduced with Ad-CMV-HI vector at a MOI of 0 (A) and 100 (B). Slides were collected after 24hrs, fixed with 1% paraformaldehyde and immunostained for insulin (A and B). Nontransduced SC do not express insulin (A), while transduced SC express insulin (brown, B).Twenty million NPSC transduced with Ad-CMV-HI vector (MOI 100) were transplanted into the renal subcapsular space of diabetic SCID mice. Graft bearing kidneys were collected at days 1 (C and D) and 10 (E and F) post-transplantation and immunostained for NPSC marker, vimentin (antibody does not cross react with mouse tissue) (brown, C and E) or insulin (brown, D and F). All the sections were counterstained with hematoxylin (blue).

Figure 3. Insulin mRNA production by MSC-1-LVEF-HI-ZsGreen cells. (A) Transduced MSC-1 cells were grown as monolayer in DMEM plus 10% fetal bovine serum plus 250 ug/ml of G418 for 9 mo. RT-PCR was performed on MSC-1-LV-HI-GFP cells (Lanes 2 and 3) or non-transduced MSC-1 cells (Lanes 4 and 5) for insulin (Lanes 2 and 4) and cyclophilin (Lanes 3 and 5). Lane 1 is the 1kb plus DNA Ladder (Invitrogen). (B) Twenty million transduced MSC-1 cells were transplanted into the renal subcapsular space of diabetic BALB/c mice. RT-PCR was performed on grafts collected at days 1 (Lanes 2 and 3), 5 (Lanes 4 and 5), 12 (Lanes 6 and 7) and 20 (Lanes 8 and 9) post-transplantation for insulin (Lanes 2, 4, 6 and 8) and cyclophilin (Lanes 3, 5, 7 and 9). Lane 1 is the 1kb DNA Ladder (Invitrogen). Care and maintenance of animals described in Figure 3B, 4C and D was performed in accordance with the Institute for Laboratory Animal Research Care and Use of Laboratory Animals, and Texas Tech University Institutional Animal Care and Use Committee-approved protocols.

Figure 4. Immunohistochemical analysis of MSC-1-LVEF-HI-ZsGreen cells and detection of insulin after transplantation. (A and B) MSC-1-LVEF-HI-ZsGreen cells were grown in culture for 9 mo as described in the legend for Figure 3A. Immunofluorescence was performed to detect GFP (A) and insulin (B) protein expression. Cells were counterstained with blue hoechst dye to detect cell nuclei. C-D) Transduced MSC-1 cells (20 million) were transplanted into the renal subcapsular space of diabetic BALB/c mice. The graft bearing kidney was collected at day 20 post-transplantation and immunostained for the MSC-1 cell marker, larger T-antigen (brown, C) and insulin (D). Insets are higher magnification of C and D. Sections were counterstained with hematoxylin (blue).