The terms blood-testis barrier (BTB) or blood-epididymis barrier (BEB), are often described as Sertoli cell-Sertoli cell tight junctions (TJs) or TJs between the epithelial cells in the epididymis, respectively. However, in reality, the BTB and BEB are much more complex than just the TJ. The focus of this minireview is to remind readers that the complete BTB and BEB are comprised of three components: anatomical, physiological, and immunological. The TJs form the anatomical (physical) barrier that restricts passage of molecules and cells from entering or exiting the lumen. The physiological barrier is comprised of transporters that regulate movement of substances in or out of the lumen, thus creating a microenvironment, which is critical for the proper development and maturation of germ cells. The immunological barrier limits access by the immune system and sequesters the majority of the autoantigenic germ cells. Combined with the overall immune-privilege of the testis, this suppresses detrimental immune responses against the autoantigenic germ cells. These three components on their own do not create a complete functional barrier; instead, it is the interaction between all three components that create a barrier of maximal competence.

The testis as an immune-privileged site allows long-term survival of allogeneic and xenogeneic transplants. Testicular Sertoli cells (SCs) play a major role in this immunoprotection and have been used to create an ectopic immune-privileged environment that prolongs survival of co-transplanted allogeneic and xenogeneic cells, including pancreatic islets and neurons. Extended survival of such grafts testifies to the immunoprotective properties of SCs. However, there is still variability in the survival rates of the co-grafted cells and rarely are 100% of the grafts protected. This emphasizes the need to learn more about what is involved in creating the optimal immunoprotective milieu. Several parameters including organization of the SCs into tubule-like structures and the production of immunomodulatory factors by SCs, specifically complement inhibitors, cytokines, and cytotoxic lymphocyte inhibitors, are likely important. In addition, an intricate interplay between several of these factors may be responsible for providing the most ideal environment for protection of the co-transplants by SCs. In this review, we will also briefly describe a novel use for the immune-privileged abilities of SCs; engineering them to deliver therapeutic proteins for the treatment of diseases like diabetes and Parkinson's disease. In conclusion, further studies and more detailed analysis of the mechanisms involved in creating the immune-protective environment by SCs may make their application in co-transplantation and as engineered cells clinically feasible.

Immune-privileged Sertoli cells survive long term after allogeneic or xenogeneic transplantation without the use of immunosuppressive drugs, suggesting they could be used as a vehicle to deliver therapeutic proteins. As a model to test this, we engineered Sertoli cells to transiently produce basal levels of insulin and then examined their ability to lower blood glucose levels after transplantation into diabetic SCID mice. Mouse and porcine Sertoli cells transduced with a recombinant adenoviral vector containing furin-modified human proinsulin cDNA expressed insulin mRNA and secreted insulin protein. Transplantation of 5–20 million insulin-expressing porcine Sertoli cells into diabetic SCID mice significantly decreased blood glucose levels in a dose-dependent manner, with 20 million Sertoli cells decreasing blood glucose levels to 9.8 ± 2.7 mM. Similar results were obtained when 20 million insulin-positive, BALB/c mouse Sertoli cells were transplanted; blood glucose levels dropped to 6.3 ± 2.4 mM and remained significantly lower for 5 days. To our knowledge, this is the first study to demonstrate Sertoli cells can be engineered to produce and secrete a clinically relevant factor that has a therapeutic effect, thus supporting the concept of using immune-privileged Sertoli cells as a potential vehicle for gene therapy.

The acute cell-mediated immune response presents a significant barrier to xenotransplantation. Immune-privileged Sertoli cells (SC) can prolong the survival of co-transplanted cells including xenogeneic islets, hepatocytes, and neurons by protecting them from immune rejection. Additionally, SC survive as allo- and xenografts without the use of any immunosuppressive drugs suggesting elucidating the survival mechanism(s) of SC could be used to improve survival of xenografts. In this study, the survival and immune response generated toward neonatal pig SC (NPSC) or neonatal pig islets (NPI), nonimmune-privileged controls, was compared after xenotransplantation into naïve Lewis rats without immune suppression. The NPSC survived throughout the study, while NPI were rejected within 9 days. Analysis of the grafts revealed that macrophages and T cells were the main immune cells infiltrating the NPSC and NPI grafts. Further characterization of the T cells within the grafts indicated that the NPSC grafts contained significantly more cluster of differentiation 4 (CD4) and cluster of differentiation 8 (CD8) regulatory T cells (Tregs) at early time points than the NPI grafts. Additionally, the presence of increased amounts of interleukin 10 (IL-10) and transforming growth factor (TGF) β and decreased levels of tumor necrosis factor (TNF) α and apoptosis in the NPSC grafts compared to NPI grafts suggests the presence of regulatory immune cells in the NPSC grafts. The NPSC expressed several immunoregulatory factors such as TGFβ, thrombospondin-1 (THBS1), indoleamine-pyrrole 2,3-dioxygenase, and galectin-1, which could promote the recruitment of these regulatory immune cells to the NPSC grafts. In contrast, NPI grafts had fewer Tregs and increased apoptosis and inflammation (increased TNFα, decreased IL-10 and TGFβ) suggestive of cytotoxic immune cells that contribute to their early rejection. Collectively, our data suggest that a regulatory graft environment with regulatory immune cells including CD4 and CD8 Tregs in NPSC grafts could be attributed to the prolonged survival of the NPSC xenografts.

Xenotransplantation has vast clinical potential but is limited by the potent immune responses generated against xenogeneic tissue. Immune-privileged Sertoli cells (SCs) survive xenotransplantation long term (>90 days) without immunosuppression, making SCs an ideal model to identify xenograft survival mechanisms. Xenograft rejection includes the binding of natural and induced antibodies and the activation of the complement cascade. Using an in vitro cytotoxicity assay, wherein cells were cultured with human serum and complement, we demonstrated that neonatal pig SCs (NPSCs) are resistant to complement mediated cell lysis and express complement inhibitory factors, membrane cofactor protein (MCP; CD46), and decay- accelerating factor (DAF; CD55) at significantly higher levels than neonatal pig islets (NPIs), which served as non-immune-privileged controls. After xenotransplantation into naive Lewis rats, NPSCs survived throughout the study, while NPIs were rejected within 9 days. Serum antibodies, and antibody and complement deposition within the grafts were analyzed. Compared to preformed circulating anti-pig IgM anti bodies, no significant increase in IgM production against NPSCs or NPIs was observed, while IgM deposition was detected from day 6 onward in both sets of grafts. A late serum IgG response was detected in NPSC (days 13 and 20) and NPI (day 20) recipients. Consistently, IgG deposition was first detected at days 9 and 13 in NPSC and NPI grafts, respectively. Interestingly, C3 was deposited at days 1 and 3 in NPI grafts and only at day 1 in NPSC grafts, while membrane attack complex (MAC) deposition was only detected in NPI grafts (at days 1–4). Collectively, these data suggest NPSCs actively inhibit both the alternative and classical pathways of complement-mediated cell lysis, while the alternative pathway plays a role in rejecting NPIs. Ultimately, inhibiting the alternative pathway along with transplanting xenogeneic tissue from transgenic pigs (expressing human complement inhibitory factors) could prolong the survival of xenogeneic cells without immunosuppression.

Conventionally, it was believed that Sertoli cells (SC) stopped proliferating at puberty and became terminally differentiated quiescent cells. However, recent studies have challenged that dogma. In this study, we transplanted nondividing SC isolated from 23- to 27-day-old postpubertal rats transduced with a recombinant adenoviral vector (containing furin-modified human proinsulin cDNA) into diabetic severe combined immunodeficiency mice. Immunostaining the grafts for cell proliferation markers, proliferating cell nuclear antigen (PCNA) and MKI67, revealed that transplanted SC within the grafts were proliferating. Possible causes for resumption of proliferation of SC could be viral transduction, cell isolation and culture, higher abdominal temperature at the transplant site, and/or transplantation. To test for these possible causes, double- immunofluorescence staining was performed for GATA4 (SC marker) and MKI67. None of the SC were positive for MKI67 in tissue collected during SC isolation and culture or at higher temperature. However, nontransduced SC stained positive for MKI67 after transplantation into rats, suggesting viral transduction was not a key factor for induction of SC proliferation. Interestingly, resumption in proliferative ability of nondividing SC was temporary, as SC stopped proliferating within 14 days of transplantation and did not proliferate thereafter. Quantification of 5-bromo-2'-deoxyuridine-labeled SC demonstrated that 7%-9% of the total transplanted SC were proliferating in the grafts. These data indicate for the first time that nondividing SC resumed proliferation after transplantation and further validate previous findings that SC are not terminally differentiated. Hence, transplantation of SC could provide a useful model with which to study the regulation of SC proliferation in vivo.

Spermatogonial stem cells (SSC) are essential for spermatogenesis and male fertility. In addition, these adult tissue stem cells can be used as vehicles for germline modification in animal models and may have application for treating male infertility. To facilitate the investigation of SSCs and germ lineage development in rats, we generated a DEAD-box helicase 4 (DDX4) (VASA) promoter-enhanced green fluorescent protein (EGFP) reporter transgenic rat. Quantitative real-time polymerase chain reaction and immunofluorescence confirmed that EGFP was expressed in the germ cells of the ovaries and testes and was absent in somatic cells and tissues. Germ cell transplantation demonstrated that the EGFP-positive germ cell population from DDX4-EGFP rat testes contained SSCs capable of establishing spermatogenesis in experimentally infertile mouse recipient testes. EGFP-positive germ cells could be easily isolated by fluorescence-activated cells sorting, while simultaneously removing testicular somatic cells from DDX4-EGFP rat pup testes. The EGFP-positive fraction provided an optimal cell suspension to establish rat SSC cultures that maintained long-term expression of zinc finger and BTB domain containing 16 (ZBTB16) and spalt-like transcription factor 4 (SALL4), two markers of mouse SSCs that are conserved in rats. The novel DDX4-EGFP germ cell reporter rat described here combined with previously described GCS-EGFP rats, rat SSC culture and gene editing tools will improve the utility of the rat model for studying stem cells and germ lineage development.

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