Differentiation of primate primordial germ cell-like cells following transplantation into the adult gonadal niche

A major challenge in stem cell differentiation validation is the availability of bioassays to prove cell types generated in vitro are equivalent to cells in vivo. In the mouse model, differentiation of primordial germ cell-like cells (PGCLCs) from pluripotent cells was validated by transplantation, leading to the generation of spermatogenesis and to the birth of offspring. Here we report the use of xenotransplantation (monkey to mouse) and homologous transplantation (monkey to monkey) to validate our in vitro protocol for differentiating male rhesus macaque PGCLCs (rPGCLCs) from rhesus macaque induced pluripotent stem cells (riPSCs). Specifically, transplantation of aggregates containing rPGCLCs into mouse and nonhuman primate testicles overcomes a major bottleneck in rPGCLC differentiation with the expression of VASA and MAGEA4, but not ENO2. These findings suggest that immature rPGCLCs once transplanted into an adult gonadal niche commit to differentiate towards late PGCs and spermatogonia-like cells but do not complete the conversion into ENO2-positive spermatogonia.

198 SUCCESSFUL TRANSPLANTATION OF BUFFALO (Bubalus bubalis) GERM CELLS TO HOMOLOGOUS RECIPIENTS

Transplantation of isolated germ cells from a fertile donor male into the seminiferous tubules of infertile recipients can result in donor germ cells-derived sperm production. This technique has the potential to be used as an alternative strategy for producing transgenic livestock with higher efficiency and less time and capital requirement than the current methods. The objective of the present study was to investigate whether the homologous transplantation of isolated buffalo germ cells could result in colonization of recipient testes. Germ cells were isolated from prepubertal buffalo testes (4–6 months of age) by using double enzymatic digestion method and filtration through 80- and 60-µm nylon mesh filters. Further enrichment was achieved by differential plating on Datura stramonium agglutinin lectin-coated dishes and after that Percoll density gradient centrifugation as descrived by van Pelt et al. (1996) with minor modifications. A discontinuous density gradient was prepared with 60, 50, 40, 36, 34, 32, 30, 28, and 20% Percoll in a 15-mL centrifuge tube. The enriched germ cells were then labelled with red fluorescent linker dye PKH26 (Sigma, St. Louis, MO, USA) as per the manufacturer’s instructions, and ~10 million cells/testis were transferred into the rete testis of 3 recipients (16–18 months of age) under ultrasonographic guidance. After 45 days, testes were surgically removed and samples were prepared for analysis of labelled cells via wet mount of seminiferous tubules and individual cells isolation. When wet mount specimen were observed under a fluorescence microscope, PKH26-positive cells were identified on the seminiferous tubule basement membrane in all 3 recipients, which indicated that these cells had successfully migrated from the tubule lumen and were likely to be donor germ cells. In freshly isolated cells, clumps of PKH26-positive cells were observed, which indicated either cell division or extensive local colonization of specific areas of the seminiferous tubules. In conclusion, we report successful homologous transplantation of germ cells in prepubertal buffalo testes. Further studies will investigate functionality of transferred testicular cells.

Medial meniscus transplantation using cyanoacrylate in rabbits

PURPOSE: To evaluate meniscal transplantation using as fixation method a synthetic glue derived from cyanoacrylate acid. METHODS: Twenty rabbits were used, of which 10 for autologous transplantation and 10 for homologous transplantation. For the autologous transplantation the meniscus was removed, and then transplanted in the same animal, using the synthetic glue. For the homologous transplantation, the study was divided into two stages: 1- Removal of the meniscus which was maintained at a temperature of 73ºC. 2- Use of cyanoacrylate acid-derived surgical adhesive for meniscal retransplantation in a different rabbit 30 days after the transplant. RESULTS: Due to complications, euthanasia had to be anticipated to the 15th day in the homologous group and to the 18th day in the autologous group. Macroscopically, knees submitted to transplantation presented whitish secretions from the surgical incision up to deep planes. Necrosis was observed in both groups. Statistical analysis has shown that mild (p=0.043) and moderate (p=0.001) complications emerged in a significantly earlier way in the homologous group, where euthanasia was also performed earlier (p=0,005). CONCLUSION: Synthetic surgical adhesives derived from cyanoacrylate acid promoted cortical to medullary bone necrosis bone in both groups.

Experimental model for composite tissue allotransplantations

In homologous transplantation or allotranplantation of limbs, the great tissue diversity causes variability in the rejection process and, consequently, its immunology is very complex. Thus, limb transplantation is the most used prototype of compound tissue transplantation among the protocols of experimental studies. Composite tissue allotransplantation represents the experimental model to study the homologous transplantation (from an individual to another) of vascularized, innervated musclecutaneous units, joints, bone or even the whole member. Groups of rats were undergone allogeneic hindlimb transplantation. The receptors were randomized and control groups were established as: Control Group A: Autograft controls (F344 rats had its limbs reimplanted) and no immunosuppressive therapy. Control Group B: Allograft controls (BN rats limbs were transplanted to F344). Composite tissue homotransplantation allows the inclusion of innervated muscle-cutaneous units, joint and bone or even the hole limb, is considerably applicable in cases of congenital absence or deformity, trauma or greater resection due to malignant tumor. For many complex deformities, these transplantations would allow a more precise reconstruction than the current reconstruction techniques.

Autologous and homologous transplantation of bovine spermatogonial stem cells

The aim of this study was to develop a method for spermatogonial stem cell transplantation into the bovine testis. Five-month-old Holstein-Friesian calves were used and half of the calves were hemicastrated to allow autologous transplantation and the other half were used for homologous transplantation. Approximately 20 g of each testis was used for cell isolation. On average 106 cells per gram of testis containing about 70% type A spermatogonia were isolated. The cells were frozen in liquid nitrogen until transplantation. Testes were irradiated locally with 10-14 Gy of X-rays to deplete endogenous spermatogenesis. At 2 months after irradiation, cells (approximately 10 x 10(6) were injected into the rete testis through a long injection needle (18 gauge), using ultrasonography and an ultrasound contrast solution. At 2.5 months after transplantation, calves were castrated and samples of testes were taken for histological examination. After 2.5 months in the irradiated non-transplanted control testes, only 45% of the tubules contained type A spermatogonia. However, after autologous spermatogonial transplantation, >80% of the tubule cross-sections contained type A spermatogonia. In addition, only 20% of the tubules of the control testes contained spermatocytes and, except for a few tubules (5%) with round spermatids, no more advanced germ cells were found. After autologous spermatogonial transplantation, about 60% of the tubules contained spermatocytes; 30% contained spermatids and in about 15% of tubules spermatozoa were found. No improvement in spermatogonial repopulation was found after homologous transplantation. The results of this study demonstrate, for the first time, successful autologous transplantation of bovine spermatogonial stem cells resulting in a complete regeneration of spermatogenesis.