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Co-transplantation of mesenchymal stem cells improves spermatogonial stem cell transplantation efficiency in mice.

ABSTRACT: BACKGROUND:Spermatogonial stem cell transplantation (SSCT) could become a fertility restoration tool for childhood cancer survivors. However, since in mice, the colonization efficiency of transplanted spermatogonial stem cells (SSCs) is only 12%, the efficiency of the procedure needs to be improved before clinical implementation is possible. Co-transplantation of mesenchymal stem cells (MSCs) might increase colonization efficiency of SSCs by restoring the SSC niche after gonadotoxic treatment. METHODS:A mouse model for long-term infertility was developed and used to transplant SSCs (SSCT, n?=?10), MSCs (MSCT, n =?10), a combination of SSCs and MSCs (MS-SSCT, n?=?10), or a combination of SSCs and TGFß1-treated MSCs (MSi-SSCT, n?=?10). RESULTS:The best model for transplantation was obtained after intraperitoneal injection of busulfan (40 mg/kg body weight) at 4 weeks followed by CdCl2 (2 mg/kg body weight) at 8 weeks of age and transplantation at 11 weeks of age. Three months after transplantation, spermatogenesis resumed with a significantly better tubular fertility index (TFI) in all transplanted groups compared to non-transplanted controls (P?

SUBMITTER: Kadam P

PROVIDER: S-EPMC6249754 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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Co-transplantation of mesenchymal stem cells improves spermatogonial stem cell transplantation efficiency in mice.

Kadam Prashant P Ntemou Elissavet E Baert Yoni Y Van Laere Sven S Van Saen Dorien D Goossens Ellen E

Stem cell research & therapy 20181121 1

<h4>Background</h4>Spermatogonial stem cell transplantation (SSCT) could become a fertility restoration tool for childhood cancer survivors. However, since in mice, the colonization efficiency of transplanted spermatogonial stem cells (SSCs) is only 12%, the efficiency of the procedure needs to be improved before clinical implementation is possible. Co-transplantation of mesenchymal stem cells (MSCs) might increase colonization efficiency of SSCs by restoring the SSC niche after gonadotoxic trea ...[more]

PMID: 30463610

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Project description:BACKGROUND:Spermatogonial stem cell transplantation (SSCT) is a promising therapy in restoring the fertility of childhood cancer survivors. However, the low efficiency of SSCT is a significant concern. SSCT could be improved by co-transplanting transforming growth factor beta 1 (TGF?1)-induced mesenchymal stem cells (MSCs). In this study, we investigated the reproductive efficiency and safety of co-transplanting spermatogonial stem cells (SSCs) and TGF?1-induced MSCs. METHODS:A mouse model for long-term infertility was used to transplant SSCs (SSCT, n?=?10) and a combination of SSCs and TGF?1-treated MSCs (MSi-SSCT, n?=?10). Both transplanted groups and a fertile control group (n?=?7) were allowed to mate naturally to check the reproductive efficiency after transplantation. Furthermore, the testes from transplanted males and donor-derived male offspring were analyzed for the epigenetic markers DNA methyltransferase 3A (DNMT3A) and histone 4 lysine 5 acetylation (H4K5ac). RESULTS:The overall tubular fertility index (TFI) after SSCT (76?±?12) was similar to that after MSi-SSCT (73?±?14). However, the donor-derived TFI after MSi-SSCT (26?±?14) was higher compared to the one after SSCT (9?±?5; P?=?0.002), even after injecting half of the number of SSCs in MSi-SSCT. The litter sizes after SSCT (3.7?±?3.7) and MSi-SSCT (3.7?±?3.6) were similar but differed significantly with the control group (7.6?±?1.0; P?<?0.001). The number of GFP+ offspring per litter obtained after SSCT (1.6?±?0.5) and MSi-SSCT (2.0?±?1.0) was also similar. The expression of DNMT3A and H4K5ac in germ cells of transplanted males was found to be significantly reduced compared to the control group. However, in donor-derived offspring, DNMT3A and H4K5ac followed the normal pattern. CONCLUSION:Co-transplanting SSCs and TGF?1-treated MSCs results in reproductive efficiency as good as SSCT, even after transplanting half the number of SSCs. Although transplanted males showed lower expression of DNMT3A and H4K5ac in donor-derived germ cells, the expression was restored to normal levels in germ cells of donor-derived offspring. This procedure could become an efficient method to restore fertility in a clinical setup, but more studies are needed to ensure safety in the long term.

Project description:Prepubertal cancer treatment leads to irreversible infertility in half of the male patients. Current in vitro spermatogenesis protocols and cryopreservation techniques are inadequate to expand spermatogonial stem/progenitor cells (SSPC) from testicles. Bone marrow derived mesenchymal stem cells (BM-MSC) bearing a close resemblance to Sertoli cells, improved spermatogenesis in animal models. We asked if a co-culture setup supported by syngeneic BM-MSC that contributes to the air-liquid interphase (ALI) could lead to survival, expansion and differentiation of SSPCs in vitro. We generated an ALI platform able to provide a real-time cellular paracrine contribution consisting of syngeneic BM-MSCs to neonatal C57BL/6 mice testes. We aimed to evaluate the efficacy of this culture system on SSPC pool expansion and spermatogenesis throughout a complete spermatogenic cycle by measuring the number of total germ cells (GC), the undifferentiated and differentiating spermatogonia, the spermatocytes and the spermatids. Furthermore, we evaluated the testicular cell cycle phases, the tubular and luminal areas using histochemical, immunohistochemical and flow cytometric techniques. Cultures in present of BM-MSCs displayed survival of ID4(+) spermatogonial stem cells (SSC), expansion of SALL4(+) and OCT4(+) SSPCs, VASA(+) total GCs and Ki67(+) proliferative cells at 42 days and an increased number of SCP3(+) spermatocytes and Acrosin(+) spermatids at 28 days. BM-MSCs increased the percentage of mitotic cells within the G2-M phase of the total testicular cell cycle increased for 7 days, preserved the cell viability for 42 days and induced testicular maturation by enlargement of the tubular and luminal area for 42 days in comparison to the control. The percentage of PLZF(+) SSPCs increased within the first 28 days of culture, after which the pool started to get smaller while the number of spermatocytes and spermatids increased simultaneously. Our findings established the efficacy of syngeneic BM-MSCs on the survival and expansion of the SSPC pool and differentiation of spermatogonia to round spermatids during in vitro culture of prepubertal mice testes for 42 days. This method may be helpful in providing alternative cures for male fertility by supporting in vitro differentiated spermatids that can be used for round spermatid injection (ROSI) to female oocyte in animal models. These findings can be further exploited for personalized cellular therapy strategies to cure male infertility of prepubertal cancer survivors in clinics.

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Co-transplantation of mesenchymal stem cells improves spermatogonial stem cell transplantation efficiency in mice.

Publications

Co-transplantation of mesenchymal stem cells improves spermatogonial stem cell transplantation efficiency in mice.

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