What is the best protocol to cryopreserve immature mouse testicular cell suspensions?

Onofre, Jaime; Faes, Katrien; Kadam, Prashant; Vicini, Elena; Pelt, Ans M.M. van; Goossens, Ellen

doi:10.1016/j.rbmo.2018.04.045

Cited by 9 publications

(9 citation statements)

References 46 publications

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“…Donor-derived spermatogenesis was found in 21.4% of successfully injected testes after SSCT which was significantly improved after MSi-SSCT (50%) but is low compared to our previous study (75%) [ 35 ]. The latter could be explained by the aggressive recipient preparation (busulfan and CdCl 2 ) leading to a more severely damaged SSC niche.…”

Section: Discussioncontrasting

confidence: 61%

“…The latter could be explained by the aggressive recipient preparation (busulfan and CdCl 2 ) leading to a more severely damaged SSC niche. But, interestingly, we could achieve up to 19% of donor-derived TFI after MSi-SSCT, while this was only 2% in the SSCT group (9% in our previous study [ 35 ]). Moreover, in all transplanted groups, endogenous spermatogenesis was restored which proves the regenerative potential of MSCs and their supportive role in re-establishing the SSC niche.…”

Section: Discussionmentioning

confidence: 57%

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

Kadam

Ntemou²,

Baert³

et al. 2018

Stem Cell Res Ther

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BackgroundSpermatogonial 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.MethodsA 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).ResultsThe 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 < 0.001). TFI after MSi-SSCT (83.3 ± 19.5%) was significantly higher compared to MS-SSCT (71.5 ± 21.7%, P = 0.036) but did not differ statistically compared to SSCT (78.2 ± 12.5%). In contrast, TFI after MSCT (50.2 ± 22.5%) was significantly lower compared to SSCT (P < 0.001). Interestingly, donor-derived TFI was found to be significantly improved after MSi-SSCT (18.8 ± 8.0%) compared to SSCT (1.9 ± 1.1%; P < 0.001), MSCT (0.0 ± 0.0%; P < 0.001), and MS-SSCT (3.4 ± 1.9%; P < 0.001). While analyses showed that both native and TGFß1-treated MSCs maintained characteristics of MSCs, the latter showed less migratory characteristics and was not detected in other organs.ConclusionCo-transplanting SSCs and TGFß1-treated MSCs significantly improves the recovery of endogenous SSCs and increases the homing efficiency of transplanted SSCs. This procedure could become an efficient method to treat infertility in a clinical setup, once the safety of the technique has been proven.Electronic supplementary materialThe online version of this article (10.1186/s13287-018-1065-0) contains supplementary material, which is available to authorized users.

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Section: Discussioncontrasting

confidence: 61%

Section: Discussionmentioning

confidence: 57%

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

Kadam

Ntemou²,

Baert³

et al. 2018

Stem Cell Res Ther

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“…In contrast to the present model, somatic cells from W/W v mice have never supported spermatogenesis, which may have interfered with epigenetic modification. SSCT using fresh SSCs resulted in significantly higher donor-derived TFI (35%), compared to using cryopreserved SSCs, which resulted only in a donor-derived TFI of only 9% ( P < 0.001) [44]. This confirms the negative impact of the cryopreservation process.…”

Section: Discussionmentioning

confidence: 86%

Does co-transplantation of mesenchymal and spermatogonial stem cells improve reproductive efficiency and safety in mice?

et al. 2019

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BackgroundSpermatogonial 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 (TGFβ1)-induced mesenchymal stem cells (MSCs). In this study, we investigated the reproductive efficiency and safety of co-transplanting spermatogonial stem cells (SSCs) and TGFβ1-induced MSCs.MethodsA mouse model for long-term infertility was used to transplant SSCs (SSCT, n = 10) and a combination of SSCs and TGFβ1-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).ResultsThe 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.ConclusionCo-transplanting SSCs and TGFβ1-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.

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“…Handling of ITT prior to cryopreservation or in vitro culture may influence testicular architecture, endocrine function, and spermatogonial proliferation. Previous studies have provided substantial data to optimize ITT cryopreservation technique [14,[25][26][27]. Due to the unique architecture and constant turnover of stem cells, prepubertal testes may be more prone to impairments in functionality due to the way they are handled, transported, and maintained.…”

Section: Discussionmentioning

confidence: 99%

Impact of Temperature and Time Interval Prior to Immature Testicular-Tissue Organotypic Culture on Cellular Niche

et al. 2020

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Cryopreservation of immature-testicular-tissue (ITT) prior to gonadotoxic treatment, while experimental, is the only recommended option for fertility preservation in prepubertal boys. The handling and manipulation of ITT before cryopreservation could influence the functionality of cells during fertility restoration, which this study explored by evaluating cellular niche and quality of mouse ITT subjected to various temperatures and time durations in vitro. ITT from 6-day-old mice were handled at ultraprofound-hypothermic, profound-hypothermic, and mild-warm-ischemic temperatures for varying time periods prior to 14-day organotypic culture. Viability, functionality, synaptonemal complex and chromatin remodeling markers were assessed. Results have shown that cell viability, testosterone level, and in vitro proliferation ability did not change when ITT were held at ultraprofound-hypothermic-temperature up to 24 h, whereas cell viability was significantly reduced (P < 0.01), when held at profound-hypothermic-temperature for 24 h before culture. Further, cell viability and testosterone levels in cultured cells from profound-hypothermic group were comparable to corresponding ultraprofound-hypothermic group but with moderate reduction in postmeiotic cells (P < 0.01). In conclusion, holding ITT at ultraprofound-hypothermic-temperature is most suitable for organotypic culture, whereas short-term exposure at profound-hypothermic-temperature may compromise postmeiotic germ cell yield post in vitro culture. This data, albeit in mouse model, will have immense value in human prepubertal fertility restoration research.

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What is the best protocol to cryopreserve immature mouse testicular cell suspensions?

Cited by 9 publications

References 46 publications

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

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

Does co-transplantation of mesenchymal and spermatogonial stem cells improve reproductive efficiency and safety in mice?

Impact of Temperature and Time Interval Prior to Immature Testicular-Tissue Organotypic Culture on Cellular Niche

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