Haploid Germ Cells Generated in Organotypic Culture of Testicular Tissue From Prepubertal Boys

Abstract

While in mice various studies have described the completion of spermatogenesis in vitro using either organotypic culture of prepubertal testicular tissue or 3D culture of isolated cells, in humans it has not been possible to achieve germ cell differentiation from immature testicular tissue (ITT). In our study, we evaluated the ability of human ITT to differentiate via a long-term organotypic culture of frozen–thawed 1 mm3 testicular fragments from five prepubertal boys in two different culture media. Tissue and supernatants were analyzed at regular intervals up to day 139. Sertoli cell (SC) viability and maturation was evaluated using immunohistochemistry (IHC) for SOX9, GDNF, anti-Mullerian hormone (AMH) and androgen receptor (AR), and AMH concentration in supernatants. Spermatogonia (SG) and proliferating cells were identified by MAGE-A4 (for SG) and Ki67 (for proliferating cells) via immunohistochemistry (IHC). Apoptotic cells were studied by active caspase 3. To evaluate Leydig cell (LC) functionality testosterone was measured in the supernatants and steroidogenic acute regulatory protein (STAR) IHC was performed. Germ cell differentiation was evaluated on Hematoxylin-Eosin histological sections, via IHC for synaptonemal complex 3 (SYCP3) for spermatocytes, Protein boule-like (BOLL) for spermatocytes and round spermatids, angiotensin-converting enzyme (ACE), protamine 2 and transition protein 1 (for elongated spermatids) and via chromogenic in situ hybridization (CISH). We reported the generation of meiotic and postmeiotic cells after 16 days of culture, as shown by the histological analyses, the presence of differentiation markers and the increase of haploid germ cells. We showed SC viability and maturation by a decrease of AMH secretion in the supernatants (p ≤ 0.001) while the number of SOX9 positive cells did not show any variation. A decrease of spermatogonia (p ≤ 0.001) was observed. The number of apoptotic cells did not vary. LC functionality was shown by the increase in STAR expression (p ≤ 0.007) and a peak in testosterone secretion, followed by a reduction (p ≤ 0.001) with stabilization. According to our knowledge, this is the first report of generation of haploid cells in human ITT. Differentiating germ cells have to be further evaluated for their ability to complete differentiation, their fecundability and epigenetic characteristics.

<p>Role of stem cells in fertility preservation: current insights</p>

“…22 These diploid SCs are able both to self-renew and give rise to differentiated haploid cells at the end of the spermatogenic process. 19 Due to the smallness of testicular biopsies taken for cryopreservation in prepubertal boys, the scarcity of SSCs in the testes, 23 the low efficiency of the transplantation process observed in mice and nonhuman primates, 24,25 and the low proportion of human haploid germ cells generated with IVM, 26 amplification of SSCs is an essential step for fertility restoration.…”

“…50 Culturing unsorted cells prior to cell selection has also been attempted, showing that 50 days in the same culture conditions followed by isolation of ITGα6 + cells resulted in a seven fold enrichment of SSCs. 43 Exclusion of articles in language other than English (26), guidelines (6), reviews (192), scientific video protocols (2), peer comments (5), PhD résumé (1) and erratum (1) Together, these results point to the need to identify the best method to propagate SSCs most efficiently. Recently, Bhang et al discovered that human endothelial TCs secreted GDNF, bFGF, stromal cell-derived factor-1 (SDF-1), macrophage inflammatory protein 2, and insulin-like growth factor-binding protein 2 and could support SSC growth for at least 150 days.…”

“…107,108 Recently, a long-term organotypic culture of human ITT able to preserve ST integrity and Leydig cell functionality and achieve Sertoli cell maturation with partial establishment of the blood-testicular barrier 109,110 eventually led to the generation of haploid germ cells. 26 As a decrease in spermatogonial numbers and only a few postmeiotic germ cells were observed, the next hurdles to overcome before clinical translation are enhancing the efficiency of the technique and demonstrating the fertilizing capacity and genetic integrity of in vitro matured cells. Recently, Ogawa developed a microfluidic culture system allowing growth of mice ITT for up to 6 months and resulting in higher spermatogenesis efficiency compared to standard organotypic culture, which could eventually address issues that have been encountered with human tissue.…”

Spermatogonial Stem Cells for In Vitro Spermatogenesis and In Vivo Restoration of Fertility

“…Frozen-thawed 1-mm 3 testicular fragments collected from prepubertal boys (aged 2-12 years) were cultured in a KSR-based medium supplemented with 5 IU/L of FSH, which after 16 days resulted in the formation of round spermatids. Interestingly, increasing the concentration of FSH from 5 to 50 IU/L failed to induce germ cell differentiation, perhaps due to desensitization of FSH receptors or adenyl cyclase in Sertoli cells [128]. However, it should be noted that currently such in vitro produced haploid male germ cells only serve as a proof-of-principle and would need be further analyzed for their fecundability and epigenetic characteristics before suggesting these techniques for potential clinical trials.…”

Fertility sparing strategies for pre- and peripubertal male cancer patients

“…Hence, explant tissue or organotypic culture that maintains such 3D-organised microenvironment seems to be the most promising approach based on successful differentiation of murine SSCs into functional sperm in vitro [40] and the generation of human haploid germ cells from testicular tissue samples of cancer patients between 2 and 12 years of age [41,42]. However, none of the reported studies demonstrated a system robust enough to differentiate human SSCs into functional sperm so far.…”