Primary culture of germ cells that portray stem cell characteristics and recipient preparation for autologous transplantation in the rhesus monkey

Abstract

Fertility preservation for prepubertal cancer patients prior to oncologic treatment is an emerging issue, and non‐human primates are considered to constitute suitable models due to the limited availability of human testicular tissues. However, the feasibility of spermatogonial stem cell (SSC) propagation in vitro and autologous testicular germ cell transplantation in vivo requires further exploration in monkeys. Herein, we characterized germ cells in macaque testes at 6 months (M), 18 M and 60 M of age, and effectively isolated the spermatogenic cells (including the spermatogonia) from macaque testes with high purity (over 80%) using combined approaches of STA‐PUT separation, Percoll gradients and differential plating. We also generated recipient monkey testes with ablated endogenous spermatogenesis using the alkylating agent busulfan in six macaques, and successfully mimicked autologous cell transplantation in the testes under ultrasonographic guidance. The use of trypan blue led to successful intratubular injection in 4 of 4 testes. Although SSCs in culture showed no significant propagation, we were able to maintain monkey testicular germ cells with stem cell characteristics for up to 3 weeks. Collectively, these data provided meaningful information for future fertility preservation and SSC studies on both non‐human primates and humans.

Germline stem cells in human

“…389,390 Germ cell transplantation in interspecies and intraspecies has been applied to zebrafish, rats, dogs, farm animals (goats, sheep, pigs, and cattle), nonhuman primates, and humans, in addition to mice. [391][392][393][394][395][396][397][398][399][400][401][402][403][404][405][406][407][408] In primates, postpubertal SSC transplantation in infertile rhesus monkeys restored functional sperm production after puberty. 409 In humans, the first clinical trial of testis-cell transplantation using cryopreserved single-cell suspension from patients' testis with lymphoma before chemotherapy was reported in 1999.…”

Favorable culture conditions for spermatogonial propagation in human and non-human primate primary testicular cell cultures: a systematic review and meta-analysis

“…Even though feeder layers in general have relatively high confluence ( Kanatsu-Shinohara et al, 2003 ), it was shown that low numbers of somatic cells (5,000 somatic cells) are able to support high numbers (up to 125.000 cells) of floating germ cells in establishing aggregates ( Gat et al, 2017 ). To prevent overgrowth of feeder cells, mitotic inactivation by applying γ-radiation ( Smith et al, 2014 ; Medrano et al, 2016 ) or mitomycin C treatment ( Murdock et al, 2019 ; Yuan et al, 2022 ) may be applied.…”

Recent Developments in In Vitro Spermatogenesis and Future Directions

“…However, such methods sometimes work less effectively in NHPs. Utilizing the STA-PUT velocity sedimentation method, Percoll gradient, and differential plating methods, researchers were able to isolate highly pure (over 80%) spermatogonia (UCHL1 + ) from rhesus tests [74]. Isolated cells were cultured on monkey fibroblast cells with defined serum-free mouse SSC culture medium supplemented [71] with 20 ng/mL GDNF, 1 ng/mL bFGF, 100 ng/mL GFRα1, 20 ng/mL EGF, 0.05 ng/µL BMP7, and 10 4 U/mL LIF [74].…”

“…Utilizing the STA-PUT velocity sedimentation method, Percoll gradient, and differential plating methods, researchers were able to isolate highly pure (over 80%) spermatogonia (UCHL1 + ) from rhesus tests [74]. Isolated cells were cultured on monkey fibroblast cells with defined serum-free mouse SSC culture medium supplemented [71] with 20 ng/mL GDNF, 1 ng/mL bFGF, 100 ng/mL GFRα1, 20 ng/mL EGF, 0.05 ng/µL BMP7, and 10 4 U/mL LIF [74]. However, the isolated spermatogonia cells did not propagate but maintained testicular germ cells (Dazl and Zbtb16) with stem cell (Fgfr3 and Utf1) characteristics for up to 3 weeks [74].…”

“…Isolated cells were cultured on monkey fibroblast cells with defined serum-free mouse SSC culture medium supplemented [71] with 20 ng/mL GDNF, 1 ng/mL bFGF, 100 ng/mL GFRα1, 20 ng/mL EGF, 0.05 ng/µL BMP7, and 10 4 U/mL LIF [74]. However, the isolated spermatogonia cells did not propagate but maintained testicular germ cells (Dazl and Zbtb16) with stem cell (Fgfr3 and Utf1) characteristics for up to 3 weeks [74].…”