Long-term health in recipients of transplanted in vitro propagated spermatogonial stem cells

Abstract

STUDY QUESTIONIs testicular transplantation of in vitro propagated spermatogonial stem cells associated with increased cancer incidence and decreased survival rates in recipient mice?SUMMARY ANSWERCancer incidence was not increased and long-term survival rate was not altered after transplantation of in vitro propagated murine spermatogonial stem cells (SSCs) in busulfan-treated recipients as compared to non-transplanted busulfan-treated controls.WHAT IS KNOWN ALREADYSpermatogonial stem cell autotransplantation (SSCT) is a promising experimental reproductive technique currently under development to restore fertility in male childhood cancer survivors. Most preclinical studies have focused on the proof-of-principle of the functionality and efficiency of this technique. The long-term health of recipients of SSCT has not been studied systematically.STUDY DESIGN, SIZE, DURATIONThis study was designed as a murine equivalent of a clinical prospective study design. Long-term follow-up was performed for mice who received a busulfan treatment followed by either an intratesticular transplantation of in vitro propagated enhanced green fluorescent protein (eGFP) positive SSCs (cases, n = 34) or no transplantation (control, n = 37). Using a power calculation, we estimated that 36 animals per group would be sufficient to provide an 80% power and with a 5% level of significance to demonstrate a 25% increase in cancer incidence in the transplanted group. The survival rate and cancer incidence was investigated until the age of 18 months.PARTICIPANTS/MATERIALS, SETTING, METHODSNeonatal male B6D2F1 actin-eGFP transgenic mouse testis were used to initiate eGFP positive germline stem (GS) cell culture, which harbor SSCs. Six-week old male C57BL/6 J mice received a single dose busulfan treatment to deplete the testis from endogenous spermatogenesis. Half of these mice received a testicular transplantation of cultured eGFP positive GS cells, while the remainder of mice served as a control group. Mice were followed up until the age of 18 months (497–517 days post-busulfan) or sacrificed earlier due to severe discomfort or illness. Survival data were collected. To evaluate cancer incidence a necropsy was performed and tissues were collected. eGFP signal in transplanted testis and in benign and malignant lesions was assessed by standard PCR.MAIN RESULTS AND THE ROLE OF CHANCEWe found 9% (95% CI: 2–25%) malignancies in the transplanted busulfan-treated animals compared to 26% (95% CI: 14–45%) in the busulfan-treated control group, indicating no statistically significant difference in incidence of malignant lesions in transplanted and control mice (OR: 0.3, 95% CI: 0.1–1.1). Furthermore, none of the malignancies that arose in the transplanted animals contained eGFP signal, suggesting that they are not derived from the in vitro propagated transplanted SSCs. Mean survival time after busulfan treatment was found to be equal, with a mean survival time for transplanted animals of 478 days and 437 days for control animals (P = 0.076).LARGE...

Comparing genome-scale DNA methylation and CNV marks between adult human cultured ITGA6+ testicular cells and seminomas to assess in vitro genomic stability

“…propagated SSCs [69]. It should be noted that the human testicular cell culture system used in this study differs from the mouse culture system in several aspects, including but not limited to the use of feeder cells.…”

Noncoding RNAs: Potential players in the self‐renewal of mammalian spermatogonial stem cells

“…As for SSCs, although a large number of ncRNAs have been screened as potential regulatory molecules for SSC self‐renewal using high‐throughput RNA sequencing and bioinformatics approaches, only a small portion of them have been validated and functionally characterized using biological experiment to uncover roles in SSCs self‐renewal, particularly lncRNAs, piRNA, and circRNAs (K. Hu et al, ; Li et al, ; X. Li, Ao, et al, ), thus there is an urgent need to reveal more ncRNAs associated with SSC self‐renewal to elucidate the molecular mechanisms underlying SSC self‐renewal. Encouragingly, great improvements of technologies have been recently made on isolation, characterization, in vitro culture, and in vivo transplantation of SSCs will provide us a robust research platform to explore the function of ncRNAs in SSC self‐renewal (X. Li, Ao, et al, ; Mulder et al, ; Takashima & Shinohara, ; Zhang, Sun, & Zou, ). Remarkably, common genome editing techniques such as TALEN and CRISPR–Cas offer a rapid, efficient, and accurate genome engineering platform for functional characterization of putative ncRNAs in the context of established various models both in vitro and in vivo (Sato et al, ; Tang et al, ).…”

“…Encouragingly, great improvements of technologies have been recently made on isolation, characterization, in vitro culture, and in vivo transplantation of SSCs will provide us a robust research platform to explore the function of ncRNAs in SSC selfrenewal (X. Li, Ao, et al, 2017;Mulder et al, 2018;Takashima & Shinohara, 2018;Zhang, Sun, & Zou, 2016). Remarkably, common genome editing techniques such as TALEN and CRISPR-Cas offer a rapid, efficient, and accurate genome engineering platform for functional characterization of putative ncRNAs in the context of established various models both in vitro and in vivo (Sato et al, 2015;Tang et al, 2018).…”

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

“…82 In addition, propagation of moue SSCs before transplantation did not increase the incidence of cancer or decrease the survival of mice that had undergone SSC transplantation. 83 In view of these encouraging results, SSC transplantation is considered a potential fertility-restoration method for future clinical application ( Figure 3). Using cadaver testes, ultrasound-guided injection in the rete testis has been determined as the best technique for cell transplantation in larger testes.…”