Spermatogonial Stem Cell Culture in Oncofertility

Introduction

Advancements in cancer therapies over the last several decades have led to a rise in pediatric cancer survival rates to about 88%¹. This increase in cancer survivorship has made it increasingly important to address factors that affect patient quality of life post-treatment, including treatment-induced gonadotoxicity and increased risk of infertility^2,3. The majority of patients that are exposed to gonadotoxic therapies experience transient azoospermia and will recover normal levels of spermatogenesis within 1-5 years post-treatment⁴. However, about 24% of patients will be rendered permanently infertile by treatment for their primary disease⁵.The extent and permanence of azoospermia depends on a combination of several factors including the primary disease diagnosis and the therapeutic regimen employed to treat the disease. Methods to predict the risk of infertility are imperfect⁶, but some guidance is available to predict treatment regimens that are associated with significant or high risk of infertility^7,8. The prospect of having biological children is important to cancer survivors and the risk of iatrogenic infertility causes psychosocial stress in these individuals⁹. Therefore, the American Society of Clinical Oncology, the American Society for Reproductive Medicine and the American Academy of Pediatrics recommend that all cancer patients and patients receiving cytotoxic treatments for hematologic conditions be counseled about the risk of infertility and about methods for fertility preservation prior to the onset of treatment^10–13.

The standard of care approach to preserve fertility in adolescent and adult male patients is cryopreservation of spermatozoa that can be used at a later time to establish pregnancy through assisted reproductive technology (ART)^14,15. This option is not available to prepubertal patients who do not produce sperm. However, several centers around the world are cryopreserving testicular tissues for young patients with anticipation that spermatogonial stem cells (SSCs) in those tissues might be used to restore fertility in the future^16–23.

SSC transplantation to restore fertility

Several new technologies have emerged over the last 25 years that may allow patients to use their cryopreserved testicular tissues to produce sperm and have biological offspring including SSC transplantation, de novo testicular morphogenesis, testicular tissue organ culture, testicular tissue grafting or xenografting and derivation of germ cells from induced pluripotent stem cells^24,25. SSC transplantation is a mature technology that may be ready for translation to the male fertility clinic. In fact, Radford and Colleagues reported a clinical trial in 1999 in which testicular cell suspensions were cryopreserved for 12 patients with Hodgkin’s disease^26,27. Seven of those patients returned to have their cryopreserved cells, including SSCs, transplanted back into their testes via injection into the rete testis space²⁷. While follow up studies on the outcome of transplantation in those cases have not been reported, the study demonstrates patient willingness to undergo an experimental SSC-based therapy to have a biological child.

Like other tissue-specific stem cells, SSCs have the potential to colonize the testicular niche and regenerate spermatogenesis. Brinster and colleagues first demonstrated this principle twenty five years ago by showing that mouse testicular cell suspensions containing SSCs could be transplanted into the seminiferous tubules of an infertile mouse recipient to restore complete spermatogenesis and fertility^28,29. This method has since been replicated in a number of mammalian species including rats, sheep, goats, pigs, bulls, dogs and primates^30–36. SSCs from all ages, newborn to adult are competent to regenerate spermatogenesis and spermatogenesis can be restored from testicular cells that have been cryopreserved for as long as 14 years^33,37–41. Thus, it appears feasible to cryopreserve testicular tissues/cells containing SSCs for prepubertal patients and recover those cells years later for autologous transplantation and regeneration of spermatogenesis.

Testicular cells are typically transplanted into the recipient testis through the rete testis space that is contiguous with all seminiferous tubules^33,42–44. SSCs migrate from the lumen of the seminiferous tubules, through the blood-testis barrier (BTB), to the basement membrane. Rac1 and β1 Integrin have been shown to be critical in SSC transmigration through the BTB and attachment to the basement membrane, respectively, in mice^45,46. Despite innate properties allowing SSCs to penetrate the BTB, the majority of transplanted cells are eliminated through phagocytosis by Sertoli cells, which may be one factor that reduces overall efficiency of the method⁴⁷. Nagano and colleagues evaluated the kinetics of SSC engraftment in the mouse testis and deduced that transplantation with 1 million testicular cells led to colonization and spermatogenesis by 19 SSCs⁴⁸. Thus, methods to isolate and enrich SSCs and expand their numbers in culture are needed to ensure robust engraftment and regeneration of spermatogenesis.

In fertility preservation centers that provide testicular tissue cryopreservation services, about 20% of testicular volume from one testis is typically biopsied although some centers allow for collection of larger volumes and/or biopsy of both teste^22,23,49. Hence, the number of SSCs obtained from small biopsies of prepubertal testes could be a limiting factor in the successful application SSC transplantation in the clinic. One way to overcome this limitation is to isolate and enrich SSCs from the testicular biopsy and expand their numbers in vitro prior to transplantation. These approaches might also be used to assess and eliminate malignant contamination as described below.

Sorting methods to isolate and enrich SSCs as well as eliminate malignant contamination

Using SSC transplantation as a functional assay, a number of cell surface markers have been identified that are conserved between murine and human spermatogonia. Murine SSCs have been shown to exhibit the phenotype GPR125⁺ (G-protein coupled receptor 125), EpCAM^low (Epithelial cell adhesion molecule), ITGA6⁺ (α6 Integrin), ITGB1⁺ (β1 Integrin), CD9⁺, THY1⁺ (CD90), GFRα1⁺ (GDNF family receptor alpha 1), MCAM⁺ (Melanoma cell adhesion molecule 1), ITGAV⁻ (αV Integrin), cKIT⁻ (CD117 or Stem cell growth factor), MHC-I⁻ (Major histocompatibility complex class I), SCA-1⁻ (stem cells antigen 1)^50–58. Characterization of human spermatogonia has identified GPR125, EpCAM, ITGA6 and GFRA1 as well as FGFR3 (Fibroblast growth factor receptor 3), SSEA4 (Stage specific embryonic antigen 4), TSPAN33 (Tetraspanin 33) as cell surface markers of human SSCs^59–61. In addition to its application in basic research, the ability to identify and enrich SSCs is important for clinical translation of SSC transplantation as a method to restore fertility. These methods could be especially valuable for patients with malignancies that may contaminate testicular cells, posing a risk of reintroducing cancer cells into patient survivors. A study using a rat model showed that transplanting a testicular cell suspension with as few as 20 leukemic cells could cause the disease to recurrence in the recipient⁶². Some studies have reported the use of multi-parametric flow cytometry methods to negatively select spermatogonia from cancer cells^58,63. Other reports used markers for both spermatogonia and cancer cells for a more stringent segregation of the 2 populations but produced conflicting results^64–66. In addition, these reports were based on the use of cancer cell lines and the efficacy of these methods in eliminating heterogenous populations of malignant cells needs to be determined.

While sorting techniques to enrich SSCs and eliminate contaminating malignant cells are promising, there is a need to develop stringent methods to test and quantify residual malignant contamination prior to autologous transplantation. PCR-based methods to detect minimal residual disease (MRD) may be employed in addition to flow cytometry approaches to increase the sensitivity of selection. Currently, there is limited information about how low level contamination detected by PCR corresponds to tumor forming capacity and hence, the absolute risk for inducing relapse remains difficult to predict^67,68. Development of human SSC culture methods may enable clonal expansion of SSCs from an enriched population providing an extra level of stringency for decontamination of patient samples⁶⁹.

Methods for enriching spermatogonia are routinely used to establish SSC culture (Table 1). Shortly after the discovery of the role of glial cell line-derived neurotropic factor (GDNF) on SSC self-renewal⁷⁰, Kanatsu-Shinohara and colleagues described a method for the long term culture of mouse SSCs. In this report, they placed testicular cells on plates coated with gelatin; the testicular somatic cells selectively adhered to the plates while germ cells remained floating and could be aspirated and plated onto secondary plates⁷¹. This approach served the dual purpose of enriching SSCs and removing testicular somatic cells that can rapidly overwhelm the cultures. After two or more rounds of differential plating, floating cells were plated on mouse embryonic fibroblasts in low serum medium supplemented with EGF, LIF, GDNF and FGF2. Subsequent studies employed fluorescence activated cell sorting (FACS) and magnetic activated cell sorting (MACS) for the cell surface marker, THY1, to enrich spermatogonia^72–74. Spermatogonial stem cell transplantation provided the experimental evidence that functional rodent SSCs could be maintained with expansion in number during long-term culture^71,73–76. Cultured SSCs not only regenerated spermatogenesis in infertile recipients, but also produced sperm that were competent to fertilize rodent oocytes and give rise to healthy offspring^71,73.

Table 1:

Literature review of reports on human SSC culture

Citation	Duration of culture	Sort/differential plating	Medium	Growth factors	Feeders or ECM	Passaging technique	End point	Type and age of donor	Claim
Sadri-Ardekani et al., 200918	15 weeks	Differential plating on plastic	MEM+10%F CS for differential plating followed by StemPro-34	20ng/ml EGF, 10ng/ml GDNF, 10ng/ml LIF, 10ng/ml bFGF	Human placental laminin	Passaged every 7-10 days using Trypsin EDTA and differential passaging if there was somatic cell overgrowth	Xenotransplants; ICC – PLZF; RT-PCR – PLZF, ITGA6, ITGB1;	Adult orchidectomy patients (n = 6)	18,000-fold increase in xenotransplant colonizing activity over 64 days in culture
Wu et al., 200978	1 week	Differential plating on Gelatin	MEMa	20ng/ml GDNF, 150ng/ml GFRA1, 1ng/ml bFGF	C166 mouse endothelial cells	Not reported	ICC – UCHL1	Prepuberatal male aged 2-10 years diagnosed with cancer (n = 2)	UCHL1 + spermatogonia can be maintained at least 19 days. No Quantification. GDNF required.
Chen et al., 200994	2 months	MACS for ITGA6	DMEM	10ng/ml GDNF, 4ng/ml bFGF, 1500 IU/ml LIF	Human embryonic stem cells derived fibroblasts (hdF)	Passaged every 4-5 days using Cell dissociation buffer or Trypsin	ICC – OCT4, SSEA1, ITGA6; RT-PCR – OCT4, STRA8, DAZL, NOTCH1, NGN3, SOX3, KIT	Fetal	Colonies maintained over 10 passages. No Quantification.
Lim et al., 201087	>6 months	Percoll selection, differential plating on plastic and collagen followed by MACS for CD9	DMEM during enrichment followed by StemPro-34	10ng/ml GDNF, 10ng/ml bFGF, 20ng/ml EGF, 10,000 U/ml LIF	Laminin	Passaged very 2 weeks using Trypsin	RT-PCR - OCT4, ITGA6, ITGB1, cKIT, TH2B, SYCP3, TP-1; MTT; TUNEL; ICC - GFRA1, CD-9, ITGA6; Alkaline phosphatase staining	Males with obstructive and non-obstructive azoospermia (n = 37)	clumps maintained and continued proliferating over 12 passages (>26 weeks). Total cells quantified.
He et al., 201089	14 days	Differential plating on plastic and MACS for GFR125	DMEM/F12 during enrichment followed StemPro-34	100ng/ml GDNF, 300ng/ml GFRA1-Fc, 10ng/ml NUDT6, 10ng/ml LIF, 20ng/ml EGF, 30ng/ml TGFB, 100ng/ml Nodal	0.1% gelatin	Not reported	ICC – GPR125, ITGA6, GFRA1, THY1	Adult organ donors (n = 5)	GPR125+ cells proliferated during two weeks in culture, but were not quantified.
Kokkinaki et al., 201190	4-5 months	Differential plating on FBS-coated dish, treatment with RBC Lysis Buffer and Dead Cell Removal Kit followed by SSEA4 MACS	StemPro-34	10ng/ml GDNF, 10ng/ml bFGF, 20ng/ml EGF, 10,000 U/ml LIF	Growth factor-reduced matrigel	Passaged manually at 1 month followed by digestion with dispase + collagenase every 10-15 days	Morphology, number of colonies and cells/colony, RT-PCR for SSC markers (PLZF, GPR125, SSEA4) and pluripotency markers (KLF4, OCT4, LIN28, SOX2, NANOG)	14, 34 and 45 year old organ donors (n = 3)	Number of colonies and number of cells per colony increased during five months in culture.
Sadri-Ardekani et al., 201177	15.5 and 10 weeks	Differential plating on plastic	StemPro-34	20ng/ml EGF, 10ng/ml GDNF, 10ng/ml LIF, 10ng/ml bFGF	Human placental laminin	Passaged every 7-10 days using Trypsin EDTA and differential passaging if there was somatic cell overgrowth	Xenotransplants; RT-PCR – PLZF, ITGA6, ITGB1, CD9, GFRA1, GPR125, UCHL1	Prepuberatal male Hodgkin lymphoma patients; 6.5 and 8 years old (n = 2)	5.6-fold increase in xenotransplant colonizing activity over 14 days in culture and 6.2-fold increase over 21 days in culture.
Nowroozi et al., 201179	18 days	Differential plating on lectin-coated plates	DMEM	Not Reported	Human Sertoli cells	Passaged every 7 days with Trypsin EDTA	ICC – OCT4, Vimentin; Alkaline phosphatase staining	Adults with non obstructive azoospermia (n = 47)	Colonies were observed over 18 days in culture. No quantification.
Liu et al., 201188	1 month	Percoll separation and differential plating on plastic	DMEM/F12	Not Reported	Human Sertoli cells	Not reported	ICC – OCT4, SSEA4; Flow cytometry – OCT4	Fetal (n = 5)	OCT4+ cells observed; timeframe uncertain. No quantification.
Mirzapour et al., 201280	5 weeks	Differential plating on lectin-coated plates	DMEM	Various concentrations of bFGF and LIF	Human Sertoli cells	Passaged every 7 days using Trypsin EDTA	Xenotransplants; Alkaline phosphatase staining; ICC – OCT4, Vimentin; RT-PCR – OCT4, NANOG, STRA8, PIWIL2, VASA	Adult males with NOA-maturation arrest (n = 20)	Tested bFGF and LIF concentrations. Colony number increased in some conditions over 30 days in culture.
Koruji et al., 201286	2 months	Differential plating on plastic	DMEM+5%F CS	20ng/ml GDNF, 10ng/ml BFGF, 10ng/ml LIF, 20ng/ml EGF	Laminin or plastic	Passaged every 5-7 days using Trypsin-EDTA	Morphology-number and diameter of colonies; RT-PCR – PLZF, DAZL, OCT4, VASA, ITGA6, ITGB1	Adult males with NOA	Clusters present after two months. Xenotransplant colonizing activity and expression of spermatogonial markers reported. No quantification.
Goharbakhsh et al., 201381	52 days	Differential plating on plastic for cells>10^6, all cells were plated is number<10^6	DMEM-F12	10ng/ml GDNF, 10ng/ml bFGF, 20ng/ml EGF, 10,000 U/ml LIF	20ul/ml laminin or 0.2% gelatin	Passaged every 7-10 days, method wasn’t mentioned	Morphological observation of EB-like colonies and ICC staining for GPR125	Azoospermic adult males (n = 12)	Clusters observed over several passages during 52 days in culture. GPR125+ cells observed at end of culture. No quantification of clusters or GPR125 cells.
Piravar, Z et al., 201382	6 weeks	Differential plating on plastic	DMEM/F12 for 16 hours then StemPro-34	10ng/ml GDNF, 20ng/ml EGF, 10ng/ml LIF	Uncoated plates for the first 14 days followed by laminin	Trypsinization every 2 weeks	qPCR for UCHL1	non-obstructive azoospermic males (n = 10)	Clusters number increased over 6-weeks of culture. UCHL1 expression observed by RT-PCR.
Akhondi, MM et al., 2013112	6 weeks	Enrichment was not performed	StemPro-34	10ng/ml GDNF, 20ng/ml EGF, 10ng/ml LIF	Not reported	Trypsinization every 10 days	ICC for Oct4; qPCR for PLZF	44 year old organ donor (n = 1)	Cluster number increased during 6-week culture. OCT4 observed by ICC at end of culture. PLZF expression observed by RT-PCR
Zheng et al., 201483	2 weeks	Differential plating on plastic and collagen	DMEM during enrichment followed by StemPro-34	20ng/ml EGF, 10ng/ml GDNF, 10ng/ml LIF, 10ng/ml bFGF	Not reported	Passaged using Trypsin when confluent	Flow cytometry - SSEA4; qRT-PCR - UTF1, FGFR3, SALL4, PLZF, DAZL, VIM, ACTA2, GATA4	Adult organ donors (n = 8)	SSEA4+ spermatogonia decreased over time in culture. VIM+, ACTA2+ somatic cells were the main cell type present after 48 days in culture
Chikhovskaya et al., 201491	2 weeks	Differential plating on plastic followed by MACS for ITGA6 and differential plating on Collagen I and Laminin	StemPro-34	20ng/ml EGF, 10ng/ml GDNF, 10ng/ml LIF, 10ng/ml bFGF	MEFs or plastic	Not reported	qPCR for PLZF, MAGEA4, CD49f, DAZL, UTF1, DDX4, TM4SF1, ACTA2; Flow cytometry for SSEA4, CD29, CD44, CD49f, CD73, CD90, CD105, HLAABC, HLADR, CD31, CD34, CD117, CD133	Adult cancer patients undergoing bilateral orchidectomy (n = 3)	Mixed cultures: rapid proliferation of testicular somatic cells and rapid decrease in PLZF+ and MAGEA4+ germ cells. Isolated spermatogonia degenerated by 2 weeks in culture.
Smith et al., 201495	21 days	FACS – CD45−, THY1−, SSEA4+	StemPro-34	20ng/ml EGF, 10ng/ml GDNF, 10ng/ml LIF, 10ng/ml bFGF	Adult human THY1 + cells	Not reported	ICC – SSEA4, VASA	Adults with normal spermatogenesis (n = 13)	Colonies expressing SSEA4and VASA were present at 21 days. No quantification.
Guo et al., 201592	2 months	Differential plating on plastic with DMEM-F12 followed by MACS for GPR125	StemPro-34	20ng/ml EGF, 10ng/ml bFGF, 10ng/ml LIF, 50ng/ml GDNF	Hydrogel Stem Easy	Not reported	Morphological observation; cell proliferation assay; ICC – GPR125, UCHL1 and THY1, PLZF; RT-PCR for GPR123, GFRa1, RET, PLZF, UCHL1, MAGEA4, SYCP3, PRM1 and TNP1 at 30 days	22-35 year old obstructive azoospermia patients (n = 40)	Colonies of grapelike cells observed at 14 days, 1 month and 2 months. Colonies stained for GPR125, THY1, UCHL1 and MAGEA4. No quantification.
Baert et al., 201584	2 months	Differential plating on plastic	StemPro-34	20ng/ml EGF, 10ng/ml GDNF, 10ng/ml LIF, 10ng/ml bFGF	no substrate	Not reported	ICC and RT-PCR - VASA, UCHL1	Vasectomy reversal patients and adult males who underwent bilateral orchidectomy due to prostate cancer (n = 6)	Single or small groups of VASA+/UCHL1 + cells detected in considerable amounts up to 1 month but infrequently after 2 months
Abdul Wahab et al., 201697	49 days	Enrichment was not performed	DMEM	80μl bFGF	Plastic	Not reported	in-well staining for ITGA6, ITGB1, CD9 and GFRA1	non-obstructive azoospermic male (n = 1)	Clusters observed until 49 days in culture. Some ITGA6+ and CD9+ cells were observed. No quantification.
Medrano et al., 201696	28 days	FACS for HLA-/EPCAM+	StemPro-34	20ng/ml EGF, 10ng/ml LIF, 10ng/ml bFGF, 10ng/ml GDNF	Testicular somatic cells	Not reported	ICC - Ki67; TUNEL; RT-PCR - UTF1, DAZL, VASA, PLZF, FGFR3, UCHL1; Elecsys Testosterone II competitive immunoassay; ELISA - Inhibin B	Adult males who underwent bilateral orchidectomy due to prostate cancer (n = 3)	VASA+/UTF1+ cells observed after 2 weeks but were rarely Ki67+ and disappeared by 4 weeks
Gat et al., 201785	12 days	Differential plating on Gelatin	DMEM-F12 and StemPro-34	20ng/ml EGF, 10ng/ml GDNF, 10ng/ml LIF, 10ng/ml bFGF	Laminin and testicular somatic cells	Passaged using Trypsin when cells were 80-90% confluent	SSC-like aggregates and targeted RNA seq for DAZL, ITGA6 and SYCP3	Adult orchidectomy patients (4 for testicular malignancies and 3 for testicular pain) and 1 adult who underwent microTESE due to NOA (n = 8)	Germ cell aggregates observed. Number impacted by medium and ratio of somatic cells to germ cells. No quantification over time.
Murdock et al., 201893	14 days	MACS for ITGA6 followed by Differential plating on Collagen I	MEMα	20ng/ml GDNF, 1ng/ml bFGF	STO, mouse and human laminin, htECM, ptECM, SIS and UBM	Passaged using Trypsin at day 7	ICC – UTF1; Flow cytometry – SSEA4, cKIT, AnnexinV and Ki-67	Adult organ donors (n = 4)	Aggregates observed. Number of UTF1+ cell declined over 14 days in culture.

Human spermatogonial stem cell culture: components and methods of analysis

In 2009, Sadri-Ardekani and colleagues reported the long-term culture of adult human SSCs¹⁸ following a protocol very similar to that described by Kanatsu-Shinohara and colleagues in their first report on mouse SSC cultures⁷¹. Specifically, differential plating was used to reduce the number of testicular somatic cells, which attached to the plate; floating germ cells were passaged onto plates coated with human placental laminin in StemPro medium supplemented with EGF, LIF, GDNF and FGF2. Using this method, the authors reported that human SSCs could be maintained for several months and expanded over 18,000-fold¹⁸. The same group later reported similar success culturing SSCs from prepubertal human testes⁷⁷.

There are now over 20 reports on human SSC culture (Table 1). Many have used differential plating on plastic, lectin, collagen or gelatin as the sole means to enrich SSCs and/or reduce testicular somatic cells prior to culture^18,77–86. Others have supplemented differential plating with Percoll gradient selection^87,88 and/or positive or negative selection for cell surface markers using FACS or MACS^87,89–93 or used FACS/MACS selection alone^94,95. Positive selection markers used for human SSC culture have included ITGA6, CD9, GPR125, SSEA4 and EPCAM. Negative selection markers have included cKIT, CD45 and THY1 (see Table 1). Interestingly, while Thy1 has been used as a positive selection marker prior to mouse SSC culture⁷³, Smith and colleagues⁹⁵ used THY1 as a negative marker of human SSCs and in fact used irradiated THY1+ human testis cells as feeders for their human SSC cultures.

Human spermatogonia, like murine spermatogonia, have been shown to require an extracellular matrix (ECM) or feeder-cell-based substrates to promote the attachment, survival and proliferation in vitro. Feeder cells that have been used to culture human SSCs include fibroblasts derived from human embryonic stem cells, human Sertoli cells, mouse embryonic fibroblasts, mouse endothelial cells, human testicular somatic cells and THY1+ testicular cells (Table 1)^{78–80,88,93–96}. ECM substrates that have been used for human SSC culture include human laminin, gelatin, Matrigel, Hydrogen Stem Easy, human and porcine testicular ECM, porcine small intestinal submucosa ECM and urinary bladder ECM^{18,77,81,82,87,90,92,93}. Most human SSC culture studies summarized in Table 1 used culture conditions similar to what was originally described by Kanatsu-Shinohara in mouse⁷¹ and Sadri-Ardekani in human¹⁸, including StemPro-34 medium supplemented with GDNF, bFGF, EGF and LIF. Some of those studies reported significant expansion of spermatogonia in culture, suggesting an evolutionary conservation of factors required for SSC maintenance and proliferation^18,77,90, although few studies have formally tested the requirement for those factors in human SSC culture^78,80. Others reported a rapid decline in the number of human spermatogonia using those conditions^83,84,91,96. The discrepancy in results can be explained in part by differences in starting cell populations and in part by different approaches to analysis of culture outcomes. Some studies reported the presence of clusters or colonies of putative spermatogonia with no attempt to quantify^79,85,94. Some studies observed spermatogonial markers or xenotransplant colonizing activity in culture but did not quantify^{78,81,86,88,89,92,95,97}. Some quantified the number of clusters/colonies or total cells in culture but did not specifically quantify spermatogonia using markers or transplantation^{77,80,82,87,90}. Finally, some studies quantified the number of cells with spermatogonial markers or xenotransplant colonizing activity over time in culture^{18,77,83,84,91,93,96}.

Challenges, Opportunities and Future Directions

Variations in the methods for 1) selection of spermatogonia prior to culture, 2) culture conditions and 3) analytical endpoints have made it difficult to compare studies or reach a consensus about optimal human SSC culture conditions. There are several cell surface markers that can be used to isolate and enrich human SSCs but none of those can produce a pure population of SSCs. Therefore, any method used to sort prior to culture will produce a heterogeneous population of cells that is likely to include testicular germ cells and somatic cells. Quantification of colony or cluster number in culture is valuable but not sufficient as a single endpoint because it is possible to produce colonies of mesenchymal cells from human testes^83,91.

Spermatogonial stem cell transplantation was the “gold-standard” assay that validated success expanding functional rodent SSCs in culture. Mouse and rat SSCs can colonize infertile mouse recipient testes and regenerate complete spermatogenesis^28,29,98. Spermatogonial stem cell transplantation in humans is not possible as a routine biological assay, but human to nude mouse xenotransplantation has emerged as a powerful tool to quantify human spermatogonia with transplantation potential. Human cells do not produce complete spermatogenesis in mouse testes, but they do migrate to the basement membrane of seminiferous tubules, proliferate to produce characteristic chains and clusters of spermatogonia and survive long-term^64,99,100.

It is recognized that not all labs will have the expertise or infrastructure for human to nude mouse xenotransplantation. There has been significant progress in the last few years identifying protein markers of undifferentiated human stem/progenitor spermatogonia (e.g., UTF1, PIWIL4, UCHL1, PLZF, SALL4, GFRA1, LPPR3, TCF3, TSPAN33 and others)^100–103. These markers can be detected and quantified at a single cell level using immunocytochemistry or flow cytometry. RT-PCR is a complementary and sensitive method to confirm the presence of spermatogonial transcripts but does not reveal protein expression or provide information about spermatogonial quantity. Similarly, markers that are expressed by spermatogonia and other somatic cell types in the testis can be misleading (e.g., ITGA6, THY1, UCHL1)^84,91,95,96. Multiparameter staining (e.g., VASA+/UCHL1+) may help to resolve these issues^84,96. Spermatogonial marker positive cells should be monitored and quantified throughout the culture period and not just the end because this will help to understand spermatogonial proliferation dynamics over time. Complementing markers of undifferentiated spermatogonia with markers of differentiation (e.g., cKIT), apoptosis (e.g., annexinV, Caspase 3) and proliferation (e.g., ki67) will help elucidate the fate of spermatogonia once they are placed in culture.

The growth requirements to maintain or expand human SSCs in culture are not known. Many studies have started with factors that were used in mouse SSC culture (any combination of GDNF, bFGF, EGF and LIF), but few have formally tested the requirement for those factors^78,80. Characterization of human germ and testicular somatic cells through single cell RNA sequencing may help shed light on additional factors or signaling pathways in human SSCs that might be manipulated in culture to promote SSC survival and/or proliferation or new markers that can be used to isolate and enrich human SSCs^101,102,104.

Mouse and rat SSCs divide once every 3-11 days in culture, similar to their in vivo proliferation dynamics. The in vivo cell cycle time of undifferentiated human A_dark and A_pale spermatogonia ranges from 1.5-8 months. If, like rodents, human SSC proliferation dynamics in culture are similar to the in vivo situation, it raises questions about whether it will ever be possible to expand human SSC numbers in culture. An emerging alternative approach could be to expand patient-derived induced pluripotent stem cells (iPSCs) in culture and then differentiate them into primordial germ cell-like cells (PGCLCs)^105–109. PGCLCs can potentially be transplanted into the testes to regenerate spermatogenesis¹¹⁰ or differentiated to sperm in vitro¹¹¹, outcomes that have been reported in mice, but not yet for any other species.

Despite the challenges outlined above, culture of human male germline stem cells, including SSCs or iPSC-derived PGCLCs, will have important applications for fundamental investigations of human germ lineage development and spermatogenesis. This is important because our current understanding of human SSC function and spermatogenesis is based primarily on data generated in mice. Knowledge generate from human germline stem cell culture could have important implications for development next generation reproductive technologies using stem cells.

Synopsis.

Infertility caused by chemotherapy or radiation treatments negatively impacts patient-survivor quality of life. The only fertility preservation option available to prepubertal boys who are not making sperm is cryopreservation of testicular tissues that contain spermatogonial stem cells (SSCs) with potential to produce sperm and/or restore fertility. SSC transplantation to regenerate spermatogenesis in infertile adult survivors of childhood cancers is a mature technology. However, the number of SSCs obtained in a biopsy of a prepubertal testis may be small. Therefore, methods to expand SSC numbers in culture prior to transplant are needed. Here we review progress with human SSC culture.

Key points:

• Chemotherapy, radiation and other medical treatments can cause permanent infertility. Sperm freezing is the standard of care method to preserve male fertility.

• Testicular tissue freezing is an experimental option to preserve the fertility of prepubertal boys and others who cannot produce sperm. Testicular tissues contain spermatogonial stem cells.

• Spermatogonial stem cell-based techniques that are currently in the research pipeline may be available in the male fertility clinic of the future.

• To facilitate clinical translation, methods are needed to isolate and enrich human spermatogonial stem cells as well as expand their numbers in culture.