Key points

Introduction

Advancements in cancer therapies over the past several decades have led to a rise in pediatric cancer survival rates to approximately 88%. This increase in cancer survivorship has made it increasingly important to address factors that affect patient quality of life posttreatment, including treatment-induced gonadotoxicity and increased risk of infertility. ^, Most patients who are exposed to gonadotoxic therapies experience transient azoospermia and will recover normal levels of spermatogenesis within 1 to 5 years posttreatment ; however, approximately 24% of patients will be rendered permanently infertile by treatment for their primary disease. 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 imperfect, but some guidance is available to predict treatment regimens that are associated with significant or high risk of infertility. ^, The prospect of having biological children is important to cancer survivors, and the risk of iatrogenic infertility causes psychosocial stress in these individuals. Therefore, the American Society of Clinical Oncology, the American Society for Reproductive Medicine, and the American Academy of Pediatrics recommend that all patients with cancer and patients receiving cytotoxic treatments for hematologic conditions be counseled about the risk of infertility and about methods for fertility preservation before the onset of treatment.

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. ^, 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.

Spermatogonial stem cell transplantation to restore fertility

Several new technologies have emerged over the past 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. ^, 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 disease. Seven of those patients returned to have their cryopreserved cells, including SSCs, transplanted back into their testes via injection into the rete testis space. Although 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 25 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. This method has since been replicated in a number of mammalian species, including rats, sheep, goats, pigs, bulls, dogs, and primates. 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. ^, 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. ^, 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. ^, Despite innate properties allowing SSCs to penetrate the BTB, most transplanted cells are eliminated through phagocytosis by Sertoli cells, which may be one factor that reduces overall efficiency of the method. 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 SSCs. 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, approximately 20% of testicular volume from one testis is typically biopsied, although some centers allow for collection of larger volumes and/or biopsy of both testes. ^{, ,} 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 before transplantation. These approaches might also be used to assess and eliminate malignant contamination, as described in the following section.

Sorting methods to isolate and enrich spermatogonial stem cells as well as eliminate malignant contamination

Using SSC transplantation as a functional assay, several 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). 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. 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 recur in the recipient. Some studies have reported the use of multiparametric flow cytometry methods to negatively select spermatogonia from cancer cells. ^, Other reports used markers for both spermatogonia and cancer cells for a more stringent segregation of the 2 populations but produced conflicting results. In addition, these reports were based on the use of cancer cell lines, and the efficacy of these methods in eliminating heterogeneous populations of malignant cells needs to be determined.

Although 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 before autologous transplantation. Polymerase chain reaction (PCR)-based methods to detect minimal residual disease may be used 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. ^, 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 samples.

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-renewal, 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, whereas germ cells remained floating and could be aspirated and plated onto secondary plates. This approach served the dual purpose of enriching SSCs and removing testicular somatic cells that can rapidly overwhelm the cultures. After 2 or more rounds of differential plating, floating cells were plated on mouse embryonic fibroblasts in low serum medium supplemented with epidermal growth factor (EGF), leukemia inhibitory factor (LIF), GDNF, and fibroblast growth factor 2 (FGF2). Subsequent studies used fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) for the cell surface marker, THY1, to enrich spermatogonia. Spermatogonial stem cell transplantation provided the experimental evidence that functional rodent SSCs could be maintained with expansion in number during long-term culture. ^, 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. ^,

Table 1

Literature review of reports on human SSC culture

Data from Refs.

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, 2009	15 wk	Differential plating on plastic	MEM+10%FCS for differential plating followed by StemPro-34	20 ng/mL EGF, 10 ng/mL GDNF, 10 ng/mL LIF, 10 ng/mL bFGF	Human placental laminin	Passaged every 7–10 d 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 d in culture
Wu et al, 2009	1 wk	Differential plating on gelatin	MEMα	20 ng/mL GDNF, 150 ng/mL GFRA1, 1 ng/mL bFGF	C166 mouse endothelial cells	Not reported	ICC – UCHL1	Prepubertal male aged 2–10 y diagnosed with cancer (n = 2)	UCHL1+ spermatogonia can be maintained at least 19 d. No quantification. GDNF required.
Chen et al, 2009	2 mo	MACS for ITGA6	DMEM	10 ng/ml GDNF, 4 ng/ml bFGF, 1500 IU/mL LIF	Human embryonic stem cells derived fibroblasts (hdF)	Passaged every 4–5 d 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, 2010	>6 mo	Percoll selection, differential plating on plastic and collagen followed by MACS for CD9	DMEM during enrichment followed by StemPro-34	10 ng/mL GDNF, 10 ng/mL bFGF, 20 ng/mL EGF, 10,000 U/mL LIF	Laminin	Passaged very 2 wk 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 nonobstructive azoospermia (n = 37)	Clumps maintained and continued proliferating over 12 passages (>26 wk). Total cells quantified.
He et al, 2010	14 d	Differential plating on plastic and MACS for GFR125	DMEM/F12 during enrichment followed StemPro-34	100 ng/mL GDNF, 300 ng/mL GFRA1-Fc, 10 ng/mL NUDT6, 10 ng/mL LIF, 20 ng/mL EGF, 30 ng/mL TGFB, 100 ng/mL Nodal	0.1% gelatin	Not reported	ICC – GPR125, ITGA6, GFRA1, THY1	Adult organ donors (n = 5)	GPR125+ cells proliferated during 2 wk in culture, but were not quantified.
Kokkinaki et al, 2011	4–5 mo	Differential plating on FBS-coated dish, treatment with RBC Lysis Buffer and Dead Cell Removal Kit followed by SSEA4 MACS	StemPro-34	10 ng/mL GDNF, 10 ng/mL bFGF, 20 ng/mL EGF, 10,000 U/mL LIF	Growth factor-reduced matrigel	Passaged manually at 1 mo followed by digestion with dispase + collagenase every 10–15 d	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 5 months in culture.
Sadri-Ardekani et al, 2011	15.5 and 10 wk	Differential plating on plastic	StemPro-34	20 ng/mL EGF, 10 ng/mL GDNF, 10 ng/mL LIF, 10 ng/mL bFGF	Human placental laminin	Passaged every 7–10 d using Trypsin EDTA and differential passaging if there was somatic cell overgrowth	Xenotransplants; RT-PCR – PLZF, ITGA6, ITGB1, CD9, GFRA1, GPR125, UCHL1	Prepuberatal male patients with Hodgkin lymphoma; 6.5 and 8.0 years old (n = 2)	5.6-fold increase in xenotransplant colonizing activity over 14 d in culture and 6.2-fold increase over 21 d in culture.
Nowroozi et al, 2011	18 d	Differential plating on lectin-coated plates	DMEM	Not reported	Human Sertoli cells	Passaged every 7 d with Trypsin EDTA	ICC – OCT4, Vimentin; Alkaline phosphatase staining	Adults with nonobstructive azoospermia (n = 47)	Colonies were observed over 18 d in culture. No quantification.
Liu et al, 2011	1 mo	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, 2012	5 wk	Differential plating on lectin-coated plates	DMEM	Various concentrations of bFGF and LIF	Human Sertoli cells	Passaged every 7 d 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 d in culture.
Koruji et al, 2012	2 mo	Differential plating on plastic	DMEM+5%FCS	20 ng/mL GDNF, 10 ng/mL bFGF, 10 ng/mL LIF, 20 ng/mL EGF	Laminin or plastic	Passaged every 5–7 d using Trypsin EDTA	Morphology-number and diameter of colonies; RT-PCR – PLZF, DAZL, OCT4, VASA, ITGA6, ITGB1	Adult males with NOA	Clusters present after 2 mo. Xenotransplant colonizing activity and expression of spermatogonial markers reported. No quantification.
Goharbakhsh et al, 2013	52 d	Differential plating on plastic for cells >10 6 , all cells were plated is number<10 6	DMEM-F12	10 ng/mL GDNF, 10 ng/mL bFGF, 20 ng/mL EGF, 10,000 U/mL LIF	20 μL/mL laminin or 0.2% gelatin	Passaged every 7–10 d, method was not mentioned	Morphologic observation of EB-like colonies and ICC staining for GPR125	Azoospermic adult males (n = 12)	Clusters observed over several passages during 52 d in culture. GPR125+ cells observed at end of culture. No quantification of clusters or GPR125 cells.
Piravar et al, 2013	6 wk	Differential plating on plastic	DMEM/F12 for 16 h then StemPro-34	10 ng/mL GDNF, 20 ng/mL EGF, 10 ng/mL LIF	Uncoated plates for the first 14 d followed by laminin	Trypsinization every 2 wk	qPCR for UCHL1	Nonobstructive azoospermic males (n = 10)	Clusters number increased over 6-wk of culture. UCHL1 expression observed by RT-PCR.
Akhondi, MM et al, 2013	6 wk	Enrichment was not performed	StemPro-34	10 ng/mL GDNF, 20 ng/mL EGF, 10 ng/mL LIF	Not reported	Trypsinization every 10 d	ICC for Oct4; qPCR for PLZF	44-year-old organ donor (n = 1)	Cluster number increased during 6-wk culture. OCT4 observed by ICC at end of culture. PLZF expression observed by RT-PCR
Zheng et al, 2014	2 wk	Differential plating on plastic and collagen	DMEM during enrichment followed by StemPro-34	20 ng/mL EGF, 10 ng/mL GDNF, 10 ng/mL LIF, 10 ng/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 d in culture
Chikhovskaya et al, 2014	2 wk	Differential plating on plastic followed by MACS for ITGA6 and differential plating on Collagen I and Laminin	StemPro-34	20 ng/mL EGF, 10 ng/mL GDNF, 10 ng/mL LIF, 10 ng/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 patients with cancer 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 wk in culture.
Smith et al, 2014	21 d	FACS – CD45-, THY1-, SSEA4+	StemPro-34	20 ng/mL EGF, 10 ng/mL GDNF, 10 ng/mL LIF, 10 ng/mL bFGF	Adult human THY1+ cells	Not reported	ICC – SSEA4, VASA	Adults with normal spermatogenesis (n = 13)	Colonies expressing SSEA4 and VASA were present at 21 d. No quantification.
Guo et al, 2015	2 mo	Differential plating on plastic with DMEM-F12 followed by MACS for GPR125	StemPro-34	20 ng/mL EGF, 10 ng/mL bFGF, 10 ng/mL LIF, 50 ng/mL GDNF	Hydrogel Stem Easy	Not reported	Morphologic observation; cell proliferation assay; ICC – GPR125, UCHL1,THY1 and PLZF; RT-PCR for GPR123, GFRa1, RET, PLZF, UCHL1, MAGEA4, SYCP3, PRM1 and TNP1 at 30 d	22–35-year-old patients with obstructive azoospermia (n = 40)	Colonies of grapelike cells observed at 14 d, 1 mo, and 2 mo. Colonies stained for GPR125, THY1, UCHL1 and MAGEA4. No quantification.
Baert et al, 2015	2 mo	Differential plating on plastic	StemPro-34	20 ng/mL EGF, 10 ng/mL GDNF, 10 ng/mL LIF, 10 ng/mL bFGF	No substrate	Not reported	ICC and RT-PCR – VASA, UCHL1	Vasectomy reversal patients and adult male patients 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 mo but infrequently after 2 mo.
Abdul Wahab et al, 2016	49 d	Enrichment was not performed	DMEM	80 μl bFGF	Plastic	Not reported	In-well staining for ITGA6, ITGB1, CD9 and GFRA1	Nonobstructive azoospermic male (n = 1)	Clusters observed until 49 d in culture. Some ITGA6+ and CD9+ cells were observed. No quantification.
Medrano et al, 2016	28 d	FACS for HLA-/EPCAM+	StemPro-34	20 ng/mL EGF, 10 ng/mL LIF, 10 ng/mL bFGF, 10 ng/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 male patients who underwent bilateral orchidectomy due to prostate cancer (n = 3)	VASA+/UTF1+ cells observed after 2 wk but were rarely Ki67+ and disappeared by 4 wk
Gat et al, 2017	12 d	Differential plating on Gelatin	DMEM-F12 and StemPro-34	20 ng/mL EGF, 10 ng/mL GDNF, 10 ng/mL LIF, 10 ng/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, 2018	14 d	MACS for ITGA6 followed by differential plating on Collagen I	MEMα	20 ng/mL GDNF, 1 ng/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 d in culture.

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