Generation of two isogenic sickle cell disease induced pluripotent stem cell lines from testicular fibroblasts.

Curative bone marrow transplantation (BMT) therapies for sickle cell disease (SCD) can cause infertility. The Fertility Preservation Program (FPP) in Pittsburgh cryopreserves testicular tissues for SCD patients prior to BMT in anticipation that those tissues can be thawed in the future and matured to produce sperm. Here, we generated and validated two isogenic patient-derived induced pluripotent stem cell (iPSC) lines from testicular biopsy fibroblasts of a 12-year-old SCD patient.

A bioengineered in situ ovary (ISO) supports follicle engraftment and live-births post-chemotherapy.

Female cancer patients who have undergone chemotherapy have an elevated risk of developing ovarian dysfunction and failure. Experimental approaches to treat iatrogenic infertility are evolving rapidly; however, challenges and risks remain that hinder clinical translation. Biomaterials have improved in vitro follicle maturation and in vivo transplantation in mice, but there has only been marginal success for early-stage human follicles. Here, we developed methods to obtain an ovarian-specific extracellular matrix hydrogel to facilitate follicle delivery and establish an in situ ovary (ISO), which offers a permissive environment to enhance follicle survival. We demonstrate sustainable follicle engraftment, natural pregnancy, and the birth of healthy pups after intraovarian microinjection of isolated exogenous follicles into chemotherapy-treated (CTx) mice. Our results confirm that hydrogel-based follicle microinjection could offer a minimally invasive delivery platform to enhance follicle integration for patients post-chemotherapy.

TCF21+ mesenchymal cells contribute to testis somatic cell development, homeostasis, and regeneration in mice.

Testicular development and function rely on interactions between somatic cells and the germline, but similar to other organs, regenerative capacity declines in aging and disease. Whether the adult testis maintains a reserve progenitor population remains uncertain. Here, we characterize a recently identified mouse testis interstitial population expressing the transcription factor Tcf21. We found that TCF21lin cells are bipotential somatic progenitors present in fetal testis and ovary, maintain adult testis homeostasis during aging, and act as potential reserve somatic progenitors following injury. In vitro, TCF21lin cells are multipotent mesenchymal progenitors which form multiple somatic lineages including Leydig and myoid cells. Additionally, TCF21+ cells resemble resident fibroblast populations reported in other organs having roles in tissue homeostasis, fibrosis, and regeneration. Our findings reveal that the testis, like other organs, maintains multipotent mesenchymal progenitors that can be potentially leveraged in development of future therapies for hypoandrogenism and/or infertility.

Autologous grafting of cryopreserved prepubertal rhesus testis produces sperm and offspring.

Testicular tissue cryopreservation is an experimental method to preserve the fertility of prepubertal patients before they initiate gonadotoxic therapies for cancer or other conditions. Here we provide the proof of principle that cryopreserved prepubertal testicular tissues can be autologously grafted under the back skin or scrotal skin of castrated pubertal rhesus macaques and matured to produce functional sperm. During the 8- to 12-month observation period, grafts grew and produced testosterone. Complete spermatogenesis was confirmed in all grafts at the time of recovery. Graft-derived sperm were competent to fertilize rhesus oocytes, leading to preimplantation embryo development, pregnancy, and the birth of a healthy female baby. Pending the demonstration that similar results are obtained in noncastrated recipients, testicular tissue grafting may be applied in the clinic.

Differentiation of primate primordial germ cell-like cells following transplantation into the adult gonadal niche.

A major challenge in stem cell differentiation is the availability of bioassays to prove cell types generated in vitro are equivalent to cells in vivo. In the mouse, differentiation of primordial germ cell-like cells (PGCLCs) from pluripotent cells was validated by transplantation, leading to the generation of spermatogenesis and to the birth of offspring. Here we report the use of xenotransplantation (monkey to mouse) and homologous transplantation (monkey to monkey) to validate our in vitro protocol for differentiating male rhesus (r) macaque PGCLCs (rPGCLCs) from induced pluripotent stem cells (riPSCs). Specifically, transplantation of aggregates containing rPGCLCs into mouse and nonhuman primate testicles overcomes a major bottleneck in rPGCLC differentiation. These findings suggest that immature rPGCLCs once transplanted into an adult gonadal niche commit to differentiate towards late rPGCs that initiate epigenetic reprogramming but do not complete the conversion into ENO2-positive spermatogonia.

Purification of GFRα1+ and GFRα1- Spermatogonial Stem Cells Reveals a Niche-Dependent Mechanism for Fate Determination.

Undifferentiated spermatogonia comprise a pool of stem cells and progenitor cells that show heterogeneous expression of markers, including the cell surface receptor GFRα1. Technical challenges in isolation of GFRα1+ versus GFRα1- undifferentiated spermatogonia have precluded the comparative molecular characterization of these subpopulations and their functional evaluation as stem cells. Here, we develop a method to purify these subpopulations by fluorescence-activated cell sorting and show that GFRα1+ and GFRα1- undifferentiated spermatogonia both demonstrate elevated transplantation activity, while differing principally in receptor tyrosine kinase signaling and cell cycle. We identify the cell surface molecule melanocyte cell adhesion molecule (MCAM) as differentially expressed in these populations and show that antibodies to MCAM allow isolation of highly enriched populations of GFRα1+ and GFRα1- spermatogonia from adult, wild-type mice. In germ cell culture, GFRα1- cells upregulate MCAM expression in response to glial cell line-derived neurotrophic factor (GDNF)/fibroblast growth factor (FGF) stimulation. In transplanted hosts, GFRα1- spermatogonia yield GFRα1+ spermatogonia and restore spermatogenesis, albeit at lower rates than their GFRα1+ counterparts. Together, these data provide support for a model of a stem cell pool in which the GFRα1+ and GFRα1- cells are closely related but show key cell-intrinsic differences and can interconvert between the two states based, in part, on access to niche factors.

Primate Primordial Germ Cells Acquire Transplantation Potential by Carnegie Stage 23.

Primordial germ cells (PGCs) are the earliest embryonic progenitors in the germline. Correct formation of PGCs is critical to reproductive health as an adult. Recent work has shown that primate PGCs can be differentiated from pluripotent stem cells; however, a bioassay that supports their identity as transplantable germ cells has not been reported. Here, we adopted a xenotransplantation assay by transplanting single-cell suspensions of human and nonhuman primate embryonic Macaca mulatta (rhesus macaque) testes containing PGCs into the seminiferous tubules of adult busulfan-treated nude mice. We discovered that both human and nonhuman primate embryonic testis are xenotransplantable, generating colonies while not generating tumors. Taken together, this work provides two critical references (molecular and functional) for defining transplantable primate PGCs. These results provide a blueprint for differentiating pluripotent stem cells to transplantable PGC-like cells in a species that is amenable to transplantation and fertility studies.

DDX4-EGFP transgenic rat model for the study of germline development and spermatogenesis.

Spermatogonial stem cells (SSC) are essential for spermatogenesis and male fertility. In addition, these adult tissue stem cells can be used as vehicles for germline modification in animal models and may have application for treating male infertility. To facilitate the investigation of SSCs and germ lineage development in rats, we generated a DEAD-box helicase 4 (DDX4) (VASA) promoter-enhanced green fluorescent protein (EGFP) reporter transgenic rat. Quantitative real-time polymerase chain reaction and immunofluorescence confirmed that EGFP was expressed in the germ cells of the ovaries and testes and was absent in somatic cells and tissues. Germ cell transplantation demonstrated that the EGFP-positive germ cell population from DDX4-EGFP rat testes contained SSCs capable of establishing spermatogenesis in experimentally infertile mouse recipient testes. EGFP-positive germ cells could be easily isolated by fluorescence-activated cells sorting, while simultaneously removing testicular somatic cells from DDX4-EGFP rat pup testes. The EGFP-positive fraction provided an optimal cell suspension to establish rat SSC cultures that maintained long-term expression of zinc finger and BTB domain containing 16 (ZBTB16) and spalt-like transcription factor 4 (SALL4), two markers of mouse SSCs that are conserved in rats. The novel DDX4-EGFP germ cell reporter rat described here combined with previously described GCS-EGFP rats, rat SSC culture and gene editing tools will improve the utility of the rat model for studying stem cells and germ lineage development.

Mouse Spermatogenesis Requires Classical and Nonclassical Testosterone Signaling.

Testosterone acts though the androgen receptor in Sertoli cells to support germ cell development (spermatogenesis) and male fertility, but the molecular and cellular mechanisms by which testosterone acts are not well understood. Previously, we found that in addition to acting through androgen receptor to directly regulate gene expression (classical testosterone signaling pathway), testosterone acts through a nonclassical pathway via the androgen receptor to rapidly activate kinases that are known to regulate spermatogenesis. In this study, we provide the first evidence that nonclassical testosterone signaling occurs in vivo as the MAP kinase cascade is rapidly activated in Sertoli cells within the testis by increasing testosterone levels in the rat. We find that either classical or nonclassical signaling regulates testosterone-mediated Rhox5 gene expression in Sertoli cells within testis explants. The selective activation of classical or nonclassical signaling pathways in Sertoli cells within testis explants also resulted in the differential activation of the Zbtb16 and c-Kit genes in adjacent spermatogonia germ cells. Delivery of an inhibitor of either pathway to Sertoli cells of mouse testes disrupted the blood-testis barrier that is essential for spermatogenesis. Furthermore, an inhibitor of nonclassical testosterone signaling blocked meiosis in pubertal mice and caused the loss of meiotic and postmeiotic germ cells in adult mouse testes. An inhibitor of the classical pathway caused the premature release of immature germ cells. Collectively, these observations indicate that classical and nonclassical testosterone signaling regulate overlapping and distinct functions that are required for the maintenance of spermatogenesis and male fertility.

High telomerase is a hallmark of undifferentiated spermatogonia and is required for maintenance of male germline stem cells.

Telomerase inactivation causes loss of the male germline in worms, fish, and mice, indicating a conserved dependence on telomere maintenance in this cell lineage. Here, using telomerase reverse transcriptase (Tert) reporter mice, we found that very high telomerase expression is a hallmark of undifferentiated spermatogonia, the mitotic population where germline stem cells reside. We exploited these high telomerase levels as a basis for purifying undifferentiated spermatogonia using fluorescence-activated cell sorting. Telomerase levels in undifferentiated spermatogonia and embryonic stem cells are comparable and much greater than in somatic progenitor compartments. Within the germline, we uncovered an unanticipated gradient of telomerase activity that also enables isolation of more mature populations. Transcriptomic comparisons of Tert(High) undifferentiated spermatogonia and Tert(Low) differentiated spermatogonia by RNA sequencing reveals marked differences in cell cycle and key molecular features of each compartment. Transplantation studies show that germline stem cell activity is confined to the Tert(High) cKit(-) population. Telomere shortening in telomerase knockout strains causes depletion of undifferentiated spermatogonia and eventual loss of all germ cells after undifferentiated spermatogonia drop below a critical threshold. These data reveal that high telomerase expression is a fundamental characteristic of germline stem cells, thus explaining the broad dependence on telomerase for germline immortality in metazoans.

DDX3Y gene rescue of a Y chromosome AZFa deletion restores germ cell formation and transcriptional programs.

Deletions of the AZFa region (AZoospermia Factor-a) region of the human Y chromosome cause irreversible spermatogenic failure that presents clinically in men as Sertoli-cell only (SCO) pathology of the testis. Deletions of the AZFa region typically encompass two genes: DDX3Y and USP9Y. However, human genetic evidence indicates that SCO is most tightly linked to deletion of DDX3Y and that deletions/mutations of USP9Y can be transmitted from one generation to the next. Here, we generated stable iPSC lines with AZFa deletions, tested complementation via introduction of DDX3Y, and assessed ability to form germ cells in vivo in a xenotransplantation model. We observed a quantifiable improvement in formation of germ cell like cells (GCLCs) from complemented donor iPSCs. Moreover, expression of UTF1, a prospermatogonial protein, was restored in cells complemented by introduction of DDX3Y on the AZFa background. Whole-genome RNA sequencing of purified GCLCs revealed an enrichment of genes involved in translational suppression and transcriptional control in DDX3Y-rescued GCLCs over mutant GCLCs, which maintained a molecular phenotype more similar to undifferentiated iPSCs. This study demonstrates the ability to probe fundamental genetics of human germ cell formation by complementation and indicates that DDX3Y functions in the earliest stages of human germ cell development.

Human germ cell formation in xenotransplants of induced pluripotent stem cells carrying X chromosome aneuploidies.

Turner syndrome is caused by complete or partial loss of the second sex chromosome and is characterized by spontaneous fetal loss in >90% of conceptions. Survivors possess an array of somatic and germline clinical characteristics. Induced pluripotent stem cells (iPSCs) offer an opportunity for insight into genetic requirements of the X chromosome linked to Turner syndrome. We derived iPSCs from Turner syndrome and control individuals and examined germ cell development as a function of X chromosome composition. We demonstrate that two X chromosomes are not necessary for reprogramming or maintenance of pluripotency and that there are minimal differences in gene expression, at the single cell level, linked to X chromosome aneuploidies. Formation of germ cells, as assessed in vivo through a murine xenotransplantation model, indicated that undifferentiated iPSCs, independent of X chromosome composition, are capable of forming germ-cell-like cells (GCLCs) in vivo. In combination with clinical data regarding infertility in women with X chromosome aneuploidies, results suggest that two intact X chromosomes are not required for human germ cell formation, qualitatively or quantitatively, but rather are likely to be required for maintenance of human germ cells to adulthood.

Germ cell transplantation into mouse testes procedure.

OBJECTIVE: To illustrate the step-by-step protocol followed to assay germ cell transplantation into the seminiferous epithelium of mouse testes. DESIGN: Video presentation of an animal model for research in reproductive and regenerative medicine. SETTING: Research laboratory. ANIMAL(S): Male nude mice (NU-Foxn1(nu)). INTERVENTION(S): Mice were chemically sterilized with alkylant compounds (busulfan) followed by gonadal microsurgery to inject donor germ cells. MAIN OUTCOME MEASURE(S): Donor cells should be labeled with reporter genes, such as green fluorescent protein (GFP), lactose operon (LacZ), or alternatively design an effective strategy with specific antibodies to track them within the recipient testes. Sperm detection in the ejaculate can also be used as a read out. However, in this case detection of the donor genotype in the sperm is mandatory to elucidate their origin. RESULT(S): In the present study we describe the complete protocol for germ cell transplant by efferent duct injection, including the preparation of recipient mice, surgery for the germ cell transplant, and analysis of recipient testes. The main strength of this technique is that it constitutes the gold standard for a functional test of the germ cell potential as only spermatogonial stem cells are able to properly colonize the seminal lumen. Both fresh and frozen/thawed testicular cells are suitable for this technique as donor germ cells. Also, enrichment of living spermatogonial stem cells, previous to the transplant, seems to improve the efficiency of colonization. For proper colonization of germ cells, the niche should be available and thus mouse strains that lack endogenous spermatogenesis such as W/W(v) mutant mice are usually used. In the case of nonmatched donor cells, seminiferous epithelium of immune-suppressed recipient mice should be germ cell depleted before the transplant. One limitation of this technique is that the procedure can take up to 3 months. Also, in contrast to the full recovery of spermatogenesis in mouse-to-mouse transplants, xenotransplantation of germ cells from phylogenetically distant species, such as humans into mouse recipients, results in colonization of donor cells and spermatogonial expansion, but fail in their spermatogenic progression due to evolutive incompatibilities with the recipient niche. Xenografting of pieces of donor testis tissue under the skin of mouse hosts is an alternative approach that is currently being investigated to try to solve this limitation. CONCLUSION(S): Transplantation of spermatogonial stem cells into the seminal lumen of mouse testes is a functional assay that defines this cellular subpopulation by its ability to colonize it. This technique can be used as a model to elucidate the insights of spermatogonial stem cells, to produce transgenic animals by genetically manipulating donor cells before transplantation, but also it has potential applications in fertility preservation in cattle and humans as it is feasible in large animals, as recent reports have demonstrated with rhesus monkeys, that recovered spermatogenesis after allogenic transplantation, and even from human cadaver testes. Therefore spermatogonial stem cells isolated from prepuberal boys, who are treated with alkylant chemotherapy, could be returned to their testis to regenerate spermatogenesis in the future.

Fluorescence- and magnetic-activated cell sorting strategies to isolate and enrich human spermatogonial stem cells.

OBJECTIVE: To determine the molecular characteristics of human spermatogonia and optimize methods to enrich spermatogonial stem cells (SSCs). DESIGN: Laboratory study using human tissues. SETTING: Research institute. PATIENT(S): Healthy adult human testicular tissue. INTERVENTION(S): Human testicular tissue was fixed or digested with enzymes to produce a cell suspension. Human testis cells were fractionated by fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS). MAIN OUTCOME MEASURE(S): Immunostaining for selected markers, human-to-nude mouse xenotransplantation assay. RESULT(S): Immunohistochemistry costaining revealed the relative expression patterns of SALL4, UTF1, ZBTB16, UCHL1, and ENO2 in human undifferentiated spermatogonia as well as the extent of overlap with the differentiation marker KIT. Whole mount analyses revealed that human undifferentiated spermatogonia (UCHL1+) were typically arranged in clones of one to four cells whereas differentiated spermatogonia (KIT+) were typically arranged in clones of eight or more cells. The ratio of undifferentiated-to-differentiated spermatogonia is greater in humans than in rodents. The SSC colonizing activity was enriched in the THY1dim and ITGA6+ fractions of human testes sorted by FACS. ITGA6 was effective for sorting human SSCs by MACS; THY1 and EPCAM were not. CONCLUSION(S): Human spermatogonial differentiation correlates with increased clone size and onset of KIT expression, similar to rodents. The undifferentiated-to-differentiated developmental dynamics in human spermatogonia is different than rodents. THY1, ITGA6, and EPCAM can be used to enrich human SSC colonizing activity by FACS, but only ITGA6 is amenable to high throughput sorting by MACS.

Fate of iPSCs derived from azoospermic and fertile men following xenotransplantation to murine seminiferous tubules.

Historically, spontaneous deletions and insertions have provided means to probe germline developmental genetics in Drosophila, mouse and other species. Here, induced pluripotent stem cell (iPSC) lines were derived from infertile men with deletions that encompass three Y chromosome azoospermia factor (AZF) regions and are associated with production of few or no sperm but normal somatic development. AZF-deleted iPSC lines were compromised in germ cell development in vitro. Undifferentiated iPSCs transplanted directly into murine seminiferous tubules differentiated extensively to germ-cell-like cells (GCLCs) that localized near the basement membrane, demonstrated morphology indistinguishable from fetal germ cells, and expressed germ-cell-specific proteins diagnostic of primordial germ cells. Alternatively, all iPSCs that exited tubules formed primitive tumors. iPSCs with AZF deletions produced significantly fewer GCLCs in vivo with distinct defects in gene expression. Findings indicate that xenotransplantation of human iPSCs directs germ cell differentiation in a manner dependent on donor genetic status.

Fate of induced pluripotent stem cells following transplantation to murine seminiferous tubules.

Studies of human germ cell development are limited in large part by inaccessibility of germ cells during development. Moreover, although several studies have reported differentiation of mouse and human germ cells from pluripotent stem cells (PSCs) in vitro, differentiation of human germ cells from PSCs in vivo has not been reported. Here, we tested whether mRNA reprogramming in combination with xeno-transplantation may provide a viable system to probe the genetics of human germ cell development via use of induced pluripotent stem cells (iPSCs). For this purpose, we derived integration-free iPSCs via mRNA-based reprogramming with OCT3/4, SOX2, KLF4 and cMYC alone (OSKM) or in combination with the germ cell-specific mRNA, VASA (OSKMV). All iPSC lines met classic criteria of pluripotency. Moreover, global gene expression profiling did not distinguish large differences between undifferentiated OSKM and OSKMV iPSCs; however, some differences were observed in expression of pluripotency factors and germ cell-specific genes, and in epigenetic profiles and in vitro differentiation studies. In contrast, transplantation of undifferentiated iPSCs directly into the seminiferous tubules of germ cell-depleted immunodeficient mice revealed divergent fates of iPSCs produced with different factors. Transplantation resulted in morphologically and immunohistochemically recognizable germ cells in vivo, particularly in the case of OSKMV cells. Significantly, OSKMV cells also did not form tumors while OSKM cells that remained outside the seminiferous tubule proliferated extensively and formed tumors. Results indicate that mRNA reprogramming in combination with transplantation may contribute to tools for genetic analysis of human germ cell development.

Eliminating malignant contamination from therapeutic human spermatogonial stem cells.

Spermatogonial stem cell (SSC) transplantation has been shown to restore fertility in several species and may have application for treating some cases of male infertility (e.g., secondary to gonadotoxic therapy for cancer). To ensure safety of this fertility preservation strategy, methods are needed to isolate and enrich SSCs from human testis cell suspensions and also remove malignant contamination. We used flow cytometry to characterize cell surface antigen expression on human testicular cells and leukemic cells (MOLT-4 and TF-1a). We demonstrated via FACS that EpCAM is expressed by human spermatogonia but not MOLT-4 cells. In contrast, HLA-ABC and CD49e marked >95% of MOLT-4 cells but were not expressed on human spermatogonia. A multiparameter sort of MOLT-4-contaminated human testicular cell suspensions was performed to isolate EpCAM+/HLA-ABC-/CD49e- (putative spermatogonia) and EpCAM-/HLA-ABC+/CD49e+ (putative MOLT-4) cell fractions. The EpCAM+/HLA-ABC-/CD49e- fraction was enriched for spermatogonial colonizing activity and did not form tumors following human-to-nude mouse xenotransplantation. The EpCAM-/HLA-ABC+/CD49e+ fraction produced tumors following xenotransplantation. This approach could be generalized with slight modification to also remove contaminating TF-1a leukemia cells. Thus, FACS provides a method to isolate and enrich human spermatogonia and remove malignant contamination by exploiting differences in cell surface antigen expression.

Granulocyte colony-stimulating factor with or without stem cell factor extends time to premature ovarian insufficiency in female mice treated with alkylating chemotherapy.

OBJECTIVE: To examine gonadal protective properties of granulocyte colony-stimulating factor (G-CSF) alone or in combination with stem cell factor (SCF) in female mice treated with high-dose alkylating chemotherapy. DESIGN: Experimental laboratory animal study. SETTING: Tertiary care academic hospital and research institute. ANIMAL(S): Six- and 8-week-old C57Bl/6 female mice. INTERVENTION(S): Adult female mice were treated with [1] cyclophosphamide and busulfan (CTx), [2] CTx + G-CSF/SCF, [3] CTx + G-CSF, or [4] normal saline and dimethyl sulfoxide (DMSO; vehicle control). MAIN OUTCOME MEASURE(S): Follicle counts, microvessel density, cellular response to DNA damage, and litter production. RESULT(S): G-CSF ± SCF increased microvessel density and decreased follicle loss in CTx-treated female mice compared with CTx-only treated female mice. Mice administered CTx alone exhibited premature ovarian insufficiency, with only 28% of mice producing two litters. However, 100% of mice receiving CTx with G-CSF + SCF, and 80% of mice receiving CTx + G-CSF alone produced at least three litters and 20% of mice in each group produced five litters. CONCLUSION(S): Treatment of mice with G-CSF decreases chemotherapy-induced ovarian follicle loss and extends time to premature ovarian insufficiency in female mice. Further studies are needed to validate these preclinical results in humans and compare efficacy with the established GnRH analogue treatments.

Spermatogonial stem cell transplantation into rhesus testes regenerates spermatogenesis producing functional sperm.

Spermatogonial stem cells (SSCs) maintain spermatogenesis throughout a man's life and may have application for treating some cases of male infertility, including those caused by chemotherapy before puberty. We performed autologous and allogeneic SSC transplantations into the testes of 18 adult and 5 prepubertal recipient macaques that were rendered infertile with alkylating chemotherapy. After autologous transplant, the donor genotype from lentivirus-marked SSCs was evident in the ejaculated sperm of 9/12 adult and 3/5 prepubertal recipients after they reached maturity. Allogeneic transplant led to donor-recipient chimerism in sperm from 2/6 adult recipients. Ejaculated sperm from one recipient transplanted with allogeneic donor SSCs were injected into 85 rhesus oocytes via intracytoplasmic sperm injection. Eighty-one oocytes were fertilized, producing embryos ranging from four-cell to blastocyst with donor paternal origin confirmed in 7/81 embryos. This demonstration of functional donor spermatogenesis following SSC transplantation in primates is an important milestone for informed clinical translation.

Separating spermatogonia from cancer cells in contaminated prepubertal primate testis cell suspensions.

BACKGROUND: Chemotherapy and radiation treatments for cancer and other diseases can cause male infertility. There are currently no options to preserve the fertility of prepubertal boys who are not yet making sperm. Cryopreservation of spermatogonial stem cells (SSCs, obtained via testicular biopsy) followed by autologous transplantation back into the testes at a later date may restore fertility in these patients. However, this approach carries an inherent risk of reintroducing cancer. METHODS: To address this aspect of SSC transplantation safety, prepubertal non-human primate testis cell suspensions were inoculated with MOLT4 T-lymphoblastic leukemia cells and subsequently sorted for cell surface markers CD90 (THY-1) and CD45. RESULTS: Cancer cells segregated to the CD90-/CD45+ fraction and produced tumors in nude mice. Nearly all sorted DEAD box polypeptide 4-positive (VASA+) spermatogonia segregated to the CD90+/CD45- fraction. In a preliminary experiment, a purity check of the sorted putative stem cell fraction (CD90+/CD45-) revealed 0.1% contamination with cancer cells, which was sufficient to produce tumors in nude mice. We hypothesized that the contamination resulted from mis-sorting due to cell clumping and employed singlet discrimination (SD) in four subsequent experiments. Purity checks revealed no cancer cell contamination in the CD90+/CD45- fraction from three of the four SD replicates and these fractions produced no tumors when transplanted into nude mouse testes. CONCLUSIONS: We conclude that spermatogonia can be separated from contaminating malignant cells by fluorescence-activated cell sorting prior to SSC transplantation and that post-sorting purity checks are required to confirm elimination of malignant cells.