Pamela Yango

Testicular Niche Required for Human Spermatogonial Stem Cell Expansion

Prepubertal boys treated with high‐dose chemotherapy do not have an established means of fertility preservation because no established in vitro technique exists to expand and mature purified spermatogonial stem cells (SSCs) to functional sperm in humans. In this study, we define and characterize the unique testicular cellular niche required for SSC expansion using testicular tissues from men with normal spermatogenesis. Highly purified SSCs and testicular somatic cells were isolated by fluorescence‐activated cell sorting using SSEA‐4 and THY1 as markers of SSCs and somatic cells. Cells were cultured on various established niches to assess their role in SSC expansion in a defined somatic cellular niche. Of all the niches examined, cells in the SSEA‐4 population exclusively bound to adult testicular stromal cells, established colonies, and expanded. Further characterization of these testicular stromal cells revealed distinct mesenchymal markers and the ability to undergo differentiation along the mesenchymal lineage, supporting a testicular multipotent stromal cell origin. In vitro human SSC expansion requires a unique niche provided exclusively by testicular multipotent stromal cells with mesenchymal properties. These findings provide an important foundation for developing methods of inducing SSC growth and maturation in prepubertal testicular tissue, essential to enabling fertility preservation for these boys.

CHARACTERIZATION OF HUMAN SPERMATOGONIAL STEM CELL MARKERS IN FETAL, PEDIATRIC, AND ADULT TESTICULAR TISSUES

Autologous spermatogonial stem cell (SSC) transplantation is a potential therapeutic modality for patients with azoospermia following cancer treatment. For this promise to be realized, definitive membrane markers of prepubertal and adult human SSCs must be characterized in order to permit SSC isolation and subsequent expansion. This study further characterizes the markers of male gonocytes, prespermatogonia, and SSCs in humans. Human fetal, prepubertal, and adult testicular tissues were analyzed by confocal microscopy, fluorescence-activated cell sorting, and qRT-PCR for the expression of unique germ cell membrane markers. During male fetal development, THY1 and KIT (C-Kit) are transient markers of gonocytes but not in prespermatogonia and post-natal SSCs. Although KIT expression is detected in gonocytes, THY1 expression is also detected in the somatic component of the fetal testes in addition to gonocytes. In the third trimester of gestation, THY1 expression shifts exclusively to the somatic cells of the testes where it continues to be detected only in the somatic cells postnatally. In contrast, SSEA4 expression was only detected in the gonocytes, prespermatogonia, SSCs, and Sertoli cells of the fetal and prepubertal testes. After puberty, SSEA4 expression can only be detected in primitive spermatogonia. Thus, although THY1 and KIT are transient markers of gonocytes, SSEA4 is the only common membrane marker of gonocytes, prespermatogonia, and SSCs from fetal through adult human development. This finding is essential for the isolation of prepubertal and adult SSCs, which may someday permit fertility preservation and reversal of azoospermia following cancer treatment.

Optimizing cryopreservation of human spermatogonial stem cells: comparing the effectiveness of testicular tissue and single cell suspension cryopreservation.

OBJECTIVE To determine whether optimal human spermatogonial stem cell (SSC) cryopreservation is best achieved with testicular tissue or single cell suspension cryopreservation. This study compares the effectiveness between these two approaches by using testicular SSEA-4+ cells, a known population containing SSCs. DESIGN In vitro human testicular tissues. SETTING Academic research unit. PATIENT(S) Adult testicular tissues (n=4) collected from subjects with normal spermatogenesis and normal fetal testicular tissues (n=3). INTERVENTION(S) Testicular tissue versus single cell suspension cryopreservation. MAIN OUTCOME MEASURE(S) Cell viability, total cell recovery per milligram of tissue, as well as viable and SSEA-4+ cell recovery. RESULT(S) Single cell suspension cryopreservation yielded higher recovery of SSEA-4+ cells enriched in adult SSCs, whereas fetal SSEA-4+ cell recovery was similar between testicular tissue and single cell suspension cryopreservation. CONCLUSION(S) Adult and fetal human SSEA-4+ populations exhibited differential sensitivity to cryopreservation based on whether they were cryopreserved in situ as testicular tissues or as single cells. Thus, optimal preservation of human SSCs depends on the patients age, type of samples cryopreserved, and end points of therapeutic applications.

Endometrial Stromal and Epithelial Cells Exhibit Unique Aberrant Molecular Defects in Patients With Endometriosis

Context: Endometriosis is a chronic inflammatory disease that causes pain and infertility in women of reproductive age. Objective: To investigate the pathologic pathways in endometrial stromal and epithelial cells that contribute to the manifestation of endometriosis. Design: In vitro cellular and molecular analyses of isolated eutopic endometrial stromal and epithelial cells. Methods: Eutopic stromal and epithelial cells from endometriotic and normal patients were isolated by fluorescence-activated cell sorting for paired sibling RNA sequencing and microRNA microarray. Aberrant pathways were identified using ingenuity pathway analysis networks and confirmed with in vitro modulation of the affected pathways in stromal and epithelial cell cultures. Results: Both stromal versus epithelial cell types and paired endometriotic versus normal samples exhibited distinct hierarchical clustering. Compared to normal samples, there were 151 and 215 differentially expressed genes in the endometriotic stromal and epithelial populations, respectively, and concomitantly 9 and 16 differentially expressed microRNAs. Overall, endometriotic stromal and epithelial cells revealed distinct defects. In endometriotic stromal cells, key decidualization genes Zinc finger E-box Binding protein 1 (ZEB1), Heart And Neural crest Derivatives expressed 2 (HAND2), WNT4, and Interleukin 15 (IL-15) were found to be downregulated and Periostin (POSTN) and Matrix Metallopeptidase 7 (MMP7) were upregulated. Specifically, ZEB1 was downregulated in stromal cells by aberrant elevation in miR-200b. In contrast, ZEB1 was found to be upregulated in endometriotic epithelial cells through associated upregulation of transforming growth factor β1 (TGFβ1), inducer of the TGFβ1–Bone Morphogenetic Protein 2 (BMP2)–MMP2–Prostaglandin-endoperoxide Synthase 2 (COX2)–ZEB1 pathway, which activates epithelial–mesenchymal transition. Conclusion: Manifestation of endometriosis involves dysregulation of unique molecular pathways within the diseased endometrial stromal and epithelial cells in the endometrium. Targeting the cell type–specific defects may offer a novel approach to treating endometriosis.

MP74-16 DEVELOPMENT OF A PRACTICAL ANIMAL MODEL FOR HUMAN SPERMATOGONIAL STEM CELL TRANSPLANTATION

probenecid but did not reach statistical significance (18 2.6% vs. 22 3.7%, p1⁄40.19, Figure 1A). The most pronounced improvement was in the grade of sperm motility. Rapid linear motility increased significantly after 4 weeks of treatment (5.0 1.3% vs. 16 3.2%, p < 0.005, Figure 1B), and continued after the cessation of treatment (5.0 1.3% vs. 15 8.3%, p < 0.005, Figure 1B). Sperm concentrations remained normal throughout the course of the study. The medication was well-tolerated by all patients. CONCLUSIONS: This is the first study to report improved sperm motility from an oral medication in men with spinal cord injury. Planned placebo-controlled trials will determine optimal dosage regimens. Probenecid holds promise as a simple, effective therapy for low sperm motility in this severely affected group of patients.