Hanna Valli

Spermatogonial Stem Cell Transplantation into Rhesus Testes Regenerates Spermatogenesis Producing Functional Sperm

Spermatogonial stem cells (SSCs) maintain spermatogenesis throughout a mans 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.

Direct Differentiation of Human Pluripotent Stem Cells into Haploid Spermatogenic Cells

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) have been shown to differentiate into primordial germ cells (PGCs) but not into spermatogonia, haploid spermatocytes, or spermatids. Here, we show that hESCs and hiPSCs differentiate directly into advanced male germ cell lineages, including postmeiotic, spermatid-like cells, in vitro without genetic manipulation. Furthermore, our procedure mirrors spermatogenesis in vivo by differentiating PSCs into UTF1-, PLZF-, and CDH1-positive spermatogonia-like cells; HIWI- and HILI-positive spermatocyte-like cells; and haploid cells expressing acrosin, transition protein 1, and protamine 1 (proteins that are uniquely found in spermatids and/or sperm). These spermatids show uniparental genomic imprints similar to those of human sperm on two loci: H19 and IGF2. These results demonstrate that male PSCs have the ability to differentiate directly into advanced germ cell lineages and may represent a novel strategy for studying spermatogenesis in vitro.

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.

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.

Germline stem cells: toward the regeneration of spermatogenesis

Improved therapies for cancer and other conditions have resulted in a growing population of long-term survivors. Infertility is an unfortunate side effect of some cancer therapies that impacts the quality of life of survivors who are in their reproductive or prereproductive years. Some of these patients have the opportunity to preserve their fertility using standard technologies that include sperm, egg, or embryo banking, followed by IVF and/or ET. However, these options are not available to all patients, especially the prepubertal patients who are not yet producing mature gametes. For these patients, there are several stem cell technologies in the research pipeline that may give rise to new fertility options and allow infertile patients to have their own biological children. We will review the role of stem cells in normal spermatogenesis as well as experimental stem cell-based techniques that may have potential to generate or regenerate spermatogenesis and sperm. We will present these technologies in the context of the fertility preservation paradigm, but we anticipate that they will have broad implications for the assisted reproduction field.

Spermatogonial Stem Cells and Spermatogenesis

This chapter reviews the development of the spermatogonial stem cell (SSC) field from the late 1800s to 2014 and envisions the fundamental and practical advances that the field will experience in the coming decades. We describe the current models of SSCs and spermatogenic lineage development in rodents, nonhuman primates, and humans to identify features that are conserved through evolution as well as species-specific differences. We describe experimental tools used to study SSCs and spermatogenic lineage development and discuss how data generated with those tools should be interpreted. We discuss the current knowledge about the molecular mechanisms that regulate SSC function and, more importantly, the knowledge gaps that will be the focus of future investigations. Finally, we describe how SSCs and the SSC transplantation technique can be exploited to produce transgenic animals, develop biopharmaceuticals, and treat male infertility.

Stem Cell Therapies for Male Infertility: Where Are We Now and Where Are We Going?

Chemotherapy and radiation treatments for cancer and other conditions can cause permanent infertility. Adult men have the option to cryopreserve a semen sample with sperm prior to treatment and use their sample in the future to have biological children using the established assisted reproductive technologies, in vitro fertilization (IVF) with intracytoplasmic sperm injection (ICSI). This option is not available to prepubertal boys who are not yet producing mature sperm. However, these boys do have spermatogonial stem cells in their testes that are poised to initiate sperm production at puberty. Centers in the United States and abroad are actively cryopreserving testicular tissue for prepubertal cancer patients, bone marrow transplant patients, and others in anticipation that stem cell therapies will be available for them in the future. This chapter reviews progress in the development of spermatogonial stem cell transplantation, testicular tissue grafting and xenografting, testicular tissue organ culture, de novo testicular morphogenesis, and pluripotent stem cell-derived gametogenesis. The prepubertal cancer patient is used throughout this chapter as a model for discussion, which intended to stimulate thinking about other applications of stem cell technologies for preserving and treating male infertility.

Whole-mount immunohistochemistry to study spermatogonial stem cells and spermatogenic lineage development in mice, monkeys, and humans.

Spermatogonial stem cells (SSCs) and undifferentiated progenitor spermatogonia in mammalian seminiferous tubules are organized in chains, connected by intracellular bridges. Clone size is generally related to stem cell potential, with shorter chains containing the majority of the stem cell population. Immunofluorescence detection of spermatogonia-specific proteins in whole-mount seminiferous tubule preparations is the only method that allows researchers to relate clone size with the molecular phenotype in spermatogenic lineage development. Here we describe in detail the method used to detect nuclear, cytoplasmic, and cell surface molecules in seminiferous tubules isolated from mouse, monkey, and human testes.

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

Abstract 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 realtime 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 spaltlike transcription factor 4 (SALL4), two markers ofmouse 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. Summary Sentence A DDX4 (VASA)-EGFP transgenic rat was produced to facilitate investigations of stem cells and germ lineage development.