Bart T. Phillips

Spermatogonial stem cell regulation and spermatogenesis

This article will provide an updated review of spermatogonial stem cells and their role in maintaining the spermatogenic lineage. Experimental tools used to study spermatogonial stem cells (SSCs) will be described, along with research using these tools to enhance our understanding of stem cell biology and spermatogenesis. Increased knowledge about the biology of SSCs improves our capacity to manipulate these cells for practical application. The chapter concludes with a discussion of future directions for fundamental investigation and practical applications of SSCs.

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.

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.

Genes involved in post-transcriptional regulation are overrepresented in stem/progenitor spermatogonia of cryptorchid mouse testes.

Gene expression and consequent biological activity of adult tissue stem cells are regulated by signals emanating from the local microenvironment (niche). To gain insights into the molecular regulation of spermatogonial stem cells (SSCs), gene expression was characterized from SSCs isolated from their cognate niches of cryptorchid (stem cell‐enriched), wild‐type, and busulfan‐treated (stem cell‐depleted) mouse testes. Quantitative assessment of stem cell activity in each testis model was determined using an in vivo functional assay and correlated with gene expression using Affymetrix MGU74Av2 microarrays and the ChipStat algorithm optimized to detect gene expression from rare cells in complex tissues. We identified 389 stem/progenitor spermatogonia candidate genes, which exhibited significant overlap with genes expressed by embryonic, hematopoietic, and neural stem cells; enriched spermatogonia; and cultured SSCs identified in previous studies. Candidate cell surface markers identified by the microarray may facilitate the isolation and enrichment of stem and/or progenitor spermatogonia. Flow cytometric analyses confirmed the expression of chemokine receptor 2 (Ccr2) and Cd14 on a subpopulation cryptorchid testis cells (α6‐integrin+, side scatterlo) enriched for SSCs. These cell surface molecules may mark progenitor spermatogonia but not SSCs because Ccr2+ and Cd14+ fractions failed to produce spermatogenesis upon transplantation to recipient testes. Functional annotation of candidate genes and subsequent immunohistochemistry revealed that proteins involved in post‐transcriptional regulation are overrepresented in cryptorchid testes that are enriched for SSCs. Comparative analyses indicated that this is a recurrent biological theme among stem cells.

The transition from stem cell to progenitor spermatogonia and male fertility requires the SHP2 protein tyrosine phosphatase

SHP2 is a widely expressed protein tyrosine phosphatase required for signal transduction from multiple cell surface receptors. Gain and loss of function SHP2 mutations in humans are known to cause Noonan and LEOPARD syndromes, respectively, that are characterized by numerous pathological conditions including male infertility. Using conditional gene targeting in the mouse, we found that SHP2 is required for maintaining spermatogonial stem cells (SSCs) and the production of germ cells required for male fertility. After deleting SHP2, spermatogenesis was halted at the initial step during which transit‐amplifying undifferentiated spermatogonia are produced from SSCs. In the absence of SHP2, proliferation of SSCs and undifferentiated spermatogonia was inhibited, thus germ cells cannot be replenished and SSCs cannot undergo renewal. However, germ cells beyond the undifferentiated spermatogonia stage of development at the time of SHP2 knockout were able to complete their maturation to become sperm. In cultures of SSCs and their progeny, inhibition of SHP2 activity reduced growth factor‐mediated intracellular signaling that regulates SSC proliferation and cell fate. Inhibition of SHP2 also decreased the number of SSCs present in culture and caused SSCs to detach from supporting cells. Injection of mice with an SHP2 inhibitor blocked the production of germ cells from SSCs. Together, our studies show that SHP2 is essential for SSCs to maintain fertility and indicates that the pathogenesis of infertility in humans with SHP2 mutations is due to compromised SSC functions that block spermatogenesis. Stem Cells 2014;32:741–753

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.

The Elusive Spermatogonial Stem Cell Marker

Commentary on Oatley et al., “Inhibitor of DNA binding 4 is expressed selectively by single spermatogonia in the male germline and regulates the self-renewal of spermatogonial stem cells.”