b1- and a6-integrin are surface markers on mouse spermatogonial stem cells

Although spermatogenesis is essential for reproduction, little is known about spermatogonial stem cells. These cells provide the basis for spermatogenesis throughout adult life by undergoing self-renewal and by providing progeny that differentiate into spermatozoa. A major impediment to our understanding of the biology of these stem cells is the inability to distinguish them from spermatogonia that are committed to differentiation. We made use of the known association of stem cells with basement membranes and our spermatogonial transplantation assay system to identify specific molecular markers on the stem cell surface. Selection of mouse testis cells with anti-b1or anti-a6-integrin antibody, but not anti-c-kit antibody, produced cell populations with a significantly enhanced ability to colonize recipient testes and generate donor cell-derived spermatogenesis. We demonstrate spermatogonial stem cell-associated antigens by using an assay system based on biological function. Furthermore, the presence of surface integrins on spermatogonial stem cells suggests that these cells share elements of a common molecular machinery with stem cells in other tissues. Spermatogenesis is a complex process that originates with and depends on spermatogonial stem cells. These primitive cells reside on the basement membrane of the seminiferous tubule and slowly proliferate to provide (a) additional stem cells by self-renewal, and (b) progeny cells that undergo significant amplification during the differentiation process to spermatozoa (1, 2). A single stem cell in the rat is capable of generating more than 4,000 spermatozoa, although many progeny cells are lost through programmed cell death during normal spermatogenesis (3–5). The differentiation process consists of three main phases: spermatogonial amplification, meiotic recombination, and spermatid differentiation. Although considerable information exists regarding differentiation stages of spermatogenesis, stem cell biology is poorly understood. The location of stem cells on the basement membrane and their general morphology are known; however, no specific biochemical or surface antigen markers have been identified (3–5). Consequently, detailed studies on the biology of this important cell have been severely handicapped. The body also contains self-renewing stem cell systems in other tissues, including hematopoietic, intestinal epithelial, and epidermal (6). All are characterized by a stem-cell population and a differentiating cell population. The number of stem cells relative to differentiating cells is low in each system; in testes there are estimated to be about two stem cells per 104 total testis cells, making these rare cells difficult to isolate and study (7). Because stem cells of different tissues show certain similarities in biological behavior, we hypothesized that they might share similar molecular properties even though the cell types or number of lineages produced during differentiation of daughter cells might be quite variable (6, 8). For instance, stem cells divide slowly and are usually on the basement membrane situated in a protected region, often described as a niche, among supporting cells. After damage by irradiation or chemicals, the stem cell is the last cell type destroyed and can regenerate the entire system in a relatively short time and in a regulated manner. Also, the stem cell often has the capacity to migrate during development, after transplantation, or when part of the population is destroyed (5, 6, 8). Thus, although the molecular mechanisms that control these events are not well understood for any of the self-renewing systems, it seems reasonable to assume that similar characteristics of stem cell activity may reflect shared molecular properties. For example, both steel factor and its surface tyrosine kinase receptor, c-kit, are critical for development and maintenance of both hematopoiesis and spermatogenesis (9), and c-kit appears on early stages of spermatogonia (10). Moreover, mice deficient in telomerase have defects in several systems that depend on stem cell self-renewal (11). These facts strongly argue for the existence of other shared molecules. Among self-renewing systems, hematopoiesis has been the best characterized. Identification of several stem cell markers now has facilitated purification of stem cells in a definitive manner (12) and led to isolation of single hematopoietic stem cells (HSC) (13). Success in this endeavor was possible only because a functional assay system was developed. Initially, donor cells were injected into irradiated animals, and spleen colony formation was assessed to indicate putative stem cells (14). Although considerable information evolved from this assay system, these cells proved to be progenitor cells rather than true stem cells. Definitive identification of the HSC was achieved by using the more laborious competitive long-term reconstitution analysis, in which transplanted cells repopulate the hematopoietic system (15). Thus, only a functional assay system capable of determining both long-term regenerative ability and adequacy of differentiated cell types is able to definitively identify the presence of a stem cell population. Recently, a system to transplant mouse testis cells from a fertile donor male to the seminiferous tubules of an infertile recipient male has been developed (16, 17). Spermatogenesis is generated from transplanted cells, and recipients are capable of transmitting the donor haplotype to progeny (17). After transplantation, primitive donor spermatogonia migrate to the basement membrane of recipient seminiferous tubules and begin proliferating (18, 19). Eventually, these cells establish stable colonies with a characteristic appearance, which expands and produces differentiating germ cells, including mature spermatozoa (18, 19). Thus, the transplanted cells selfrenew and produce progeny that differentiate into fully functional spermatozoa. By definition, only stem cells could produce this result. Therefore, the spermatogonial transplantation system provides a method to determine the spermatogonial stem cell potential of any donor cell population. In The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. PNAS is available online at www.pnas.org. Abbreviations: ECM, extracellular matrix; PE, R-phycoerythrin; Xgal, 5-bromo-4-chloro-3-indolyl b-D-galactoside. *To whom reprint requests should be addressed at: School of Veterinary Medicine, University of Pennsylvania, 3850 Baltimore Avenue, Philadelphia, PA 19104.