Identification and enrichment of spermatogonial stem cells displaying side‐population phenotype in immature mouse testis

Abstract

In mammals, spermatogenesis is maintained by spermatogonial stem cells (SSC). In their niche, SSC divide to self-maintain and to produce a transit-amplifying population that eventually enters the meiotic cycle to give rise to spermatozoa. The low number of SSC and the lack of specific markers hinder their isolation and enrichment. Stem cells in several adult tissues can be identified by using their verapamil-sensitive Hoechst dye-effluxing properties, which define the characteristic "side population" (SP). Here we show, by multicolor flow cytometric analysis, that immature mouse testis contains a "side-population" (T-SP), which is Sca-1pos, Ep-CAMpos, EE2 pos, alpha6-integrin pos, and alpha(v)-integrin neg. A 13-fold enrichment in SSC activity was observed when sorted T-SP cells from ROSA 26 mice were transplanted in busulfan-treated mouse testis. Whereas an incomplete range of spermatogenic stages was encountered two months after transplantation of unsorted testicular cells, the transplantation of T-SP cells generated all associations of mouse germ cells representing the full range of spermatogenic stages. These data suggest that Hoechst staining and cell sorting might provide a novel approach to SSC enrichment in mammals.

Sca-1 expression identifies stem cells in the proximal region of prostatic ducts with high capacity to reconstitute prostatic tissue

“…Sca-1 is expressed by stem cells from a variety of origins including hematopoietic tissue, heart, mammary gland, skin, muscle, and testis (10)(11)(12)(13)(14)(15). Although a ligand for Sca-1 has not been identified, Sca-1 appears to be important for the self-renewal of mesenchymal (28) and hematopoietic (29) stem cells.…”

“…We have shown that cells with stem cell features (quiescence and high proliferative potential) are concentrated in the proximal region of prostatic ducts (9). By using FACS analysis, we determined whether the expression of three antigens, Sca-1, ␣6 integrin, and Bcl-2, known to be expressed by stem cells of other origins (10)(11)(12)(13)(14)(15)(16)(17)(18)(20)(21)(22), differs between the proximal and remaining ductal regions.…”

“…1 A and B and Table 1). Because high levels of Sca-1 are found on purified populations of other types of stem cells (14,15,26,27), we determined the location of cells with high MFI for Sca-1. The proximal region of ducts contained 7.2-fold more cells with high levels of Sca-1 (Sca-1 ϩ cells with fluorescence intensities in the upper one-third) than the remaining regions (Table 1; P Ͻ 0.00001; Fig.…”

“…Sca-1 is expressed by stem͞progenitor cells from a variety of tissues, such as hematopoietic tissue (10), cardiac tissue (11), mammary gland (12), skin (13), muscle (14), and testis (15). ␣6 integrin (CD49f) is expressed by primitive cells in the liver (16) and skin (17), and anti-␣6 integrin antibodies have been used to enrich for spermatogonial stem cells from mouse testis (18).…”

Side population purified from hepatocellular carcinoma cells harbors cancer stem cell-like properties

“…[19][20][21] SP cells are detected by their own ability to efflux Hoechst 33342 dye through an adenosine triphosphate (ATP)-binding cassette (ABC) membrane transporter. Recently, SP cells were also used in an attempt to isolate a stem cell-like fraction in cancer cells.…”