Enrichment and In Vitro Culture of Spermatogonial Stem Cells from Pre-Pubertal Monkey Testes

Abstract

Spermatogonial stem cells (SSCs) are essential for spermatogenesis throughout the lifespan of the male. However, the rarity of SSCs has raised the need for an efficient selection method, but little is known about culture conditions that stimulate monkey SSC proliferation in vitro. In this study, we report the development of effective enrichment techniques and in vitro culturing of germ cells from pre-pubertal monkey testes. Testis cells were analyzed by fluorescence-activated cell sorting techniques and were transplanted into the testes of nude mice to characterize SSCs. Thy-1-positive cells showed a higher number of colonies than the unselected control after xenotransplantation. Extensive colonization of monkey cells in the mouse testes indicated the presence of highly enriched populations of SSCs in the Thy-1-positive sorted cells. Furthermore, monkey testis cells were enriched by differential plating using extracellular matrix, laminin, and gelatin, and then cultured under various conditions. Isolation of monkey testicular germ cells by differential plating increased germ cell purity by 2.7-fold, following the combinational isolation method using gelatin and laminin. These enriched germ cells actively proliferated under culture conditions involving StemPro medium supplemented with bFGF, GDNF, LIF, and EGF at 37 °C. These results suggest that the enrichment and in vitro culture method proposed in the present study for harvesting a large number of functionally active monkey SSCs can be applied as the basis for efficient in vitro expansion of human SSCs.

Keywords: Enrichment; Monkey spermatogonial stem cells; in vitro culture.

Fig. 1

Characteristics of pre-pubertal monkey testis cells. A Hematoxylin and eosin (H&E) staining of donor testis (left). Immunohistochemical analysis of PLZF (green) within cross-sections of seminiferous tubules from pre-pubertal monkey testes (right). Histological sections were counterstained with DAPI (blue), indicating the nuclei of all cells in the section. Arrows indicate PGP 9.5-expressing germ cells. Scale Bar = 50 μm. B Testis cells were sorted into two fractions (G1, G2) based on light-scattering properties by fluorescence-activated cell sorting (FACS) and PI-positive (dead) cells were excluded before the analysis (top). The cells in the G2 fraction, excluding the PI-positive cells, were analyzed to determine the percentage of cells positive for undifferentiated spermatogonia markers (E-cadherin, EP-CAM, Thy-1, SSEA-1, and SSEA-4), and the differentiating germ cell marker c-kit, compared with that of unselected controls (n = 3). The vertical lines represent the range of fluorescence in the controls. C Immunocytochemical detection of undifferentiated spermatogonia promyelocytic leukemia zinc finger (PLZF) in FACS-isolated cell fractions. The percentages of PLZF-positive cells were 10.9 ± 3.8, 66.3 ± 4.9, 3.6 ± 1.6, 59.4 ± 9.4, and 3.9 ± 2.2% in the unselected, Thy-1-positive, Thy-1-negative, SSEA-1-positive, and SSEA-4-negative populations, respectively. Values are depicted as the mean ± SEM, n = 3. Bars with different letters are statistically significant. (Color figure online)

Fig. 2

Germ cell transplantation of Thy-1-positive and SSEA-4-positive cells separated from monkey testis cells. A–D PKH26-labeled (red) unselected, Thy-1-positive, and SSEA-4-positive monkey testis cells formed colonies consisting of chains of cells in the recipient mouse testes. Scale bar = 200 μm. E The number of colonies determined by PKH26-labeling was 96.0 ± 5.1, 340.0 ± 140.0, and 257.1 ± 114.3 in the unselected, Thy-1-positive, and SSEA-4-positive cell populations transplanted into the recipient mice, respectively. Values represent the mean ± SEM. Bars with different letters are statistically significant. (Color figure online)

Fig. 3

Immunofluorescence detection of PLZF in pre-pubertal monkey testis cells enriched after differential plating. A–F PLZF (green) expression in whole testis (initial), Percoll-selected, differential plating (gelatin, laminin, or combination of gelatin and laminin)-selected populations. The cells were counterstained with DAPI (blue). Scale bar = 50 μm. G The percentages of PLZF-positive cells were 17.2 ± 3.3, 14.3 ± 0.7, 25.0 ± 3.4, 30.5 ± 6.0, 30.5 ± 1.0, 45.8 ± 0.7, 2.6 ± 0.4, 5.1 ± 0.8, and 3.7 ± 0.4% in the initial, Percoll upper, Percoll bottom, unbound (gelatin, laminin, and combination of gelatin and laminin), and bound population (gelatin, laminin, and combination of gelatin and laminin), respectively. Values represent the mean ± SEM, n = 3. Bars with different letters are statistically significant. (Color figure online)

Fig. 4

Verification of growth efficiency of monkey germ cells by in vitro culturing under various conditions. A, B Effects of the media on the maintenance of monkey germ cells. Monkey testis cells were cultured in various media, including Stempro, mSFM, rSFM, and 2 × rSFM. The data represent the number of A total testis cells or B germ cells. B A significantly higher number of germ cells was observed in a culture group with Stempro (1.9 ± 0.4 × 10⁴). C, D Effects of temperatures on in vitro culture of monkey germ cells. The number of testis cells and germ cells (17.0 ± 2.1 × 10³ cells and 3.2 ± 0.7 × 10³ cells, respectively) cultured at 37 °C were significantly greater than that for cells (5.3 ± 0.9 × 10³ cells and 0.5 ± 0.1 × 10³ cells, respectively) cultured at 34 °C. E, F Effects of growth factors on the maintenance of monkey germ cells. F A significantly higher number of germ cells was observed in the culture condition with all 4 growth factors (5.0 ± 1.1 × 10³ cells). Values represent the mean ± SEM, n = 3. Bars with different letters are statistically significant