Spermatogonial stem cells share some, but not all, phenotypic and functional characteristics with other stem cells -- Data Supplement - Supporting Information -- Proceedings of the National Academy of Sciences

Supporting information for Kubota et al. (2003) Proc. Natl. Acad. Sci. USA, 10.1073/pnas.0631767100

Supporting Materials and Methods
Cell Staining with Antibodies and Hoechst 33342.
Dissociated testis cells were suspended (5 × 10⁶ cells per ml) in Dulbecco’s PBS supplemented with 1% FBS/10 mM Hepes/1 mM pyruvate/antibiotics (50 units/ml penicillin and 50 μg/ml streptomycin)/1 mg/ml glucose (PBS-S). All antibodies were obtained from Pharmingen, unless otherwise stated. For FACS analysis of MHC-I expression, cells were incubated with β2 microglobulin (β2M) antibody (S19.8) and biotin-conjugated anti-H-2K^b/H-2D^b antibodies (28-8-6) for 20 min on ice and washed twice with excess PBS-S. All staining and washing were performed with a similar protocol. Cells were stained with Alexa Fluor 647-conjugated goat anti-mouse IgG_2b antibody (647-IgG_2b, Molecular Probes) for β2M and Alexa Fluor 488-conjugated streptavidin (488-SAv, Molecular Probes) for H-2K^b/H-2D^b. Prior to FACS, 1 μg/ml propidium iodide (PI, Sigma) was added to the cell suspensions to exclude dead cells. For FACS experiments with four-color staining, cells were stained with anti-β2M and biotin-conjugated anti-Thy-1 antibodies (53-2.1), followed by staining with R-phycoerythrin (PE)-conjugated c-kit antibody (2B8), PE-Texas red-conjugated goat anti-mouse IgG antibody (Caltag, South San Francisco, CA), 488-SAv, and allophycocyanin (APC)-conjugated α6-integrin antibody (GoH3). For surface marker analysis of MHC-I^–Thy-1⁺, cells were stained with anti-β2M and biotin-conjugated anti-Thy-1, followed by 647-IgG_2b, 488-SAv, and PE-conjugated antibodies against αv-integrin (RMV-7), Sca-1 (E13-161.7), CD34 (RAM34), or CD24 (M1/69). Hoechst 33342 staining was performed as described (1) with slight modifications for testis cells. Testis cells or bone marrow cells were resuspended at 10⁶ cells per ml in prewarmed DMEM containing 2% FBS, 10 mM Hepes, antibiotics, and 4 μg/ml Hoechst 33342 (Sigma) and were incubated for 90 min at 35ΊC. This condition consistently identified a SP in testis cells. When verapamil was used, cell suspensions were stained as described in the presence of 50 μM verapamil (Sigma). For FACS analysis of surface markers of SP, cells were stained with PE-conjugated antibodies against Sca-1, CD45 (30-F11), CD34, Thy-1, c-kit, or CD43 (S7) after Hoechst staining. Anti-β2M antibody and Alexa Fluor 488-conjugated goat anti-mouse IgG were used for two-color staining of SP cells. One microgram of PI per milliliter was added to the cell suspension prior to FACS analysis. Fluorescence-Activated Cell Sorting.
Flow cytometric sorting and analysis were performed by a dual-laser FACStar Plus (BD Biosciences) equipped with Coherent Enterprise II laser (Coherent; 488 nm and 351/353 nm) and air-cooled helium neon laser (Spectra-Physics; 633 nm) made available through the Cancer Center Flow Cytometry and Cell Sorting Shared Resource at the University of Pennsylvania. Fluorochrome-conjugated antibodies or streptavidin were excited at 488 nm for Alexa Fluor 488, PE, and PE-Texas red, or at 633 nm for Alexa Fluor 647 and APC. Their fluorescence emissions were collected with a 530/30 for Alexa Fluor 488, 575/26 for PE, 610/20 for PE-Texas red, and 660/32 for APC. Hoechst 33342 was excited with 351/353 nm ultraviolet laser. The fluorescence emission was collected with a 450/20 filter (Hoechst blue) and a 675 LP (Hoechst red) filter (1). A 610 DMSP was used to separate these emission wavelengths. A second laser (488 nm) was used to excite Alexa Fluor 488 or PE-conjugated antibodies. Cells were sorted into 5-ml polypropylene tubes (Falcon 2063) containing 4 ml of PBS containing 10% FBS, 10 mM Hepes, 1 mM pyruvate, antibiotics, and 1 mg/ml glucose. An aliquot of stained cells in each experiment was not used for FACS (unsorted control). The sorted and unsorted control cells were centrifuged and resuspended in 3 ml of Ham’s F10 supplemented with 10% FBS/50 μM 2-mercaptoethanol/10 mM Hepes/antibiotics (F10-S). The tubes were gassed with 5% CO₂ and stored overnight at 4ºC. Bone Marrow Cell Transplantation and Analysis of Recipient Mice.
Bone marrow cells were suspended (5 × 10⁸ cells per ml) in Hank’s buffered saline solution with 2% FBS and 10 mM Hepes. Approximately 20 μl of cell suspension was transplanted into the jugular vein of immunologically compatible newborn (days 2–5 postpartum) W⁵⁴/W^V recipient mice (The Jackson Laboratory). Neonatal W mice were used to avoid irradiation and allow a high level of donor engraftment (2). After 8–9 months, cells were isolated from spleen, bone marrow, and testis of recipient mice to determine the number of GFP⁺ donor cells by FACS. Spleen cells were stained with CD45 antibody and analyzed for expression of CD45 and GFP. Bone marrow cells and testis cells were stained with Hoechst 33342, followed by staining with CD45 to analyze SP cells in the tissues. Red blood cells in spleen and bone marrow cell suspensions were lysed before staining.

1. Goodell, M. A., Brose, K., Paradis, G., Conner, A. S. & Mulligan, R. C. (1996) J. Exp. Med. 183, 1797–1806.

2. Capel, B., Hawley, R., Covarrubias, L., Hawley, T. & Mintz, B. (1989) Proc. Natl. Acad. Sci. USA 86, 4564–4568.