Generation of endometrial regenerative cells
Menstrual blood was collected from a healthy female subject after menstrual blood flow initiated. Collection was performed in a urine cup and then transferred into a 5 ml tube with 0.2 ml amphotericin B (Sigma-Aldrich, St Louis, MO), 0.2 ml penicillin/streptomycin (Sigma 50 ug/nl) and 0.1 ml EDTA-Na2 (Sigma) in phosphate buffered saline (PBS). Mononuclear cells derived from menstrual blood were separated by Ficoll-Paque (Fisher Scientific, Portsmouth NH) according to the instruction and washed in PBS. Cells were subsequently cultured in a Petri dish (Corning, Acton, MA) containing DMEM medium supplemented with 1% penicillin/streptomycin, 1% amphotericin B, 1% glutamine and 20% FBS (completed DMEM). Media was changed the next day. Adherent cells were detached by trypsin and cultured in a T75 flask (Fisher Scientific, Portsmouth NH) at 1 × 105 cells. The cells were then subcultured and passaged twice a week. Cloning of cells was accomplished by plating cells at a concentration of approximately 1 cell per well in 96 well plates (Corning, Acton, MA).
Phenotypic characterization
For fluorescent antibody cell surface staining cells were washed with HBSS+2%BSA two times and incubated with the specific antibody at concentrations recommended by the respective manufacturer. Cells were incubated for 20 min and analyzed either under fluorescent microscope or flow cytometry. The antibodies used were: CD markers, SSEA-4, Stro-1, HLA-ABC and HLA-DR. These were purchased from BD Pharmingen, Ancell, Stem Cell Technologies, eBioscience, Chemicom, Miltenyi Biotech and R&D Systems. For intracellular staining by the antibodies without any conjugate, cells were washed twice in Hank's solution with 2% BSA and fixed with 4% Formalin for 1 hour. Subsequently cells were washed twice in 0.5% Tween20 and 0.1% Triton X-100 in PBS (T-PBS). Primary antibodies were added to T-PBS at the concentrations recommended by the manufacturer. Incubation was performed for 30 min. Cells were then washed twice in T-PBS. Corresponding secondary antibodies with fluorescent conjugates were subsequently diluted in T-PBS at the concentrations suggested by the manufacture instructions. Incubate was performed for 20 min and cells were analyzed using fluorescent microscopy or flow cytometry. The intracellular antibodies used were Oct-4, Nanog and telomerase (clone Y182, hTert: Abcam).
Karyotypic analysis
FPB cells were sent to NeoDiagnostix, Inc. (Rockville MD) for karyotypic analysis. Cells were harvested at 70–80% confluency and resuspended in 10 microliters of colcemid per ml of media. Cells were incubated at 37°C for 3–6 hrs after which cells were resuspended in 0.5 ml medium and mixed with 0.075 M KCl to a volume of 10 ml. After incubation for 10–15 min at 37°C in a waterbath cells were resuspended to in a total of 10 ml fixative (methonal:acetic acid as 3:1). Staining with DAPI for G-banding was performed by equilibrating the slides in 0.3 M sodium citrate, containing 3 M NaCl for 5 min and subsequent addition of 2 drops of Antifade with DAPI per slide prior to visualization.
Proteomic analysis
Conditioned media from different cell lines was sent to RayBiotech, Inc (Norcross GA) for cytokine array analysis. According to the company's sample preparation instructions, the media were changed to DMEM with 0.2% fetal calf serum. Each flask was rinsed with 10 ml of this media and refilled to 7 ml. After culture for two days, the media was removed and centrifugation at 2000 rpm for 10 minutes was performed to remove cellular debris and frozen at -70°C for shipping. The cell number in culture was used to calculate the cytokine yield (pg) per million cells. DMEM with 0.2% fetal calf serum (control media) with no cells was sent for the analysis as well.
Differentiation
Adipogenic differentiation
ERC were seeded at a concentration of 4 × 10[4] cells/ml in an 8 well chamber slide (Lab-Tek, Campbell, CA) with 0.5 ml media per well. When the cells reached 100% confluence they were transferred to Adipogenic Induction Media (Cambrex, East Rutherford, NJ) and cultured for 10 days with media changes every 3–4 days. Control cells were cultured in completed DMEM media. Cells are subsequently stained with AdipoRed (Cambrex) and visualized under fluorescent microscopy.
Osteogenic differentiation
ERC were seeded at a concentration of 1 × 10[4] cells/ml in an 8 well chamber slide (Lab-Tek) with 0.5 ml completed DMEM media per well. After the cells adhere overnight, the medium is changed to the Osteogenic Induction media (Cambrex). Cultures were cultured for 21 days with medium changes every 3–4 days. Control cells were cultured in complete DMEM. Cells were stained with Alizarin Red (ScholAr Chemistry, West Henrietta, NY) and visualized.
Endothelial differentiation
ERC cells were seeded at a concentration of 1.9 × 10[4] cells/ml in an 8 well chamber slide (Lab-Tek) with 0.5 ml complete DMEM per well. After the cells were cultured overnight the media was changed to the Endothelial Induction media (Cambrex). Cells were cultured for 21 days with media changes every 3–4 days. Control cells were cultured in complete DMEM. Cells are stained with anti-CD34 and anti-CD62 (Ancell) followed by fluourescently tagged secondary antibody.
Neurogenic differentiation
ERC cells were seeded at a concentration of 1.6 × 10 [4] cells/ml in an 8 well chamber slide (Lab-Tek) with 0.5 ml complete DMEM. After the cells adhered overnight, the media was changed to the NPMM neural induction media (Cambrex #CC-3209) and supplemented with 1% penicillin/streptomycin, 0.2 mM glutamax (Invitrogen) and hFGA-4 (Sigma F8424, 20 ng/ml). Cultures were cultured in induction or control complete DMEM media for 21 days with media changes every 3–4 days. Cells were stained with GFAP (Sigma) and Nestin (Chemicon), conjugated goat anti-mouse antibody (Bethyl Montgomery, Texas).
Pulmonary epithelial differentiation
ERC were seeded at a concentration of 2 × 10 [4] cells/ml on 8 well chamber slides (Lab-Tek) with 0.5 ml complete DMEM per well. When the cells reach 100% confluency the media was changed to induction medium (SAGM, Cambrex). Cultures were cultured for 10 days with media changes every 3–4 days. Control cells were cultured in complete DMEM media alone. Cells were stained with ProSP-C (Chemicon) plus conjugated Goat Anti-rabbit (Invitrogen).
Hepatic/pancreatic differentiation
ERC were seeded at a concentration of 2 × 10[4] cells/ml in an 8 well chamber slide (BD Biosciences #354630) with 0.5 ml CM20 per well. After the cells adhere overnight, the medium is changed to the induction medium (Cambrex) supplemented with hepatocyte growth factor (40 ng/ml), b-FGF (20 ng/ml), hFGF-4 (20 ng/ml), SCF (40 ng/ml) (all from Sigma). Cultures were maintained for 30 days with media changes every 3–4 days. Cells were stained with antibodies to Albumin (R&D #MAB1455) and insulin and developed plus secondary goat Anti-mouse (Bethyl #A90-216F) and mouse anti-rat (Serotec), respectively.
Cardiogenic and myogenic differentiation
8 well chamber slides were pre-coated with fibronectin (Sigma #F2006) and ERC were seeded at a concentration of 1.9 × 10[4] cells/ml. After overnight culture adherent cells were treated with complete DMEM containing 10 μM 5-Azacytidine (Sigma) for 24 hours. Subsequently the cells were cultured for 14 days in Skeletal Muscle Growth Medium (Cambrex) supplemented with 100 ng/ml b-FGF (Sigma). Cells were stained with Alpha-Actinin (Abcam) for myocyte and Skeletal Myosin (Abcam, Cambridge MA) for skeletal myocyte. For the cardiogenic differentiation, cultures are allowed to develop for 40 days with medium changes every 3–4 days and stained with Troponin I (Abcam #AB19615) plus conjugated Goat Anti-mouse (Bethyl #A90-216F). In some experiments cells were grown as hanging drop cultures as described [28] in order to visualize beating. Briefly, 30–50 μl of cells were placed on a lid of a petri-dish (Becton Dickinson Falcon #35–3002) and 5–9 ml sterile PBS to bottom of dish to maintain a humidified environment. Beating cells were detected after 5 days.