SUPPLEMENTAL METHODS
BMDSC and CD133 cell isolation from apheresis
Ten aphaeresis samples were obtained from PR patients and mononuclear cells were isolated by standard Ficoll-based centrifugation protocols. Briefly, 4 mL of aphaeresis was diluted to a final volume of 7 mL and added to 3 mL of Ficoll-Paque (GE Healthcare, Uppsala, Sweden) to undergo a 400xg centrifugation for 30 min at RT. Then the interphase containing BMDSC mononuclear cells was carefully recovered. Cells were washed twice with PBS and resuspended in RPMI 1640 medium supplemented with 10% human serum albumin (HSA) and 1X Penicillin/Streptomycin (P/S).
The viable CD133-positive population was then isolated with a MACS Separator using the CD133 MicroBead Kit-Hematopoietic Tissue (MiltenyiBiotec, GmbH, BergischGladbach, Germany). Briefly, BMDSC were labeled with FcR Blocking Reagent and incubated with CD133 Microbeads for 30 min at 4ºC according to the manufacturer’s recommendations. Cell suspension was then applied to an autoMACS Pro Separator for fully automated separation, yielding the unlabeled negative fraction, which was discarded, while the magnetically labeled positive fraction (viable CD133+ cells) was collected in 10%HSA-RPMI 1640 medium. The selection was performed up to the collection of a minimum of 3x105 cells per sample. Cell viability was assessed after CD133+ selection by flow cytometry with 7-aminoamictomycin (7-AAD) staining following standard procedures.
Cell labeling
BMDSC, CD133+ and PBMNC cells were labeled with Molday ION Rhodamine B (MIRB; BioPAL, Worcester, MA) by a non-transfection-based method before injection. Briefly, 1x106 cells resuspended in 1 mL of 10%HSA-1xP/S supplemented RPMI 1640 were incubated for 18 hours, at 37ºC, 5% CO2 with50 µg/mL MIRB. Then cells were washed and viable cells were countedbyTrypan blue staining (1:10 dilution) in a hemocytometer,andresuspended in PBS with calcium and magnesium for injection. MIRB-labeling was evaluated in a 100 uL aliquot using a FC500 MCL Flow Cytometer (Beckman-Coulter, CA, USA) where Rhodamine B signal was measured at 575 nm.
Cell visualization and engraftment
Cell engraftment was assessed by intracellular iron deposit visualization after treatment with MIRB using Prussian blue ACCUSTAIN IRON STAIN (HT20; Sigma-Aldrich). Mouse ovaries, human ovarian grafts, spleen, liver, and kidney sections from treated animals were screened for the presence of labeled-BMDSC and PBMNC.
Human ovarian cortex samples
After obtaining written informed consent, 7 human ovarian cortex biopsies by cesarean section from PRwomen underwent ovum donation because of PR and several failed attempts with their own oocytes (43±4yr). Ovarian biopsies (1 x 1cm approximately) were washed with serum-free M199 medium (Sigma, St. Louis MI), dissected by removing medulla with a scalped blade (21)and cryopreserved by slow-freezing until use. A small piece of each biopsy was fixed and used as pre-xenograft control.
Human ovarian cortex xenograft procedure
Animals were anesthetized by isoflurane and ovariectomized following standard procedures. One human OCfragment (5x4-5mm, 1mm thickness) was intraperitoneally grafted with a Premilene 6-0 suture (B/Braun, Melsungen AG, Germany). Incisions were closed using Monosyn 5-0 suture (Ethicon, New Brunswick, NJ).
Follicular counts
Ovarian formalin-fixed samples were paraffin-embedded and cut into 4-μm thick sections. Every 5th (mouse) or 10th(human graft) section was stained with hematoxylin-eosin (H&E). Only morphologically normal follicles were counted when the oocyte nucleus was present. Follicles were counted and classified as previously described (22).
Vascularization
Vascularization of mouse ovaries was assessed by double immunofluorescence with isolectin B4,specifically labeling endothelial cells and alpha-smooth muscle actin (α-SMA) to stain mature vessels. Briefly, Griffoniasimplicifoliaisolectin B4 (Vector Laboratories, Burlingame, USA), at the 1:50 dilution, was incubated for 18 hours at 4ºC. Then, a mouse monoclonal anti-α-SMA antibody (Sigma, St. Louis MI), at the 1:200 dilution, was used for 30 min at room temperature (RT). For human ovarian xenografts, a mouse monoclonal anti-CD31 antibody (JC70A, Dako Denmark A/S, Glostrup, Denmark), at the 1:150 dilution during 60 min was used to detect blood vessels. For the secondary antibody incubation a biotin/streptavidin reaction was used (ENVISION method Dako Denmark A/S, Glostrup, Denmark), and staining was detected with 3,30-diaminobenzidine (DAB).
In both studies, 4 representative sections were assayed per sample. High-magnification images (20×) were obtained in a bright field microscope with a fluorescence module(LEICA DM4000B) and a digital camera attached (LEICADFC450C, Leica Microsystems GmbH, Germany). Samples were analyzed using the Image Pro-plus software (Mediacybernetics; Carlsbad, CA). For mouse ovaries, lectin-positive areas were considered as vessels; in the human grafts vessels were determined by positive CD31 immunostaining. Microvessel density (MVD) was determined as vessel positive area by section.
Relative gene expression
Vascularization was also studied by mouse/human CD31 expression at mRNA level by real time PCR. The mRNA was obtained from mouse ovaries and human ovarian xenografts (30mg) of each group with the RNeasy Mini Kit (Qiagen) following the manufacturer´s protocol. cDNA was synthesized with Advantage® RT-for-PCR Kit (Clontech Laboratories, Inc). Real-Time PCR with specific Taqman probes for mouse and human CD31 (Mm01242576_m1 and Hs01065282_m1, respectively) were used. PCR reaction mixtures were prepared with 100 ng of cDNA as a template in the 1xTaqman Gene Expression Master Mix following the manufacturer´s protocol. Then samples were amplified in the 7900HT Fast PCR system (Applied Byosistems, Life Technologies Inc., Gaithersburg, MD, USA). PCR conditions consisted in an initial activation of uracyl-N-glycosylase at 50°C for 2 min., followed by AmpliTaq Gold activation at 95°C for 10 min. Then samples were cycled 40 times by denaturation at 95°C for 15 seconds, and an annealing extension at 60°C for 1 min per cycle.Each sample was assayed in triplicate. The relative expression was obtained by the comparative Ct method (DDCt) with the 18S reporter gene.
Cell proliferation and apoptosis
To establish the ovarian status of analyzed samples, proliferative and damagedstromal cells and follicles were quantified by Ki-67 immunostaining and TUNELassay, respectively. Briefly, a polyclonal rabbit anti-Ki67 antibody (ab15580, Abcam, Cambridge, UK at the 1:300), or a monoclonal mouse anti-human Ki-67 antibody (MIB-1, Dako Denmark A/S, Glostrup, Denmark at the 1:100 dilution), were incubated at RT for 1h. A biotin/streptavidin reaction was used for the secondary antibody incubation (LSAB method Dako Denmark A/S, Glostrup, Denmark), and staining was detected with DAB. The negative controls were an isotype-matched rabbit or mouse immunoglobulin (Ig)G at 1:50 dilution (DakoCytomation) and a section of proliferative endometrium was used as a positive control.
DNA fragmentation was detected and quantified by the TUNEL assay using the TMR red in situ cell death detection kit (Roche Diagnostics, Germany). For the negative controls samples were incubated with Label solution (without terminal transferase) instead of TUNEL reaction mix. For the positives samples were treated with DNase I (2000U/mL during 10min.
To quantify cell proliferation and damage, three sections per sample were analyzed for Ki-67 or TUNEL signal. High magnification images were obtained and analyzed by Image Pro-Plus as described above.
Estradiol (E2) production
E2 production was determined with the Estradiol EIA kit (Cayman Chemical Company, Michigan, USA) following the manufacturer´s protocol in undiluted plasma. All samples were run in triplicate and results expressed in pg/mL according to the standard curve. The LOD of the assay was 20pg/mL and interference with other steroid hormones was < 0.01%. For 102.4 pg/mL, the intra- and inter-assay variations were 13.0% and 8.2%, respectively.