SUPPLEMENTAL DATA

MSC isolation and expansion
Most studies demonstrate that mesenchymal stromal cells (MSCs) from various tissues share their principal immunomodulatory and regenerative properties (Hoogduijn et al., Stem Cell Dev 2006; 15:49-60; Kern et al., Stem Cell 2006; 24:1294-301). MSC from bone marrow, however, are the most extensively characterized and have already been used to suppress the alloresponse in primates (Bartholomew et al., Exp Hematol 2002; 30: 42-8; Beggs et al., Cell Transplant 2006; 15:711-21) and have been safely applied in first clinical trials with bone marrow transplant patients (Le Blanc et al., Lancet 2008; 371:1579-86). Thirty-five ml of bone marrow aspirates were collected. Nucleated cells (361x106 TNC) were resuspended in Minimum Essential Medium Alpha e (Gibco-Invitrogen, Carlbad, CA, USA), supplemented with 2UI/ml Heparin (Pharmatex, Milan, Italy), and 5% human platelet lysate (hPL) with approximately 1x103/mL of platelets. Cells were seeded in one 1-CellSTACK chamber (Corning, NY, USA) at a density of 5x105 TNC/cm2. After 2-3 days, the supernatant containing non-adherent cells was removed. 50% medium change was performed twice weekly until approximately 80% confluence was reached. After 13 days, cells were detached using animal-origin free TrypLETM Express (Gibco-Invitrogen, Carlbad, CA, USA). Cells recovered from the first trypsinization were subsequently replated at 200 cells/cm2 in 5-CellSTACK chambers (Corning, NY, USA). After 12 days, cells were harvested and cryopreserved in human AB plasma containing 10% DMSO in clinical-grade cryopreservation bags (Fresenius Medical Care, Bad Homburg, Germany). Aliquots of final cell product and pooled culture supernatants were removed before freezing to perform all quality control tests (see below). MSCs were stored in liquid nitrogen until the day of the infusion into the kidney transplant recipient. The MSC dose was based on that used to control steroid-resistant GVHD in patients receiving hematopoietic stem cell transplantation (Le Blanc K, et al Lancet 2008; 371:1579-86).

MSC quality controls and release criteria 
hMSC viability was evaluated by 7-amino-actinomycin D (7-AAD) exclusion measured by a FACScan instrument (Becton Dickinson, San Josè CA). Criterion for release was viability >80%. Viability values were confirmed by Trypan blue exclusion.
The phenotype of hMSC was analyzed by staining with CD45-FITC, CD34-PE, CD14-FITC, HLA-DR-FITC, CD90-FITC, CD73-PE, HLA-ABC-PE (BD Biosciences, San José, CA), CD105-PE (Caltag Laboratories, Burlingame CA) or isotype-matched IgG-FITC and IgG-PE control antibodies (BD Biosciences). Cell products with the following phenotype were considered adequate for release: CD45/CD14/CD34 <10%, CD73/CD105/CD90 >70%. Cells were also differentiated into osteogenic and adipogenic lineages as previously described (Capelli C. et al: Bone Marrow Transplant 2007; 40:785-91; Capelli C et al, Cytotherapy 2009, 11:403-13).
Sterility tests were performed according to the European Pharmacopeia (Ph.Eur. 2.6.27, Microbiological Control of Cellular Products), using an automated detection system (BactAlert/3D, Biomerieux, Marcy l'Etoile, France) and 14 day incubation. Endotoxin measurement was performed using the Limulus Amebocyte Lysate (PBI S.p.A. Milano, Italy) according to the European Pharmacopeia (Ph.Eur. 2.6.14, Bacterial Endotoxins). Possible Mycoplasma contamination was also detected using the validated culture method and fluorescence method with the Vero indicator fibroblast cell line, and with positive controls, according to the European Pharmacopeia (Ph. Eur. 2.6.7, Mycoplasmas). All these microbiological tests were negative.

MSC cytogenetic analysis
For cytogenetic analysis, 1000 cells/cm2 of the product prior to final freezing were seeded in T25 flasks (BD Falcon, Milan, Italy). Metaphases of cells were Q-banded and karyotyped according to standard procedures (Caspersson T et al., Chromosoma 1970; 30:215-27). Karyogram was performed using the Meta System Program (Zeiss, Oberkochen, Germany). Final MSC preparation showed normal karyotype.
Clonogenic assay
Final hMSC were plated at a density of 1.000 cells/35mm Petri-dish (BD Falcon, Milan, Italy) in Methylcellulose media HSC-CFU basic (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). After a 14 days incubation, colony-forming unit cells (CFU-C) were scored under an inverted microscope. Positive control consisted of the Ewing’s sarcoma PDE-02 cell line. No CFU-C was detected in 4/4 cases while the PDE-02 Ewing’s sarcoma cell line gave rise to clearly detectable colonies in the same conditions (20% clonogenic cells, data not shown). 

In vivo tumorigenesis 
To test for tumor formation potential of ex-vivo propagated hMSC, 4 female NOD/SCID mice were injected with clinical-scale expanded hMSC at P2. Three mice were infused with 106 hMSC intravenously and one mouse was injected subcutaneously with the same cell dose. Two mice were both infused intravenously and injected subcutaneously with 106  human fibrosarcoma PDE-02 cells line as positive control. At first signs of illness, the mice were sacrificed and underwent histological analysis. The design of the study was approved by the Local Ethical Committee.
None of the animals injected with hMSC developed a visible or microscopically detectable tumor over a 90-day observation period.

MSC safety issue
Somatic cell therapy with MSC offers wide perspectives due to their in vivo immunosuppressive activity but need strict Good Manufacturing Practice (GMP) compliance and respect of all the required authorisations due to the experimental nature of these studies, particularly in non oncologic and young patients. We have adhered to scientifically defined criteria for MSC identification as recently stated by the International Society of Cell Therapy (ISCT) (Dominici M. et al., Cytotherapy 2006, 8: 315-317). Safety has been judged on the basis of negativity for all the tested contaminants and lack of genetic lesions at cytogenetic analysis. In our laboratory, normal karyotype was found in 20 metaphases on 40 consecutive separated batches of MSC. So far, in several studies, in vivo transforming events of MSC have never been observed in any studied patient (more than 500) (Lucchini et al., Biol Blood Marrow Transplant 2010: in press; Ball et al., Blood 2007: 110:2764-67; Le Blanc et al., Lancet 2008; 371:1579-86; Le Blanc et al., Leukemia 2007; 21: 1733-38; Muller et al., Blood Cells Mol Dis 2008; 40:25-32; Ning et al., Leukemia 2008: 22:593-99; Ringden et al., Transplantation 2006; 81:1390-97; Ringden et al., Leukemia 2007; 21:2271-76; von Bonin et al.; Bone Marrow Transplant  2009, 43:245-51; and others) or in experimental animals (Bernardo et al., Cancer Res 2007; 67:9142-49; Miura et al., Stem Cells 2006; 24:1095-03; Schallmoser et al., Transfusion 2007; 47:1436-46; Sessarego et al., Haematologica 2008; 93:339-46.) with the exception of studies on exceedingly long term cultured cells (more than 4 months) (Rubio et al., Cancer Res 2005; 65:3035-39) and in a study in NOD mice receiving autologous MSC (Fiorina P, et al, J Immunol 2009; 183:993-1004).

Histology and immunohistochemistry
Paraffin-embedded sections of kidney tissue stained with Massons’ trichrome, hematoxylin and eosin and periodic acid-Schiff were evaluated by an independent pathologist. For immunofluorescence of graft infiltrating CD4, CD8, CD14 cells and granulocytes, snap-frozen renal tissue was cut at 3 mm thickness, air-dried, fixed with acetone and incubated with the following antibodies: FITC-conjugated mouse anti-human CD4 (5 mg/ml, cloneSK3; BD Pharmingen), mouse anti-human CD8 (2 g/ml, clone C8/144B, Dako) followed by donkey anti mouse Cy3-conjugated secondary antibody (8.7 g/ml Jackson Immunoresearch); FITC-conjugated mouse anti-human CD14 (10 mg/ml, clone M5E2; BD Pharmingen), mouse anti-human granulocytes (1:100, clone MOM/3F12/F2, Thermo scientific), followed by donkey anti mouse Cy3-conjugated secondary antibody (8.7 g/ml Jackson Immunoresearch). Immunofluorescence staining was analyzed by an inverted confocal laser scanning microscope (LS 510 Meta, Zeiss). Graft infiltrating B cells and macrophages were analyzed by immunoperoxidase technique on Dubosq-Brazil fixed, paraffin embedded renal sections by using anti-human CD20 (15 mg/ml, clone L26; Dako), and mouse anti-human CD68 (1.9 g/ml, clone PG-M1, Dako) antibodies, respectively. Renal sections (2 mm) were de-paraffinized, hydrated and incubated for 30 minutes with 0.3% H2O2 in methanol to quench endogenous peroxidase. Tissues were treated with proteinase-K (20mg/ml, Sigma-Aldrich, Milan, Italy) for 10 minutes at 37°C. Antigen retrieval was performed by boiling sections using microwave (twice for 5 min in citrate buffer 10 mM, pH 6 at operating frequency of 2450 MHz and 600W power output). After incubation with a specie-specific biotinylated secondary horse anti-mouse antibody (15 mg/ml, Vector Laboratories) and diaminobenzidine (DAB, Merck, Darmstadt, Germany) substrate solution, slides were counterstained with hematoxylin, dehydrated in graded alcohols, mounted with cover slips and observed by light microscopy (Olympus, BH2-RFCA, Melville, NY, USA). Complement deposition was evaluated on frozen kidney sections by using the specific FITC-conjugated rabbit anti-human C3c antibody.
MSC were localized by double staining with FITC-conjugated mouse anti-human CD44 (10 mg/ml, clone G44-26, BD) and anti-human CD105 (15 g/ml, clone 266, BD Pharmingen). The double immunofluorescence staining was analyzed by an inverted confocal laser scanning microscope (LS 510 Meta; Zeiss). 

Figure 1. Ultrasound image of native kidneys from patient 2 taken before kidney transplantation showing small cortical cysts bilaterally.