Expression levels of b-pix mrna in ALS-MSCs and C-MSCs.

Transcription

1 Supplementary Data SUPPLEMENTARY FIG. S1. The neurological functioning of ischemic rats depends on the origin of the transplanted MSCs. The scores of healthy rats in tests on forelimb placing, forelimb use asymmetry, and corner turn were 1.0, 0.0, and 0.5, respectively. No significant differences were observed between the 5 groups prior to implantation. However, the scores for forelimb use asymmetry and corner turn tests were significantly better in the C-MSC, huc-msc, and UCB-MSC groups (n = 10 in each group) than in the ALS-MSC group (n = 10) 21 days after implantation, and the scores on the forelimb placing test were significantly higher in the C-MSC, huc-msc, and UCB-MSC groups (n = 10 in each group) than in the ALS-MSC group (n = 10) 7 days after implantation. Data are means SD of 5 or more independent experiments. *P value < 0.05 compared with the PBS group. MSCs, mesenchymal stromal cells; ALS, amyotrophic lateral sclerosis; huc, human umbilical cord; UCB, umbilical cord blood; C-MSCs, control healthy MSCs; MCAO, MCA occlusion.

2 SUPPLEMENTARY FIG. S2. Migratory abilities of ALS- MSCs and C-MSCs. In vitro cell migration assays show that the migratory capacity of ALS-MSCs (n = 7) is substantially lower than that of C-MSCs (n = 5). SUPPLEMENTARY FIG. S3. Expression levels of b-pix mrna in ALS-MSCs and C-MSCs.

3 SUPPLEMENTARY FIG. S4. Confirmation of the role of b-pix in neurological improvement by MSC implantation. To assess functional improvement by b-pix in vivo, post-msc-transplant stroke rats underwent neurological examination daily. Rats (n = 10) that received b-pix knockdown C-MSCs did not demonstrate any significant functional recovery, whereas rats (n = 10) that received control C-MSCs exhibited improvement (A). Rats (n = 10 in each group) that received b-pix overexpressing or control ALS-MSCs did not show any improvement of neurological function comparable (B). (*P < 0.05 compared with the group implanted with b-pix depleted C-MSCs).

4 SUPPLEMENTARY FIG. S5. Comparison of the protein levels of several trophic factors in ALS-MSCs and C-MSCs. SUPPLEMENTARY FIG. S6. mrna levels of several trophic factors in ALS-MSCs and C-MSCs.

5 SUPPLEMENTARY FIG. S8. Levels of VEGF, IGF, and GDNF in ALS-MSCs and C-MSCs in infarcted hemispheres 3 days after direct injection of MSCs into the infarcted areas. Six additional rats were killed 3 days after the injection of ALS-MSCs (n = 3) or C-MSCs (n = 3), and the protein contents of homogenates taken from the hemispheres with ischemic infarcts were quantified with a BCA protein kit. Using commercially available kits for VEGF (Human VEGF Quantikine ELISA Kit; R&D Systems), IGF (Human IGF Quantikine ELISA Kit; R&D Systems), and GDNF (GDNF human ELISA Kit; Abcam), levels of VEGF, IGF, and GDNF were measured. All samples were tested in triplicate. The levels of VEGF, IGF, and GDNF, which were significantly higher in C-MSCs than in ALS-MSCs under in vitro condition, were also higher in the hemispheres injected with C-MSCs than with ALS-MSCs. [*P < 0.05; Wilcoxon Scores (rank sums)]. IGF, insulin-like growth factor. Reference: Pritchett J, C Wright, L Zeef and B Nadarajah. (2007). Stromal derived factor-1 exerts differential regulation on distinct cortical cell populations in vitro. BMC Dev Biol 7:31. SUPPLEMENTARY FIG. S7. Effect of b-pix overexpression on neurotrophic factor secretion by MSCs. A total of ALS-MSCs or C-MSCs were plated in 96-well plates. After incubation for 24 h, culture supernatants were divided into triplicate 200 ml samples, and stromal cell derived factor-1a (SDF-1a) (A), vascular endothelial growth factor (VEGF) (B), and brain-derived neurotrophic factor (BDNF) (C) levels were measured in the supernatants, with the appropriate ELISA kits (R&D Systems) [12]. [*P < 0.05 when compared with the C-MSCs, Wilcoxon Scores (rank sums)]. SDF-1a, stromal cell derived factor-1a. SUPPLEMENTARY FIG. S9. Concentrations of SDF-1a in normal areas and periinfarct areas 2 weeks after infarct. Three additional rats were killed 2 weeks after the induction of infarcts, and the protein contents of homogenates of the periinfarct areas and the corresponding areas on the contralateral hemispheres were measured with a BCA protein kit (Pierce). Using a commercially available kit for rat SDF-1a (Quantikine Human SDF-1a; R&D Systems) [33], we measured the concentrations of SDF-1a. All samples were tested in triplicate. SDF-1a was significantly higher in the periinfarct areas than in the normal areas. [*P < 0.05, Wilcoxon Scores (rank sums)].

6 Supplementary Table S1. Median Values of Surface Antigen Expression in Various Mesenchymal Stromal Cells Patient no. CD29 + CD44 + CD73 + CD105 + CD34 + CD45 + HLA-DR + ALS-MSCs (Sporadic) A A A A A A ALS-MSCs (Familial) A C-MSCs C C C C C UCB-MSCs U U U U huc-mscs hu hu hu hu Value; %. Cells were labeled with the following antihuman antibodies: CD45-phycoerythrin (PE), CD44-fluorescein isothiocyanate (FITC) (DakoCytomation), CD73-PE (BD Pharmingen), CD34-PE, CD29-FITC, CD49C-PE, CD54-FITC, CD105-FITC, CD106-FITC, HLA-DR-FITC, and PE- and FITC-conjugated isotype controls (Serotec). Labeled cells were analyzed by flow cytometry (Calibur) [8]. MSCs, mesenchymal stromal cells; ALS, amyotrophic lateral sclerosis; huc, human umbilical cord; UCB, umbilical cord blood; C-MSCs, control healthy MSCs. Supplementary Table S2. Migration-Associated Genes No. Unigene GeneBank Symbol Description 1 Hs NM_ APC Adenomatous polyposis coli 2 Hs NM_ BRMS1 Breast cancer metastasis suppressor 1 3 Hs NM_ CCL7 Chemokine (C-C motif) ligand 7 4 Hs NM_ CD44 CD44 molecule (Indian blood group) 5 Hs NM_ CDH1 Cadherin 1, type 1, E-cadherin (epithelial) 6 Hs NM_ CDH11 Cadherin 11, type 2, OB-cadherin (osteoblast) 7 Hs NM_ CDH6 Cadherin 6, type 2, K-cadherin (fetal kidney) 8 Hs NM_ CDKN2A Cyclin-dependent kinase inhibitor 2A (melanoma, p16, inhibits CDK4) 9 Hs NM_ CHD4 Chromodomain helicase DNA binding protein 4 10 Hs NM_ COL4A2 Collagen, type IV, alpha 2 11 Hs NM_ CST7 Cystatin F (leukocystatin) 12 Hs NM_ CTBP1 C-terminal binding protein 1 13 Hs NM_ CTNNA1 Catenin (cadherin-associated protein), alpha 1, 102 kda 14 Hs NM_ CTSK Cathepsin K 15 Hs NM_ CTSL1 Cathepsin L1 16 Hs NM_ CXCL12 Chemokine (C-X-C motif) ligand 12 (stromal cell derived factor 1) 17 Hs NM_ CXCR4 Chemokine (C-X-C motif) receptor 4 18 Hs NM_ DENR Density-regulated protein 19 Hs NM_ EPHB2 EPH receptor B2 20 Hs NM_ ETV4 Ets variant 4 21 Hs NM_ EWSR1 Ewing sarcoma breakpoint region 1 22 Hs NM_ FAT1 FAT tumor suppressor homolog 1 (Drosophila) 23 Hs NM_ FGFR4 Fibroblast growth factor receptor 4 24 Hs NM_ FLT4 Fms-related tyrosine kinase 4 25 Hs NM_ FN1 Fibronectin 1 26 Hs NM_ FXYD5 FXYD domain containing ion transport regulator 5 27 Hs NM_ GNRH1 Gonadotropin-releasing hormone 1 (luteinizing-releasing hormone) 28 Hs NM_ KISS1R KISS1 receptor 29 Hs NM_ HGF Hepatocyte growth factor (hepapoietin A; scatter factor) (continued)

7 Supplementary Table S2. (Continued) No. Unigene GeneBank Symbol Description 30 Hs NM_ HPSE Heparanase 31 Hs NM_ HRAS V-Ha-ras Harvey rat sarcoma viral oncogene homolog 32 Hs NM_ HTATIP2 HIV-1 Tat interactive protein 2, 30 kda 33 Hs NM_ IGF1 Insulin-like growth factor 1 (somatomedin C) 34 Hs NM_ IL18 Interleukin 18 (interferon-gamma-inducing factor) 35 Hs NM_ IL1B Interleukin 1, beta 36 Hs.846 NM_ IL8RB Interleukin 8 receptor, beta 37 Hs NM_ ITGA7 Integrin, alpha 7 38 Hs NM_ ITGB3 Integrin, beta 3 (platelet glycoprotein IIIa, antigen CD61) 39 Hs NM_ CD82 CD82 molecule 40 Hs NM_ KISS1 KiSS-1 metastasis-suppressor 41 Hs NM_ KRAS V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog 42 Hs NM_ RPSA Ribosomal protein SA 43 Hs NM_ MCAM Melanoma cell adhesion molecule 44 Hs NM_ MDM2 Mdm2 p53 binding protein homolog (mouse) 45 Hs NM_ MET Met protooncogene (hepatocyte growth factor receptor) 46 Hs NM_ METAP2 Methionyl aminopeptidase 2 47 Hs NM_ MGAT5 Mannosyl (alpha-1,6-)-glycoprotein beta-1,6-n-acetylglucosaminyltransferase 48 Hs.2258 NM_ MMP10 Matrix metallopeptidase 10 (stromelysin 2) 49 Hs NM_ MMP11 Matrix metallopeptidase 11 (stromelysin 3) 50 Hs.2936 NM_ MMP13 Matrix metallopeptidase 13 (collagenase 3) 51 Hs NM_ MMP2 Matrix metallopeptidase 2 (gelatinase A, 72 kda gelatinase, 72 kda type IV collagenase) 52 Hs NM_ MMP3 Matrix metallopeptidase 3 (stromelysin 1, progelatinase) 53 Hs.2256 NM_ MMP7 Matrix metallopeptidase 7 (matrilysin, uterine) 54 Hs NM_ MMP9 Matrix metallopeptidase 9 (gelatinase B, 92 kda gelatinase, 92 kda type IV collagenase) 55 Hs NM_ MTA1 Metastasis associated 1 56 Hs NM_ MTSS1 Metastasis suppressor 1 57 Hs NM_ MYC V-myc myelocytomatosis viral oncogene homolog (avian) 58 Hs NM_ MYCL1 V-myc myelocytomatosis viral oncogene homolog 1, lung carcinoma derived (avian) 59 Hs NM_ NF2 Neurofibromin 2 (merlin) 60 Hs NM_ NME1 Nonmetastatic cells 1, protein (NM23A) expressed in 61 Hs NM_ NME2 Nonmetastatic cells 2, protein (NM23B) expressed in 62 Hs.9235 NM_ NME4 Nonmetastatic cells 4, protein expressed in 63 Hs NM_ NR4A3 Nuclear receptor subfamily 4, group A, member 3 64 Hs NM_ PLAUR Plasminogen activator, urokinase receptor 65 Hs NM_ PNN Pinin, desmosome associated protein 66 Hs NM_ PTEN Phosphatase and tensin homolog 67 Hs NM_ RB1 Retinoblastoma 1 68 Hs NM_ RORB RAR-related orphan receptor B 69 Hs NM_ SET SET nuclear oncogene 70 Hs NM_ SMAD2 SMAD family member 2 71 Hs NM_ SMAD4 SMAD family member 4 72 Hs NM_ SRC V-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog (avian) 73 Hs NM_ SSTR2 Somatostatin receptor 2 74 Hs NM_ SYK Spleen tyrosine kinase 75 Hs NM_ TCF20 Transcription factor 20 (AR1) 76 Hs NM_ TGFB1 Transforming growth factor, beta 1 77 Hs NM_ TIMP2 TIMP metallopeptidase inhibitor 2 78 Hs NM_ TIMP3 TIMP metallopeptidase inhibitor 3 79 Hs NM_ TIMP4 TIMP metallopeptidase inhibitor 4 80 Hs NM_ TNFSF10 Tumor necrosis factor (ligand) superfamily, member Hs NM_ TP53 Tumor protein p53 82 Hs NM_ TRPM1 Transient receptor potential cation channel, subfamily M, member 1 83 Hs NM_ TSHR Thyroid stimulating hormone receptor 84 Hs NM_ VEGFA Vascular endothelial growth factor A 85 Hs NM_ B2M Beta-2-microglobulin 86 Hs NM_ HPRT1 Hypoxanthine phosphoribosyltransferase 1 87 Hs NM_ RPL13A Ribosomal protein L13a (continued)

8 Supplementary Table S2. (Continued) No. Unigene GeneBank Symbol Description 88 Hs NM_ GAPDH Glyceraldehyde-3-phosphate dehydrogenase 89 b-pix 90 Hs NM_ ACTB Actin, beta 91 N/A SA_00105 HGDC Human Genomic DNA Contamination 92 N/A SA_00104 RTC Reverse transcription control 93 N/A SA_00104 RTC Reverse transcription control 94 N/A SA_00104 RTC Reverse transcription control 95 N/A SA_00103 PPC Positive PCR control 96 N/A SA_00103 PPC Positive PCR control 97 N/A SA_00103 PPC Positive PCR control PCR, polymerase chain reaction. Supplementary Table S3. Body Weights of Groups of Sprague-Dawley Rats ALS-MSCs (n = 20) C-MSCs (n = 20) UCB-MSCs (n = 20) huc-mscs (n = 20) PBS (n = 10) Age (weeks) Body weight (g) Supplementary Table S4. Physiological Variables Before (Preischemia), During (Ischemia), and 30 min After Occlusion of the Middle Cerebral Arteries (Reperfusion) Group MABP (mmhg) ph PaCO 2 (mmhg) PaO 2 (mmhg) Haematocrit (%) CBF (% of preischemia of saline) Rectal temperature ( C) ALS-MSCs (n = 20) Preischemia Ischemia Reperfusion C-MSCs (n = 20) Preischemia Ischemia Reperfusion UCB-MSCs (n = 20) Preischemia Ischemia Reperfusion huc-mscs (n = 20) Preischemia Ischemia Reperfusion PBS (n = 10) Preischemia Ischemia Reperfusion Values are means SEM. MABP, mean arterial blood pressure; CBF, cerebral blood flow.

9 Supplementary Table S5. Surface Marker Expression on ALS-MSCs and C-MSCs After Genetic Modification Vehicle C-MSCs Vehicle ALS-MSCs b-pix knockdown C-MSCs b-pix overexpressed ALS-MSCs CD CD CD CD CD CD CD49C CD CD HLA-DR Cells were labeled with the following antihuman antibodies: CD45-PE, CD44-FITC (DakoCytomation), CD73-PE (BD Pharmingen), CD34- PE, CD29-FITC, CD49C-PE, CD54-FITC, CD105-FITC, CD106-FITC, HLA-DR-FITC, and PE- and FITC-conjugated isotype controls (Serotec). Labeled cells were analyzed by flow cytometry (Calibur) [8]. ALS-MSCs and C-MSCs have the same CD45 - CD34 - CD29 + CD73 + CD105 + CD44 + HLA-DR - phenotype. This phenotype was not altered by b-pix knockdown in C-MSCs or b-pix overexpression in ALS-MSCs.