SUPPLEMENTARY INFORMATION

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1 SUPPLEMENTARY INFORMATION Pleiotrophin Regulates the Expansion and Regeneration of Hematopoietic Stem Cells Heather A Himburg 1, Garrett G Muramoto 1 *, Pamela Daher 1*, Sarah K Meadows 1, J. Lauren Russell 1, Phuong Doan 1, Jen-Tsan Chi 1,2, Alice B Salter 1, William E. Lento 3, Tannishtha Reya 3, Nelson Chao 1,4, John P Chute 1,3 1 Division of Cellular Therapy, Department of Medicine, Duke University, Durham, NC; 2 Department of Molecular Genetics and Microbiology, Duke University, Durham, NC; 3 Department of Pharmacology and Cancer Biology, Duke University, Durham, NC; 4 Department of Immunology, Duke University, Durham, NC *GGM and PD contributed equally to this manuscript Supplementary Figures 1 5 Supplementary Tables 1, 2

2 Supplementary Fig. 1

3 Supplementary Fig. 2 a

4 b HUBEC CD45.1 CD CD45.2 B220 Mac-1/Gr-1 Thy1.2 HUBEC + anti-ptn CD45.2 B220 Mac-1/Gr-1 Thy1.2

5 c * * ^ ^ ^

6 Supplementary Fig. 3 * **

7 Supplementary Fig. 4

8 Supplementary Fig. 5 a * ^ **

9 Supplementary Fig. 5 b *

10 Supplementary Table 1. Genes overexpressed by HUBECs Fold Change Symbol Name SCG2 secretogranin II (chromogranin C) IGFBP1 insulin-like growth factor binding protein APOE apolipoprotein E PTN pleiotrophin CX3CL1 chemokine (C-X3-C motif) ligand 1 or fractalkine OLFML2A olfactomedin-like 2A TNFRSF11B tumor necrosis factor receptor superfamily, member 11b (osteoprotegerin) HAPO hemangiopoietin LGALS3BP lectin, galactoside-binding, soluble, 3 binding protein 12.1 CXCL12 stromal cell-derived factor IGFBP2 insulin-like growth factor binding protein 2, 36kDa IGFBP3 insulin-like growth factor binding protein SEMA3B sema domain, immunoglobulin domain, secreted, (semaphorin) 3B

11 Supplementary Table 2. CRU frequencies in BM 34 - KSL cells and their progeny BM source Cell Dose No. CRU 95% Confidence Engrafted Estimate Interval Day of 9 1 in 39 1/21 1/ KSL 30 6 of of 7 TSF 10 2 of 9 1 in 58 1/31 1/ of of 9 TSF + PTN 10 6 of 8 1 in 10 1/5 1/ of of 9 BM 34 - KSL cells (CD ) or their progeny following 7 day culture were transplanted at limiting dilutions into lethally irradiated C57Bl6 (CD ) mice along with 1 x 10 5 host BM MNCs in a competitive repopulating assay. At 12 weeks post-transplant, PB was collected from all recipient mice and flow cytometric analysis was performed to measure CD donor-derived cell repopulation in the recipient mice. Positive engraftment was defined as > 1% CD cells in the recipient mice. Poisson statistical analysis using the maximum likelihood estimator was performed to estimate the CRU frequency in each group 20,39.

12 Supplementary Figure Legends Supplementary Figure 1 Transplantation of PTN-treated BM HSCs facilitates the engraftment of neutrophils and platelets in lethally irradiated mice. 100% of mice (13 of 13) transplanted with BM 34 - KSL cells (black line) died by day +18. A fraction of mice transplanted with the progeny of TSF cultures (blue line) or TSF + PTN cultures (red line) survived through day +25 (4/13 in each group). Mice transplanted with the progeny of TSF + PTN culture demonstrated increased neutrophil counts at day +21 and +25 and increased platelet count at day +21 (n=13 mice per group, means + SEM). Supplementary Figure 2 PTN signaling is necessary for HUBEC-mediated HSC expansion. (a) Scatter plots show the percentages of total CD donor cells and donor-derived B-lymphoid, myeloid, and T cell populations in the PB in all mice transplanted with 30 BM 34 - KSL cells or their progeny following 7 day culture with HUBECs + TSF + IgG or HUBECs + TSF + anti-ptn. Mice transplanted with the progeny of TSF + HUBECs + IgG cultures demonstrated significantly higher total CD cell engraftment (P=0.03) and engraftment of B- lymphoid (B-220 +, P=0.004) and myeloid cells (Mac-1/Gr-1 +, P=0.01) compared to mice transplanted with the same dose of day 0 BM 34 - KSL cells (mean + SEM, n=7-10/group). Conversely, mice transplanted with the progeny of BM 34 - KSL cells cultured with TSF + HUBECs + anti-ptn demonstrated significant reduction in total CD cell, B-lymphoid, myeloid, and T cell (Thy ) engraftment compared to mice transplanted with the progeny of TSF + HUBECs + IgG (mean

13 + SEM, n=7-10/group, P=0.004, P=0.0001, P=0.002, P=0.001, respectively; one tailed t test). (b) Representative flow cytometric analysis is shown of PB donorderived (CD ) multilineage engraftment at 12 weeks post-transplant in a mouse transplanted with the progeny of TSF + HUBECs + IgG cultures versus a mouse transplanted with the progeny of TSF + HUBECs + anti-ptn. Percentages of total are shown in each quadrant. (c) Inhibition of PTN signaling prevents HUBEC-mediated expansion of LT-HSCs. Donor CD cell engraftment was persistently higher in mice transplanted with the progeny of BM 34 - KSL cells following HUBECs + TSF culture (striped bars) as compared to mice transplanted with the same dose of day KSL cells (black bars), with significant differences at weeks 8 and 12 (mean + SEM, n=7-10/group, *P=0.004 and *P=0.03, respectively). Mice transplanted with the progeny of 34 - KSL cells cultured with HUBECs + TSF + anti-ptn (gray bars) demonstrated significantly decreased CD cell engraftment at 8, 12 and 24 weeks post-transplant compared to mice transplanted with the progeny of 34 - KSL cells cultured with HUBECs + TSF (mean + SEM, n=7-10/group, ^P=0.0001, ^P=0.002, and ^P=0.002 for weeks 8, 12, and 24 respectively). Supplementary Figure 3 PTN does not signal through β-catenin. BM 34 - KSL cells ( cells/well) from flox-β-catenin mice (gray bars) and β-catenin -/- (LoxP,LoxP;Vav-cre) mice (black bars) were plated in culture with TSF alone or TSF ng/ml PTN x 7 days. No differences were observed in the

14 amplification of %KSL cells in culture between the flox-β-catenin group and the β-catenin -/- group (means + SD, n=3, *P=0.04 and **P=0.04). Supplementary Figure 4 Systemic administration of PTN augments the regeneration of BM LT-HSCs in irradiated mice. (Top) PB CD donor cell engraftment is shown at 12 weeks in CD mice (dots) transplanted with BM cells from mice that were irradiated with 700 cgy and then treated with saline or PTN x 7 days. (Bottom) Mice transplanted with BM cells from PTN treated mice displayed increased B220 (B cell), Mac-1 (myeloid) and Ter119 (erythroid) lineage engraftment compared to mice transplanted with saline-treated BM cells. Horizontal bars represent mean levels of engraftment in each condition (n=9/group). Supplementary Figure 5 Systemic administration of PTN increases phenotypic BM stem/progenitor cell content in non-irradiated mice. (a) Adult C57Bl6 mice were irradiated with 300 cgy TBI and then treated with saline, 2 µg GCSF or 2µg PTN IP x 7 days. At day +7, total BM cells, BM KSL cells and BM CFCs were measured in each group. * P=0.01 versus saline group, ^ P=0.01 versus saline group, **P=0.02 and P=0.01 versus saline and GCSF groups (n=5 mice/group, means + SD). (b) Non-irradiated adult C57Bl6 mice were treated IP daily x 7 days with saline or 2 μg PTN. At day +7, total BM cells, KSL cells, CFCs and LTC-ICs were measured in each group. PTN-treated mice demonstrated significantly increased BM KSL cells compared to saline-treated mice, but no

15 difference in total BM cells, CFCs or LTC-ICs. * P=0.009 versus saline group (n=5 mice/group, means + SD). Supplementary Methods HSC side population analysis and replating assay Hoechst staining of the cultured progeny of BM 34 - KSL cells was performed following a previously described protocol 21. Cells were analyzed on a BD FACSVantage using UV laser excitation at 350 nm and Hoechst Red and Blue emission was measured to identify side population cells on the basis of dye efflux of Hoechst 33342, as previously described 24. Greater than 100,000 events were analyzed per sample to maximize ability to detect side population cells. For the 14 day re-plating experiment, BM 34 - KSL cells were plated in a 96 well U-bottom plate at 1000 cells/well in either TSF or TSF ng/ml PTN. Cells were collected at day 7, pelleted, resuspended in PBS with 10%FBS and 1% pen/strep, and sorted for collection of 34 - KSL cells KSL cells were then replated into a 96 well plate with TSF alone or TSF + PTN culture media. Homing analysis BM Sca-1 + lin - cells from CD B6.SJL mice were injected via tail vein infusion at a dose of 4 x 10 4 cells into lethally irradiated CD C57BL6 recipients or cultured for 7 days in TSF or TSF with 100 ng/ml PTN. The cultured progeny of the identical dose of Sca-1 + lin - cells were also injected into lethally irradiated CD recipients. BM cells from recipient mice were collected at 24 hours post

16 cell infusion. Homing capacity was determined by the percentage of donor CD cells present in the recipient BM at 24 hours post-infusion. Neutrophil and platelet engraftment assay Lethally irradiated C57Bl6 mice were transplanted with either a limiting dose (100 cells) of BM 34 - KSL cells from C57Bl6 donor mice or the progeny of the identical dose of cells cultured for 7 days with TSF or TSF ng/ml PTN. PB complete blood counts (CBCs) were measured prior to irradiation and twice weekly starting at day +8 post-irradiation. Blood samples were analyzed on a HEMAVET 950FS hematology analyzer. Quantitative RT-PCR and Direct ELISA RT-PCR analyses of PTN in ECs and HES-1 and PTEN in BM KSL cells and FACS-sorted KSL cells following culture were performed using a 2-step RT-PCR reaction as previously described 39. Conditioned medium was generated from HUBECs and non-brain ECs as previously described 11,20 and ELISA for PTN was performed following manufacturer s guidelines. Analysis of PI3-kinase and Notch signaling For analysis of RPTPβ/ζ expression on hematopoietic cells, cytospins of BM MNCs were generated (~10,000 cells/slide). Rat anti-rptpβ/ζ (BD) or rat IgG was added and a FITC anti-rat secondary antibody was utilized. Flow cytometric analysis was performed on BM KSL cells to confirm RPTPβ/ζ expression. PI3-

17 kinase signaling in BM 34 - KSL cells was inhibited using 10 µm of the PI3-kinase inhibitor, LY (Cell Signaling Technology, Inc). Notch signaling was antagonized using 30 µm of γ-secretase inhibitor II (Calbiochem). The inhibitors were added to cultures of 500 BM 34 - KSL cells supplemented with TSF or TSF + PTN. Transgenic β-catenin -/- (loxp,loxp;vav-cre) mice were a gift from T. Reya, Duke University. Immunofluorescence analysis for the activated β-catenin was performed using cytospins of BM KSL cells or their progeny and staining with antibody against non-phosphorylated β-catenin (Clone 8E7, Upstate Biotechnology, Lake Placid, NY) or isotype control, and goat anti-mouse alexafluor 488 (BD) 34. Administration of PTN to non-irradiated mice and following 300 cgy Non-irradiated, adult C57Bl6 mice were treated with saline or 2 µg PTN IP daily x 7 days and effects on total BM cells, BM KSL cells, BM CFCs and LTC-ICs were measured at day +7. Adult C57Bl6 mice were also irradiated with 300 cgy TBI and treated identically with saline or PTN x 7 days and BM cells, BM KSL cells and BM CFCs were measured in each group at day +7.