Cell Stem Cell, Volume 8 Supplemental Information Prostaglandin E2 Enhances Human Cord Blood Stem Cell Xenotransplants and Shows Long-Term Safety in Preclinical Nonhuman Primate Transplant Models Wolfram Goessling, Robyn S. Allen, Xiao Guan, Ping Jin, Naoya Uchida, Michael Dovey, James M. Harris, Mark E. Metzger, Aylin C. Bonifacino, David Stroncek, Joseph Stegner, Myriam Armant, Thorsten Schlaeger, John F. Tisdale, Leonard I. Zon, Robert E. Donahue, and Trista E. North Inventory of Supplemental Information Figure S1, related to Figure 1 Representative FACS plots showing gating for apoptosis (A) and proliferation (B) analysis depicted graphically in Figure 1E, F. Figure S2, related to Figure 2 Results of preliminary short-term hematopoietic transplantation experiments to determine incubation period (A) and media (B) to be utilized in hcb xenotransplantation assays shown in Figure 2C,D. Figure S3, related to Figure 2 Representative FACS plots showing gating for hcd45 content for chimerism analysis summarized graphically in Figure 2C,D. Figure S4, related to Figure 2 Representative FACS plots showing gating for multilineage contribution of control (A) and dmpge2 (B) BM samples with tabular summary of total average (+/- SD) lineage distribution (C) from mice depicted in Figure 2C,D with greater than 1% chimerism. Figure S5, related to Figure 2 Graphical summary of xenotransplantation experiments using CD34+ hcb following treatment with dmpge2, which correlates with the whole hcb transplants in Figure 2C,D. Figure S6, related to Figure 4 FACS plots illustrating the gating controls and the contribution of DMSO or dmpge2- treated GFP and YFP-transduced cells to total PB of each of the rhesus monkey undergoing autologous transplantation at the representative time point indicated, which correlates to the multilineage longitudinal distribution study shown in Figure 4A,B.
Figure S7, related to Figure 5 Heat map representation of microarray analysis of human and rhesus MPBSCs at fixed intervals post dmpge2 or control treatment related to gene list content depicted in Figure 5 and Supplemental Table 3. Table S1, related to Figure 2 Average and standard deviation of individual colony types following control or dmpge2 treatment at fixed intervals post exposure related to data shown graphically in Figure 2A,B. Table S2, related to Figure 4 EGFP and EYFP transduction efficiencies for each autologous MPBSC unit utilized in the rhesus dmpge2 transplantation protocol for which results are shown in Figure 4. Table S3, related to Figure 5 Commonly regulated gene lists corresponding to microarray data shown in the scatter plots and Venn diagram in Figure 5: Tab A- Human MPBSCs at 10 and 50uM PGE2 vs Monkey at 50uM dmpge2; Tab B- Human MPBSCs at 10 vs 50uM dmpge2; Tab C- Human MPBSCs at 10uM dmpge2 vs Monkey at 50uM dmpge2; Tab D- Human MPBSCs vs Monkey at 50uM dmpge2. Available online. Table S4, related to Figure 6 Genes indicative of commonly UP (Tab A) and DOWN (Tab B) regulated Ingenuity pathways found in Human MPBSCs at 10 and 50uM PGE2 vs Monkey at 50uM dmpge2 as depicted in the Venn diagram and highlighted in a tabular form in Figure 6. Available online. Table S5, related to Figure 6 Commonly UP (Tab A) and Down (Tab B) regulated Ingenuity pathways found in Human MPBSCs at 10 vs 50uM dmpge2 as depicted in the Venn diagram in Figure 6. Available online. Table S6, related to Figure 6 Commonly UP (Tab A) and Down (Tab B) regulated Ingenuity pathways found in Human MPBSCs at 10uM dmpge2 vs Monkey at 50uM dmpge2 as depicted in the Venn diagram in Figure 6. Available online. Table S7, related to Figure 6 Commonly UP (Tab A) and Down (Tab B) regulated Ingenuity pathways found in Human MPBSCs vs Monkey at 50uM dmpge2 as depicted in the Venn diagram in Figure 6. Available online. Supplemental Experimental Procedures 1, related to Quantitative PCR methods List of primers used in qpcr characterization of human and rhesus CD34+ HSCs.
Supplemental Experimental Procedures 2, related to Autologous Competitive Transplant Study methods Schematic representation of the non-human primate autologous transplant protocol.
Figure S1 (related to Figure 1) dmpge2 limits apoptosis and enhances proliferation of CD34+ hcb cells in vitro hcb cells are incubated with 1μM dmpge2 and analyzed at fixed intervals; representative examples are shown. A- AnnexinV, a marker of apoptosis, was diminished 6 hrs after dmpge2 incubation. B- EdU incorporation, marking cellular proliferation, was increased at 6 hrs following dmpge2 treatment.
Figure S2 (related to Figure 2) Alteration in dmpge2 exposure length and incubation media does not effect spleen colony formation A,B- ckit+sca1+lin- cells were isolated from murine bone marrow and transplanted into lethally irradiated C57Bl/6 mice. Results depicted as mean with standard deviation. 5000 cells transplanted, n=10/group; t-test, * p<0.001 versus control. A- An incubation time of 1 vs. 2hrs had no measurably distinct effect on the enhancement of spleen colony formation by dmpge2. B- Alteration in the incubation media: PBS, dextran/albumin or Dulbecco s minimal essential medium (DMEM), had no effect on the ability of dmpge2 to enhance spleen colony formation.
Figure S3 (related to Figure 2) Chimerism for hcd45 in the peripheral blood and bone marrow of NOD/SCID mice A,B- Sublethally irradiated NOD/SCID mice were transplanted with either control- or dmpge2-treated hcb. Chimerism for hcd45+ cells was determined by FACS analysis in PB (A) and BM (B). Representative FACS plots are shown, indicating the increase in hcd45+ cells in the mice that received dmpge2-treated hcb.
Figure S4 (related to Figure 2) Multilineage analysis of BM chimerism reveals a positive effect of dmpge2 on human stem and progenitor, myeloid and T-cell repopulation CD34+ hcb was transplanted into sublethally irradiated NOD/SCID mice. BM from
transplant recipients with >1% hcb chimerism was analyzed at 3 months post transplantation for multilineage distribution of human cells. A,B- Representative FACS plots of BM multilineage analysis of (A) control-treated and (B) PGE2-treated hcb recipients. C- Summary and statistical analysis (t-test) of average (+/-SD) human contribution to individual blood lineages across recipients of control-treated (n=5) and dmpge2-treated (n=15) hcb.
Figure S5 (related to Figure 2) dmpge2 enhances CD34+ hcb proliferation engraftment in vivo A,B- CD34+ hcb was transplanted into sublethally irradiated NOD/SCID mice. PB was analyzed at 1 and 3 months post transplantation, recipient BM at 3 months. A- Individual chimerism of mice receiving control CD34+ hcb (red squares) or dmpge2-treated hcb (blue circles). The mean is indicated by a solid horizontal line; a dashed line indicates the cut-off of 1% for positive engraftment. PB- 1 month: control 1.20851.2821, dmpge2 2.55051.8441; t-test, p=0.0055; 3 months: control 1.10781.0181, dmpge2 2.3051.194; t-test, p=0.0013; BM- control 0.63060.8486, dmpge2 1.92821.4809; t-test, p=0.0018. B- Cumulative summary depicting the percentage of all animals with hcb engraftment: PB- 1 month: control 9/20, dmpge2 26/20; Fisher s exact test (one-tailed) p=0.0484; 3 months: control 8/18, dmpge2 15/18; Fisher s exact test (one-tailed) p=0.013; BMcontrol 7/17, dmpge2 14/17; Fisher s exact test (one-tailed) p=0.032.
Figure S6 (related to Figure 4) Representative PB EGEP/EYFP FACS analysis of individual rhesus macaques at the most recent analysis post-reinfustion. A- Control FACS plots showing fluorescence intensity of unstimulated/untransduced rhesus PB or transduced (EGFP OR EYFP) rhesus cell lines. B- FACS analysis for individuals 1 and 2 of the pre-treatment protocol, at day 391 (M1)
and day 355 (M2) post-reinfusion respectively; DMSO or dmpge2 treatment is indicated. C- FACS analysis for individuals 3, 4 and 5 of the post-treatment protocol, at day 328 (M3), day 279 (M4) and day 193 (M5) post-reinfusion respectively; DMSO or dmpge2 treatment is indicated.
Figure S7 (related to Figure 5) Heat map representation of the human vs. non-human primate MPBSC microarray analysis, showing all treatment groups.
Table S1: Limiting-dilution H4434 methylcellulose hcb differentiation assay Exposure Treatment Cell Density Ery Mac G GM GEMM [min] Avg SD Avg SD Avg SD Avg SD Avg SD 12 DMSO 2000 13.3 1.2 14.0 1.7 16.7 4.0 2.3 1.5 6.0 2.6 dmpge2 2000 12.7 3.1 21.0 7.0 24.3 5.5 3.3 0.6 8.7 3.5 DMSO 800 4.3 0.6 5.7 0.6 6.3 1.5 1.3 0.6 2.7 0.6 dmpge2 800 3.0 1.0 10.7 1.5 9.7 2.1 1.3 0.6 2.3 0.6 DMSO 320 1.3 0.6 3.0 1.0 3.7 1.5 1.0 1.0 2.0 1.0 dmpge2 320 1.7 1.2 2.3 1.5 5.0 2.0 0.7 0.6 0.7 1.2 30 DMSO 2000 7.3 2.3 18.3 6.5 20.7 3.1 1.3 0.6 6.0 1.7 dmpge2 2000 7.7 1.5 22.0 3.0 29.7 7.6 5.3 1.5 8.7 3.8 DMSO 800 2.0 1.0 3.0 0.0 6.3 0.6 1.7 0.6 1.7 1.2 dmpge2 800 5.7 2.1 7.7 2.1 14.7 3.1 1.7 0.6 2.3 0.6 DMSO 320 1.3 1.5 1.7 0.6 3.7 2.1 0.3 0.6 1.0 1.0 dmpge2 320 1.3 1.2 4.0 2.0 7.7 3.1 1.7 1.2 2.3 0.6 60 DMSO 2000 10.7 2.9 29.0 2.6 36.3 4.5 5.0 1.7 9.3 2.1 dmpge2 2000 10.0 1.0 30.7 4.9 49.7 12.1 5.3 0.6 13.0 1.0 DMSO 800 4.3 2.1 13.3 4.5 15.3 2.5 1.7 0.6 4.0 1.7 dmpge2 800 4.3 1.5 19.0 3.6 22.3 4.2 2.7 0.6 4.7 1.5 DMSO 320 2.0 2.0 7.0 1.0 6.0 0.0 0.3 0.6 0.7 0.6 dmpge2 320 2.3 1.2 10.3 2.1 9.0 1.7 1.3 1.2 3.0 1.0 90 DMSO 2000 8.7 4.7 21.7 2.5 41.0 6.9 3.7 1.5 7.0 1.0 dmpge2 2000 12.0 3.5 29.0 2.6 45.7 3.5 4.7 1.5 9.3 1.2 DMSO 800 5.3 0.6 10.0 2.0 18.7 0.6 3.0 1.0 2.3 1.2 dmpge2 800 3.3 1.2 11.3 3.5 22.7 7.5 2.3 1.5 3.7 1.2 DMSO 320 1.3 0.6 4.7 2.5 4.0 4.0 1.3 0.6 1.3 0.6 dmpge2 320 0.7 1.2 6.0 3.0 8.0 1.0 2.3 0.6 1.7 1.5 180 DMSO 2000 15.0 4.0 25.3 1.5 38.3 8.5 5.0 2.0 11.3 1.2 dmpge2 2000 15.7 2.3 37.7 7.6 43.0 6.2 5.0 2.0 9.7 5.7 DMSO 800 5.0 2.0 13.7 1.2 12.3 4.9 1.7 0.6 3.0 0.0 dmpge2 800 5.7 3.5 18.0 3.6 18.3 4.7 1.3 0.6 2.7 2.9 DMSO 320 2.0 1.0 5.3 2.1 5.7 3.5 0.0 0.0 1.7 1.2 dmpge2 320 1.3 0.6 9.3 4.7 12.0 4.6 1.0 1.7 1.0 1.0 360 DMSO 2000 5.3 2.1 20.7 5.0 25.3 0.6 1.0 1.0 2.3 1.5 dmpge2 2000 8.3 1.2 30.7 4.5 32.0 6.1 3.3 0.6 7.7 4.2 DMSO 800 1.7 2.1 9.0 1.0 10.3 0.6 0.0 0.0 1.0 1.0 dmpge2 800 1.0 1.0 16.3 1.2 15.7 4.6 1.0 1.0 1.3 0.6 DMSO 320 0.7 1.2 5.0 2.6 6.7 4.5 0.3 0.6 1.0 1.0 dmpge2 320 0.7 1.2 5.0 2.6 6.0 5.6 0.3 0.6 0.7 0.6
Table S2: Transduction efficiency of Monkey CD34+ MPBSC Animal ID Vector Treatment Efficiency [%] Std. Dev. [%] Pre-Transduction Treatment M1 chiv-egfp dmpge2 50μM 50.7 0.8 chiv-eyfp XVIVO 10 45.8 2.5 M2 chiv-egfp XVIVO 10 37.5 0.5 chiv-eyfp dmpge2 50μM 35.1 1.1 Post-Transduction Treatment M3 chiv-egfp dmpge2 50μM 48.4 0.4 chiv-eyfp XVIVO 10 43.7 0.3 M4 chiv-egfp XVIVO 10 27.3 1.1 chiv-eyfp dmpge2 50μM 25.8 0.1 M5 chiv-egfp XVIVO 10 23.1 0.5 chiv-eyfp dmpge2 50μM 19.8 0.8
Supplemental Experimental Procedures 1: qpcr analysis for human and monkey genes was performed using the primer pairs listed. HUMAN Forward Primer Reverse Primer Actin GCACAGAGCCTCGCCTTT GGAATCCTTCTGACCCATGC EP1 GATGGTGGGCCAGCTTGTC GCCACCAACACCAGCATTG EP2 GTGCTGACAAGGCACTTCATGT TGTTCCTCCAAAGGCCAAGTAC EP3 AAGGCCACGGCATCTCAGT TGATCCCCATAAGCTGAATGG EP4 CTTGGAGGCAGGAATTTGCTT AAAGTCCTCAGTGAGGTGGTGTCT SCL ATGAGATGGAGATTACTGATG GCCCCGTTCACATTCTGCT Runx1A/B CGTGCACATACATTAGTAGCACTACCTTTG CTTCCACGAATCTTGCTTGCAGAGGTTAAG Runx1C GAAGTCTGAACCCAGCATAGTGGTCAGCAG GTGGACGTCTCTAGAAGGATTCATTCCAAG CCND1 GTGCTGCGAAGTGGAAACC ATCCAGGTGGCGACGATCT CCNE1 CCACACCTGACAAAGAAGATGATGAC GAGCCTCTGGATGGTGCAATAAT BCLXL AACTGGGTCTCCTGACTTGG AAGGACAGTGTTGGCAAAGG BAX GGCGGCAATCATCCTCTG TGCTTCAGGGTTTCATCCAG BCL2 GGTCATGTGTGTGGAGAGCG GGTGCCGGTTCAGGTACTCA CDNK1A GTCACTGTCTTGTACCCTTGTG CGGCGTTTGGAGTGGTAGAAA FAS AGCTTGGTCTAGAGTGAAAA GAGGCAGAATCATGAGATAT BAD CGAGTGAGCAGGAAGACTCCA AGGAGTCCACAAACTCGTCACT CDK1 CCATTTTGCCAGAAATTCGT TTTTCAGAGCTTTGGGCACT HDAC1 TAAATTCTTGCGCTCCATCC AACAGGCCATCGAATACTGG COX1 TGGCTGGGCGTGCTAGAGGTT CAGCCTGCGTGAGGTGTGTCACT COX2 GGAACACAACAGAGTATGCG AAGGGGATGCCAGTGATAGA PTGES GGTCTTGGGTTCCTGTATGGT GGGACTCAAACCTTGGGAGG CREM CTCTAGTGCAGTTACCTTCGGG ACCTGAACGGTGTGTATTTCTG CREB CCAGCAGAGTGGAGATGCAG GGGCTAATGTGGCAATCTGTG PDE4D CATGTCACCCAAGACCATGTC GAGAAGTTGCTACGGACGCT CXCR4 CCTATGCAAGGCAGTCCATGT GGTAGCGGTCCAGACTGATGA VEGF CCAATCGAGACCCTGGTG CACACAGGATGGCTTGAAGA IL8 ACCAGCAGTACGTGATCGG CTTCTCGCGGAAGCTGATGTT NCAD CCATCAAGCCTGTGGGAATC GCAGATCGGACCGGATACTG BMP4 TTCCTGGTAACCGAATGCTGA CCCTGAATCTCGGCGACTTTT CD41 GATGAGACCCGAAATGTAGGC TCAGTCTTTTCTAGGACGTTCCA EPHB2 GGACTGGTACTATACCCACAGAT TGTCTGCTTGGTCTTTATCAACC VAV1 CAGCAGTGGGAAGCACAAAG GTCACGGGCGCAGAAGTC GLUT1 GATTGGCTCCTTCTCTGTGG TCAAAGGACTTGCCCAGTTT FOS TGCCTCTCCTCAATGACCCTGA ATAGGTCCATGTCTGGCACGGA HIF1A GCTTCCTTCGATCAGTTGTC TCAGTGGTGGCAGTGGTAGT EVI1 TTGCCAAGTAACAGCTTTGCTG CCAAAGGGTCCGAATGTGACTT BMI1 CTGGTTGCCCATTGACAGC CAGAAAATGAATGCGAGCCA FLT3 TTTACCCCACTTTCCAATCACAT CGAGTCCGGGTGTATCTGAAC MMP9 GTGCTGGGCTGCTGCTTTGCTG GTCGCCCTCAAAGGTTTGGAAT IKAROS GACGCACTCCGTCATTAAAGA CGACGTTACTTGCTAGTCTGTC STAT5 AGATGCTGGCCGAGGTCAAC AGACTTGGCCTGCTGCTCAC FOXO1 AGTCTGAGGTGCTGGCGGAG GGTGGTGGCGTATCAGAGGTG MONKEY Forward Primer Reverse Primer Actin GCACAGAGCCTCGCCTTT GGAATCCTTCTGACCCATGC EP1 GATGGTGGGCCAGCTTGTC GCCACCAACACCAGCATTG EP2 GTGCTGACAAGGCACTTCATGT TGTTCCTCCAAAGGCCAAGTAC EP3 AAGGCCACGGCATCTCAGT TGATCCCCATAAGCTGAATGG EP4 CTTGGAGGCAGGAATTTGCTT AAAGTCCTCAGTGAGGTGGTGTCT
Supplemental Experimental Procedures 2: Schematic representation of rhesus macaque autologous transplantation protocol: alternations in DMSO or dmpge2 treatment either pre- (A) or post- (B) differential fluorophore transduction prior to reinfusion are indicated by the paired dashed and solid lines.