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Blood, Vol. 114, Issue 3, 693-701, July 16, 2009
Multipotent adult progenitor cells can suppress graft-versus-host disease via prostaglandin E2 synthesis and only if localized to sites of allopriming
Blood Highfill et al.
114: 693
Supplemental materials for: Highfill et al
Files in this Data Supplement:
- Figure S1. Characterization of MAPC (JPG, 348 KB)
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(A) MAPCs isolated from B6 mice were differentiated into cells of mesodermal lineage and functionally tested for their ability to produce lipid droplets (adipocytes, Oil Red O), calcium deposition (osteocytes, Alizarin Red S), and accumulate collagen (chondrocytes, alkaline phosphatase). (B) Undifferentiated MAPCs (insert) or MAPCs directed to differentiate in vitro into cells representative of three germ layers, were stained for expression of CD31 and VWF (endothelium), HNF and albumin (endoderm), and GFAP and NF200 (neuroectoderm) and analyzed using confocal microscopy. In all images (except HNF) nuclear staining using DAPI is visualized as blue. (C) Undifferentiated MAPCs were examined for their expression of specific surface markers using flow cytometry. Isotype controls are shaded red. (D) RNA was isolated and cDNA was synthesized from undifferentiated MAPCs and murine embryonic stem cells. RT-PCR was used to examine the expression of Oct3/4 and Rex-1. No Template Control (NTC) and HPRT served as negative and positive controls, respectively. (E) Chromosomal analysis of MAPC shows a normal 40, XX karyotype.
- Figure S2. MAPC inhibit ongoing allo-responses (JPG, 18 KB)
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B6>BALB/c MLR cultures were performed and B6 MAPCs were titrated in at 1:10 ratios on days 0, 1, 2, and 3. Cultures were pulsed on the indicated days and harvested 16 hours later. Proliferation was assessed as a measure of 3H-thymidine uptake.
- Figure S3. Effects of PGE2 and IDO on T-cell proliferation (JPG, 68 KB)
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(A) RT-PCR verified that MAPC express PGE synthase. No template control (NTC) and HPRT were used as negative and positive controls. (B) Supernatant from MAPC cultures or MAPCs pretreated with 5uM indomethacin overnight and washed were analyzed for the production of PGE2 via ELISA (Day 7 shown). (C) MLR cocultures were arranged using 200 µM 1-methyl tryptophan and 5 µM indomethacin in the culture media either alone or in combination to assess the contribution of IDO and PGE2 on MAPC mediated suppression, respectively. MLR were pulsed with 3H-thymidine on the indicated days and harvested 16 hours later.
- Figure S4. Persistence of MAPC delivered intra-splenically (JPG, 168 KB)
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(A) BALB/c mice were lethally irradiated and given 106 T-cell–depleted BM cells plus 5 × 105 MAPC-DL IS. Individual mice and organs (top left-GI tract, top right-spleen, middle left-lung, middle right-LN, bottom left-femur, bottom right-liver) were monitored using bioluminescent imaging to determine the location of MAPCs on day 2, week 1, week 2, and week 3. (B) Weight curve from mice in Fig. 5C.
- Figure S5. Localization of MAPC to the spleen after “in vivo MLR” (JPG, 78.3 KB)
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5 × 105 MAPC-DL were injected IS along with 15 × 106 T cells. Day 4 bioluminescent imaging of spleens and lymph nodes from 6 mice revealed that MAPC remained within the spleen and had not migrated out to lymphoid tissue.
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