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Blood, Vol. 105, Issue 4, 1815-1822, February 15, 2005
Human mesenchymal stem cells modulate allogeneic immune cell responses
Blood Aggarwal and Pittenger
105: 1815
Supplemental materials for: Aggarwal and Pittenger, Vol 105, Issue 4, 1815-1822
Files in this Data Supplement:
- Figure S1. Human MSCs alter dendritic cell (DC) phenotype (JPEG, 60 KB)
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Human MSCs were co-cultured with isolated DCs (hMSC/DC1 ratio,1:1 or 1:10) under DC maturation conditions (IL-4 plus GM-CSF). Following this incubation, the non-adherent cells were harvested, washed extensively, and irradiated to block further proliferation. The irradiated DCs were further used as stimulators for naive T cells from a third-party donor as responders (DC-to-naive T-cell ratio, 1:100). The incubation was carried out for 6 days in the presence of rhIL-2, after which the non-adherent cell population was stimulated with PHA (2.5 µg/mL) for an additional 16 hours. The levels of IFN- for DC1 and IL-4 for DC2 were examined. Results from 1 experiment show that DC1s, when pre-incubated with hMSCs, induce naive T cells to decrease IFN- production, while DC2s incubated with hMSCs produced 3-fold–increased IL-4 as compared with controls.
- Figure S2A. hMSCs increase TReg cell proportions but do not impact functionality (JPEG, 32 KB)
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hMSCs result in an increase in TReg-cell proportions. Glucocorticosteroid-induced TNF receptor family member (GITR) is a cell-surface marker expressed on the TReg-cell population. Human MSCs were incubated with human PBMCs in the presence of rhIL-2 for 3 days. Following incubation, the non-adherent cell population was stained with PE-CD4 and FITC-GITR antibodies. Results from 1 of 2 experiments show increased GITR expression in PBMCs pre-incubated with hMSCs.
- Figure S2B. hMSCs increase TReg cell proportions but do not impact functionality (JPEG, 30 KB)
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TReg cells generated in the presence or absence of hMSCs are functionally similar. The TReg-cell population (CD4+CD25+) generated from PBMCs or MSCs+PBMCs (MSC/PBMC ratio, 1:10) cultures was isolated using a 2-step magnetic isolation procedure. These cells were irradiated to block any further proliferation and used as stimulators in a proliferation assay, where responders were allogeneic PBMCs (stimulator-responder ratio, 1:100) in the presence of PHA (2.5 µg/mL). The culture was carried out for 48 hours, after which (3H) thymidine was added and incorporated radioactivity counted after 24 hours. Results showed that functionally, the TReg population generated in the presence of MSCs (lane 3) was similar to that generated in the absence of MSCs (lane 2).
- Figure S3A. Impact of COX inhibitors on PGE2 secretion (JPEG, 25 KB)
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COX inhibitors block PGE2 secretion in hMSCs. Human MSCs were incubated with various concentrations of 2 COX inhibitors: COX-2 inhibitor NS-398 and COX-1/2 inhibitor indomethacin. The inhibitor concentrations are in µM; PGE2 levels were determined following a 24-hour culture of hMSCs with the inhibitors. Data from 1 of 2 experiments performed in triplicate are shown.
- Figure S3A. Impact of COX inhibitors on PGE2 secretion (JPEG, 25 KB)
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MSC-mediated PGE2 secretion is associated with increased COX-2 expression. Human MSCs were co-cultured with hPBMCs (MSC/PBMC ratio, 1:10) in a 12-well transwell plate for 24 hours. RNA was collected from both the upper and lower chambers and probed with primers specific for COX-1 and COX-2 in a quantitative RT-PCR reaction using Quantitect SYBR Green kit (Qiagen, Valencia, CA). Human MSCs expressed significantly higher levels of COX-2. When PBMCs were co-cultured in the presence of hMSCs, there was an increase greater than 3-fold in COX-2 expression in hMSCs. Data from 1 of 2 experiments are shown.
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