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Blood, Vol. 113, Issue 1, 46-57, January 1, 2009
Mesenchymal stem cells induce mature dendritic cells into a novel Jagged-2–dependent regulatory dendritic cell population
Blood Zhang et al.
113: 46
Supplemental materials for: Zhang et al
Files in this Data Supplement:
- Document 1. Supplemental materials and methods (PDF, 102 KB)
- Table S1. Oligonucleotides used in the construction of shRNA expression vectors (PDF, 587 KB)
- Figure S1. The map of RNAi expression vector (JPG, 29.9 KB)
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The eGFP coding sequence amplified from the plasmid pEGFPN1 and a U6 promoter from pSilencer2.1-U6 hygro (Ambion) were cloned into the retroviral vector pMSCVneo (Clontech). The retroviral vector pMSCVneo was designated as a new shRNA expression vector pMSCVneo/eGFP-U6.
- Figure S2. MSC-DCs derived mostly from maDCs, but not from imDCs or contaminating progenitors (JPG, 152 KB)
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(A) The cell composition of purified maDCs. Three cell populations, CD11chiIahi, CD11clowIalow and CD11clowIaneg, are maDCs, imDCs and contaminating progenitors respectively. The percentage of CD11chiIahi, CD11clowIalow and CD11clowIaneg are 93.7%, 4.45% and 1.75% respectively. The data are presented as mean ± s.d. of five independent experiments. CD11chiIahi indicates a high level expression of CD11c and Ia molecules; CD11clowIalow, a low level expression of CD11c and Ia molecules; CD11clowIaneg, a low level expression of CD11c but negative expression of Ia molecules. (B) The growth curves of imDCs, maDCs or BMNCs cocultured with MSCs. The imDCs, maDCs and BMNCs were respectively seeded onto MSC monolayers at the concentration of 5000 cells/well in a 24-well plate, harvested and counted by 0.4% trypan blue exclusion method at different time points. Data are mean ± s.d. of triplicate wells. (C) Apoptotic analysis of the purified maDCs cocultured with MSCs. The maDCs were harvested and stained with Annexin V-FITC and PI to analyze apoptotic cells by flow cytometry at different time points during cocultivation with MSCs. Annexin V+PI− cells represents the apoptotic cells. Data are presented as mean ± s.d. of five independent experiments. *, P<0.05 (top panel). A representative of five independent experiments showed that the percentage of Annexin V+PI−cells decreased from 12.1% on day 1 to 5.1% on day 5 (bottom panel).
- Figure S3. Both imDCs and maDCs lack expression of F4/80 or CD68 (JPG, 41.5 KB)
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The imDCs and maDCs generated in vitro were analyzed by flow cytometry for the expression levels of macrophage specific marker F4/80 or CD68. BM-derived macrophages (BMDM) were used as positive control. One of at least 3 independent experiments with similar results is shown. Dotted lines indicate background staining.
- Figure S4. Phenotypic analysis of MSCs cocultured with maDCs (JPG, 53.2 KB)
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The MSCs cocultured with maDCs for 15 days were harvested for phenotypic analysis, and results showed that MSCs still expressed high level of Flk-1, CD29 and CD44, but negative for myeloid lineage markers CD11b and CD11c. One of at least 5 independent experiments with similar results is shown. Dotted lines, background staining.
- Figure S5. The up-regulated Jagged-2 expression of MSC-DCs stimulated by LPS (JPG, 28.3 KB)
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MSC-DCs were collected and incubated with LPS (0.5 µg/ml) for 72 hours, then analyzed by FACS for the expression of Jagged-1, Jagged-2 and Delta-1. Dotted lines indicate background staining. Numbers in histograms indicate the mean fluorescence of each DC population. One of at least three independent experiments with similar results is shown.
- Figure S6. Specific Jagged-2 RNAi down-regulates IL-10 and TGF-β secretion by MSC-DCs and promotes IL-2 and IFN-γ production by lymphocytes (JPG, 48.5 KB)
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(A) No changes of Jagged-1 or Delta-1 were detected in Jagged-2 RNAi assay. After 5 days of transfection with the Jagged-2 RNAi retrovirus, the total cellular protein extract of MSC-DCs was separated for analysis of Jagged-1, Jagged-2 and Delta-1 expression. The data are representative of three independent experiments. (B) Knockdown of Jagged-2 resulted in a decreased IL-10 and TGF-β production, but an increase of IL-12. The maDCs, MSC-DCs, Jagged-2-siRNA-MSC-DCs, or Jagged-2-siRNA-MSC-DCs stimulated by 0.5 µg/ml LPS were cultured for 24 hours, and the levels of IL-10, TGF-β and IL-12 in supernatants were detected by ELISA. Data are mean ± s.d. of triplicate wells. *, P <0.05. **, P<0.01. (C) The levels of IL-2 and IFN-γ secreted by lymphocytes increased after knockdown of Jagged-2. Lymphocytes (H-2Kb) were cocultured for 5 days with maDCs (H-2Kd) in the presence of MSC-DCs (H-2Kd) or Jagged-2-RNAi-MSC-DCs (H-2Kd), and concentrations of IFN-γ and IL-2 in each well were assayed by ELISA. Data are mean ± s.d. of triplicate wells. *, P <0.05.
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