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Human Embryonic Stem Cell-extracts Inhibit the Differentiation and Function of Monocyte-derived Dendritic Cells

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Abstract

Embryonic stem cells (ESC) possess inherent properties of immune privilege with the capacity to evade allogeneic immune responses. Moreover, ESCs have been shown to prevent immune activation in response to third party antigen presenting cells in vitro and have the capacity to promote allograft survival in vivo. However, clinical use of human ESCs to treat immunological disorders may risk teratoma or ectopic tissue formation. Here, we show that cellular extracts from both human and mouse ESCs retain the immune modulatory properties of intact cells. ESC-extracts that contained 12–24 μg of total protein effectively prevented T cell proliferation in allogeneic mixed lymphocyte reactions (MLR), whereas control fibroblast extracts did not affect proliferation. Cellular mechanisms underlying hESC extract-mediated immune modulation involve the maturation of monocyte derived dendritic cells (mDC). hESC extract-treated mDCs had reduced surface expression of co-stimulatory and maturation markers CD80, HLA-DR and CD83 and secreted lower levels of IL12p40. Accordingly, hESC extract-treated DCs were found to be poor stimulators of purified allogeneic T cells compared to those DCs treated with vehicle or fibroblast extracts. Our results demonstrate that ESC extracts retain the immune modulatory properties of ESCs and for the first time demonstrates that ESC derived factors can inhibit human mDC maturation and function.

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Acknowledgements

We are grateful to Drs. A. Nagy, J. Rossant, M. Gertsenstein, K. Vinterstein, M. Mileikovsky and J. Draper for providing the CA1 hESC line; to Dr. P. W. Zandstra’s group for distributing the CA1 cell line; to Drs. Andrew Makrigiannis, Lionel Filion and Nadine Tatton for critical reading of the manuscript and to our laboratory members for experimental assistance. We would like to extend our special thanks to Dr. Ashok Kumar and his laboratory members for use of equipment, reagents and technical support. This work was supported by operating grants from the Canadian Institutes of Health Research (CIHR) MOP-158235 and Bickel Foundation, CIHR New Investigator Awards MSH-166732 (L.W.) and MSH-196457 (D.A), an Early Research Award from the Ontario Government to L.W and a Research Award from the Department of Medicine, University of Ottawa (D.A) . K.M. is supported by the Canadian Blood Services (CBS) Graduate Fellowship Scholarship program and D.A. is an Adjunct Scientist with CBS.

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The authors declare no competing financial interests.

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Authors and Affiliations

  1. Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada

    Kanishka Mohib, David Allan & Lisheng Wang

  2. Regenerative Medicine Program, Ottawa Health Research Institute, University of Ottawa, Ottawa, Ontario, Canada

    David Allan & Lisheng Wang

  3. Ottawa Institute of Systems Biology, University of Ottawa, Ottawa, Ontario, Canada

    Lisheng Wang

  4. Roger Guindon Hall, 451 Smyth Rd., Ottawa, Ontario, Canada, K1H 8M5

    Lisheng Wang

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  1. Kanishka Mohib
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Correspondence to Lisheng Wang.

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Supplementary Figure 1

hESC extracts used in all experiments were cell free. hESCs were harvested from cell culture plates with collagenase IV followed by cell dissociation buffer to obtain a single cell suspension. Subsequently, hESCs were washed twice with ice cold PBS and centrifuged at 400g for 6 minutes at 4 C. After washing, the cells were re-suspended in lysis buffer (see materials and methods). At this point the cells were incubated on ice for 30 minutes and sonicated until the cells were completely lysed. The sonicated cells were centrifuged at 15000g for 15 minutes at 4°C to remove cell debris. The supernatant (soluble cell-free fraction) was transferred to a new tube and used in all experiments. (A-C) Images represent 10ul of soluble fractions from 3 different batches of hESC-extracts that were mixed with trypan blue (1:1) and analyzed on hemocytometer (100x). All images were captured using Zeiss Invertoskop 40C. (D) H9 cells prior to sonication (100x). (PDF 2348 kb)

Supplementary Figure 2

hESC extract-treated DCs supplemented with IL-12p40 are poor stimulators of allogeneic T cells. Primary human monocytes were isolated from peripheral blood mononuclear cells by negative selection using immunomagnetic beads. Subsequently, 5.0 x 105 monocytes were cultured in the presence of 500U/mL of GM-CSF and IL-4 in order to induce them to differentiate into dendritic cells. The cells also received either 0.15mg/mL (final concentration) of hESC extracts (hESC EXT) or equivalent volume of vehicle on day 0. Fresh media were added every 2 days containing fresh cytokines and 0.075mg/mL of hESC EXT or vehicle on day 2, 4, and 6. To induce maturation, on day 6 the cells received 20ng/mL of TNF-alpha in addition to IL-4 and GM-CSF. Some cultures were also supplemented with 10ng/ml of IL-12p40 in addition to the other cytokines during the maturation step. Immature cells that did not receive TNF-alpha were harvested on day 8 like their mature counterparts. mDCs were treated with mitomycin C and cultured with 1 x 105 purified CD3+ allogeneic T cells at a ratio of 1:100. T cell proliferation was allowed to proceed for 3 days and tritiated thymidine was added for an additional 16 to 18 hours. Cell proliferation is displayed as mean counts per minute (CPM) of triplicate wells ± SD. (PDF 93 kb)

Supplementary Figure 3

Cell cycle analysis of oneway MLR. A decrease in the number of cells entering the S phase was observed after treatment with hESC extracts (A) in comparison to vehicle control (B). One way MLR were carried out with PBMC obtained from healthy volunteers. One set of donor cells were treated with 50ug/mL of mitomycin C to serve as stimulators while the second set of donor cells were used as responders. MLRs were carried out in the presence of hESC extracts (A) or vehicle control (B). MLRs were allowed to proceed for 7 days. Cells were harvested and fixed with 10% formalin in PBS for 15 minutes, permeabilized with 0.5% Triton X-100 in PBS for 15 minutes. The cells were incubated with 0.5mg/ml of RNAse A and 7AAD for 30 minutes and analyzed by flow cytometry. Data analysis was carried out with Multi Cycle AV software (Phoenix flow systems Inc.). (PDF 273 kb)

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Mohib, K., Allan, D. & Wang, L. Human Embryonic Stem Cell-extracts Inhibit the Differentiation and Function of Monocyte-derived Dendritic Cells. Stem Cell Rev and Rep 6, 611–621 (2010). https://doi.org/10.1007/s12015-010-9185-7

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