Mesenchymal stem cells (MSCs) are being increasingly recognized for their hypoimmunogenic and immunosuppressive properties. These include the expression of a wide array of surface and secreted proteins, which inhibit CD4+, CD8+, and NK cells, while promoting regulatory cell phenotypes. For this reason, MSCs are being tested for treating disorders involving inflammation and activation of the adaptive immune response. Thus far, however, the results of clinical trials have been inferior to expectations based on in vitro studies. This may be due to the cells not reaching the affected site and not optimally exhibiting their immunomodulatory phenotype. In fact, numerous studies suggest that MSCs may require exposure to microenvironmental cues to become immunosuppressive. Since inflammation and hypoxia are common to several tolerogenic microenvironments (e.g. tumors, placenta), we explored combined IFN-γ and hypoxia priming of human MSCs. After 48h of stimulation, several immunosuppressive factors were upregulated, most dramatically IDO and PDL1 (by IFN-γ) and COX2 and HLA-G (by hypoxia). Gene expression changes persisted for one-week post stimulus removal. While IFN-γ stimulation predictably upregulated expression of classical MHC proteins, allogeneic reaction with human PBMCs was not observed. In fact, MSCs preconditioned with IFN-γ/hypoxia suppressed expansion of both CD4+ and CD8+ T cell populations in response to CD2/CD3/CD28 bead stimulation to a greater extent than unconditioned MSCs. These in vitro data suggest that it may be possible to create uniformly immunosuppressive populations of MSCs for use in cell therapies. To investigate whether there is also a greater potential for cell homing following combined stimulation, we studied changes in chemokine receptor expression, and observed a consistently greater expression of CCR1 and CCR7 relative to other chemokine receptors. Future studies will explore the immunosuppressive and homing potential of preconditioned MSCs in both graft-vs-host and host-vs-graft transplant models.

Homing (biology)

Priming (agriculture)

Cell therapy

10.1016/j.bbmt.2015.11.496

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EGCG reduces viability and VEGF-D expression of IBC stem-like cells in vitro.

D. Mineva Nora Eric Paulson K.P. Stephen Amy Sallee Sonenshein Gail E.

10.1371/journal.pone.0073464.g006

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WNT enhances acinar progenitor activity through LRP6 signaling.

M. Lisa St. Laurent Jessica Wronski Ania Caballero Silvia R.L. Stephen

LRP6

Progenitor

10.1371/journal.pone.0111442.g002

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Protection of Organ Vasculature By Endothelial Overexpression of HLA-G

Biology of Blood and Marrow Transplantation (2016)

Holly Wobma P. Stephen Jennifer S. Fang Hema Vasavada Raimon Duran‐Struuck

Solid organ transplantation is a definitive treatment for organ failure. While great progress has been made in preventing and treating acute rejection, transplantation is still limited by chronic rejection. Extending the donor pool to organs from older individuals has helped curb the overwhelming shortage, but these organs are more likely to be rejected even acutely. One element that is common to both acute and chronic rejection is the involvement of vasculature. Since endothelial cells can express MHC Class I and II proteins, serve as the gateway for migration of leukocytes to the periphery, and interact with smooth muscle cells, they are promising therapeutic targets for changing the natural history of graft survival. As an approach to reducing the vasculopathy of graft rejection, we sought to alter endothelium to overexpress the non-classical major histocompatibility protein HLA-G. This molecule is found in many situations of immune tolerance (cancer, placenta, viruses) and inhibits CD4+, CD8+, NK, and dendritic cells, while increasing regulatory T-cell populations. Furthermore, soluble HLA-G levels have been correlated with graft outcome in the literature. To test the feasibility of HLA-G mediated immunoprotection, the first step was to develop an in situ model in which human umbilical arteries were either partially stripped of endothelium and reseeded with HLA-G+ endothelial cells or directly transduced with an HLA-G5 encoding lentivirus. The HLA-G5 isoform was chosen because, as a secreted form, it may enable the transduction of only a small subset of endothelial cells, which could then serve as nodes protecting the graft by continuous local "drug secretion". We have achieved a greater than one-million fold increase in HLA-G expression in purified HLA-G5+ endothelial cells compared to untransduced controls, demonstrating the feasibility of this approach. Protein production has been verified by immunofluorescence, Western blot, and ELISA. We are currently testing the ability of these cells to inhibit lymphocyte proliferation in both monolayer co-culture assays and by circulating PBMCs through our umbilical artery bioreactor.

10.1016/j.bbmt.2015.11.865

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