Hypoxia-induced downregulation of cyclooxygenase 2 leads to the loss of immunoprivilege of allogeneic mesenchymal stem cells.

Allogeneic mesenchymal stem cells (MSCs) from young and healthy donors are reported to hold the potential to treat several immunological and degenerative disorders. However, recent data from animal studies and clinical trials demonstrate that immunogenicity and poor survival of transplanted MSCs impaired the efficacy of cells for regenerative applications. It is reported that initially immunoprivileged under in vitro conditions, MSCs are targeted by the host immune system after transplantation in the ischemic tissues in vivo. We performed in vitro (in MSCs) and in vivo (in the rat model of myocardial infarction [MI]) studies to elucidate the mechanisms responsible for the change in the immunophenotype of MSCs from immunoprivileged to immunogenic under ischemic conditions. We have recently reported that a soluble factor prostaglandin E2 (PGE2) preserves the immunoprivilege of allogeneic MSCs. In the current study, we found that PGE2 levels, which were elevated during normoxia, decreased in MSCs following exposure to hypoxia. Further, we found that proteasome-mediated degradation of cyclooxygenase-2 (COX2, rate-limiting enzyme in PGE2 biosynthesis) in hypoxic MSCs is responsible for PGE2 decrease and loss of immunoprivilege of MSCs. While investigating the mechanisms of COX2 degradation in hypoxic MSCs, we found that in normoxic MSCs, COP9 signalosome subunit 5 (CSN5) binds to COX2 and prevents its degradation by the proteasome. However, exposure to hypoxia leads to a decrease in CSN5 levels and its binding to COX2, rendering COX2 protein susceptible to proteasome-mediated degradation. This subsequently causes PGE2 downregulation and loss of immunoprivilege of MSCs. Maintaining COX2 levels in MSCs preserves immunoprivilege in vitro and improves the survival of transplanted MSCs in a rat model of MI. These data provide novel mechanistic evidence that PGE2 is downregulated in hypoxic MSCs which is responsible for the post-transplantation rejection of allogeneic MSCs. Therefore, our data suggest that the new strategies that target CSN5-COX2 signaling may improve survival and utility of transplanted allogeneic MSCs in the ischemic heart.

Hypoxia-induced increase in Sug1 leads to poor post-transplantation survival of allogeneic mesenchymal stem cells.

Allogeneic mesenchymal stem cells (MSCs) from young and healthy donors are immunoprivileged and have the potential to treat numerous degenerative diseases. However, recent reviews of clinical trials report poor long-term survival of transplanted cells in the recipient that turned down the enthusiasm regarding MSC therapies. Increasing evidence now confirm that though initially immunoprivileged, MSCs eventually become immunogenic after transplantation in the ischemic or hypoxic environment of diseased tissues and are rejected by the host immune system. We performed in vitro (in rat and human cells) and in vivo (in a rat model) investigations to understand the mechanisms of the immune switch in the phenotype of MSCs. The immunoprivilege of MSCs is preserved by the absence of cell surface immune antigen, major histocompatibility complex II (MHC-II) molecule. We found that the ATPase subunit of 19S proteasome "Sug1" regulates MHC-II biosynthesis in MSCs. Exposure to hypoxia upregulates Sug1 in MSCs and its binding to class II transactivator (CIITA), a coactivator of MHC-II transcription. Sug1 binding to CIITA in hypoxic MSCs promotes the acetylation and K63 ubiquitination of CIITA leading to its activation and translocation to the nucleus, and ultimately MHC-II upregulation. In both rat and human MSCs, knocking down Sug1 inactivated MHC-II and preserved immunoprivilege even following hypoxia. In a rat model of myocardial infarction, transplantation of Sug1-knockdown MSCs in ischemic heart preserved immunoprivilege and improved the survival of transplanted cells. Therefore, the current study provides novel mechanisms of post-transplantation loss of immunoprivilege of MSCs. This study may help in facilitating better planning for future clinical trials.

Application of Ti3 C2 MXene Quantum Dots for Immunomodulation and Regenerative Medicine.

Inflammation is tightly linked to tissue injury. In regenerative medicine, immune activation plays a key role in rejection of transplanted stem cells and reduces the efficacy of stem cell therapies. Next-generation smart biomaterials are reported to possess multiple biologic properties for tissue repair. Here, the first use of 0D titanium carbide (Ti3 C2 ) MXene quantum dots (MQDs) for immunomodulation is presented with the goal of enhancing material-based tissue repair after injury. MQDs possess intrinsic immunomodulatory properties and selectively reduce activation of human CD4+ IFN-γ+ T-lymphocytes (control 87.1 ± 2.0%, MQDs 68.3 ± 5.4%) while promoting expansion of immunosuppressive CD4+ CD25+ FoxP3+ regulatory T-cells (control 5.5 ± 0.7%, MQDs 8.5 ± 0.8%) in a stimulated lymphocyte population. Furthermore, MQDs are biocompatible with bone marrow-derived mesenchymal stem cells and induced pluripotent stem cell-derived fibroblasts. Finally, Ti3 C2 MQDs are incorporated into a chitosan-based hydrogel to create a 3D platform with enhanced physicochemical properties for stem cell delivery and tissue repair. This composite hydrogel demonstrates increased conductivity while maintaining injectability and thermosensitivity. These findings suggest that this new class of biomaterials may help bridge the translational gap in material and stem cell-based therapies for tissue repair and treatment of inflammatory and degenerative diseases.

Hypoxia-induced 26S proteasome dysfunction increases immunogenicity of mesenchymal stem cells.

Bone marrow-derived allogeneic (donor derived) mesenchymal stem cells (MSCs) are immunoprivileged and are considered to be prominent candidates for regenerative therapy for numerous degenerative diseases. Even though the outcome of initial allogeneic MSCs based clinical trials was encouraging, the overall enthusiasm, of late, has dimmed down. This is due to failure of long-term survival of transplanted cells in the recipient. In fact, recent analyses of allogeneic MSC-based studies demonstrated that cells after transplantation turned immunogenic and were subsequently rejected by host immune system. The current study reveals a novel mechanism of immune switch in MSCs. We demonstrate that hypoxia, a common denominator of ischemic tissues, induces an immune shift in MSCs from immunoprivileged to immunogenic state. The immunoprivilege of MSCs is preserved by downregulation or the absence of major histocompatibility complex class II (MHC-II) molecules. We found that 26S proteasome-mediated intracellular degradation of MHC-II helps maintain the absence of MHC-II expression on cell surface in normoxic MSCs and preserves their immunoprivilege. The exposure to hypoxia leads to dissociation of 19S and 20S subunits, and inactivation of 26S proteasome. This prevented the degradation of MHC-II and, as a result, the MSCs became immunogenic. Furthermore, we found that hypoxia-induced decrease in the levels of a chaperon protein HSP90α is responsible for inactivation of 26S proteasome. Maintaining HSP90α levels in hypoxic MSCs preserved the immunoprivilege of MSCs. Therefore, hypoxia-induced inactivation of 26S proteasome assembly instigates loss of immunoprivilege of allogeneic mesenchymal stem cells. Maintaining 26S proteasome activity in mesenchymal stem cells preserves their immunoprivilege.

Graphene Oxide-Gold Nanosheets Containing Chitosan Scaffold Improves Ventricular Contractility and Function After Implantation into Infarcted Heart.

Abnormal conduction and improper electrical impulse propagation are common in heart after myocardial infarction (MI). The scar tissue is non-conductive therefore the electrical communication between adjacent cardiomyocytes is disrupted. In the current study, we synthesized and characterized a conductive biodegradable scaffold by incorporating graphene oxide gold nanosheets (GO-Au) into a clinically approved natural polymer chitosan (CS). Inclusion of GO-Au nanosheets in CS scaffold displayed two fold increase in electrical conductivity. The scaffold exhibited excellent porous architecture with desired swelling and controlled degradation properties. It also supported cell attachment and growth with no signs of discrete cytotoxicity. In a rat model of MI, in vivo as well as in isolated heart, the scaffold after 5 weeks of implantation showed a significant improvement in QRS interval which was associated with enhanced conduction velocity and contractility in the infarct zone by increasing connexin 43 levels. These results corroborate that implantation of novel conductive polymeric scaffold in the infarcted heart improved the cardiac contractility and restored ventricular function. Therefore, our approach may be useful in planning future strategies to construct clinically relevant conductive polymer patches for cardiac patients with conduction defects.

Early passaging of mesenchymal stem cells does not instigate significant modifications in their immunological behavior.

BACKGROUND: Bone marrow-derived allogeneic mesenchymal stem cells (MSCs) from young healthy donors are immunoprivileged and their clinical application for regenerative medicine is under evaluation. However, data from preclinical and initial clinical trials indicate that allogeneic MSCs after transplantation provoke a host immune response and are rejected. In the current study, we evaluated the effect of an increase in passage number in cell culture on immunoprivilege of the MSCs. Since only limited numbers of MSCs can be sourced at a time from a donor, it is imperative to expand them in culture to meet the necessary numbers required for cell therapy. Presently, the most commonly used passages for transplantation include passages (P)3-7. Therefore, in this study we included clinically relevant passages, i.e., P3, P5, and P7, for evaluation. METHODS: The immunoprivilege of MSCs was assessed with the mixed leukocyte reaction assay, where rat MSCs were cocultured with peripheral blood leukocytes for 72 h. Leukocyte-mediated cytotoxicity, apoptosis (Bax/Bcl-xl ratio), leukocyte proliferation, and alterations in cellular bioenergetics in MSCs were assessed after the coculture. Furthermore, the expression of various oxidized phospholipids (oxidized phosphatidylcholine (ox-PC)) was analyzed in MSCs using a lipidomic platform. To determine if the ox-PCs were acting in tandem with downstream intracellular protein alterations, we performed proteome analysis using a liquid chromatography/mass spectrometry (LC/MS) proteomic platform. RESULTS: Our data demonstrate that MSCs were immunoprivileged at all three passages since coculture with leukocytes did not affect the survival of MSCs at P3, P5, and P7. We also found that, with an increase in the passage number of MSCs, leukocytes did not cause any significant effect on cellular bioenergetics (basal respiration rate, spare respiratory capacity, maximal respiration, and coupling efficiency). Interestingly, in our omics data, we detected alterations in some of the ox-PCs and proteins in MSCs at different passages; however, these changes were not significant enough to affect their immunoprivilege. CONCLUSIONS: The outcome of this study demonstrates that an increase in passage number (from P3 to P7) in the cell culture does not have any significant effect on the immunoprivilege of MSCs.

Methods for Long-Term Storage of Murine Bone Marrow-Derived Mesenchymal Stem Cells.

This chapter is based on a simplified method to validate the current preservation procedure of mesenchymal stem cells (MSCs). Currently, there are various media available for freezing and thus preserving the MSCs, making it hard to decide which agent will be apt for cellular requirements. The study describes the effect of two different compositions of freezing media used in regular cell culture experiments, on the morphology, proliferation, and doubling rate of MSCs. Commonly used agents for the cryopreservation of MSCs include DMSO (Dimethyl Sulfoxide) and FBS (Fetal Bovine Serum) and DMEM (Dulbecco's Modified Eagle Medium). To ascertain that the currently used agents do not lead to major changes in the MSC morphology and proliferation, the cells are frozen using the above-mentioned agents in different groups and then their effects analyzed. Thus, the chapter helps to decide what reagents can suit the MSCs, hence minimizing the laboratory to laboratory variability of their characteristics.

Rational drug design.

In this article, current knowledge of drug design is reviewed and an approach of rational drug design is presented. The process of drug development is challenging, expensive, and time consuming, although this process has been accelerated due to the development of computational tools and methodologies. The current target based drug design approach is incomplete because most of the drugs developed by structure guided approaches have been shown to have serious toxic side effects. Otherwise these drugs would have been an ideal choice for the treatment of diseases. Hence, rational drug design would require a multidisciplinary approach. In this regard, incorporation of gene expression technology and bioinformatics tools would be indispensable in the structure based drug design. Global gene expression data and analysis of such data using bioinformatics tools will have numerous benefits such as efficiency, cost effectiveness, time saving, and will provide strategies for combination therapy in addition to overcoming toxic side effects. As a result of incorporation of gene expression data, partial benefit of the structure based drug design is slowly emerging and rapidly changing the approach of the drug development process. To achieve the full benefit of developing a successful drug, multidisciplinary approaches (approaches such as computational chemistry and gene expression analysis, as discussed in this article) would be necessary. In the future, there is adequate room for the development of more sophisticated methodologies.