Darling Rojas-Canales

Interferon-Gamma Modification of Mesenchymal Stem Cells: Implications of Autologous and Allogeneic Mesenchymal Stem Cell Therapy in Allotransplantation

Bone marrow-derived mesenchymal stem cells (MSC) have unique immunomodulatory and reparative properties beneficial for allotransplantation cellular therapy. The clinical administration of autologous or allogeneic MSC with immunosuppressive drugs is able to prevent and treat allograft rejection in kidney transplant recipients, thus supporting the immunomodulatory role of MSC. Interferon-gamma (IFN-γ) is known to enhance the immunosuppressive properties of MSC. IFN-γ preactivated MSC (MSC-γ) directly or indirectly modulates T cell responses by enhancing or inducing MSC inhibitory factors. These factors are known to downregulate T cell activation, enhance T cell negative signalling, alter T cells from a proinflammatory to an anti-inflammatory phenotype, interact with antigen-presenting cells and increase or induce regulatory cells. Highly immunosuppressive MSC-γ with increased migratory and reparative capacities may aid tissue repair, prolong allograft survival and induce allotransplant tolerance in experimental models. Nevertheless, there are contradictory in vivo observations related to allogeneic MSC-γ therapy. Many studies report that allogeneic MSC are immunogenic due to their inherent expression of major histocompatibility (MHC) molecules. Enhanced expression of MHC in allogeneic MSC-γ may increase their immunogenicity and this can negatively impact allograft survival. Therefore, strategies to reduce MSC-γ immunogenicity would facilitate “off-the-shelf” MSC therapy to efficiently inhibit alloimmune rejection and promote tissue repair in allotransplantation. In this review, we examine the potential benefits of MSC therapy in the context of allotransplantation. We also discuss the use of autologous and allogeneic MSC and the issues associated with their immunogenicity in vivo, with particular focus on the use of enhanced MSC-γ cellular therapy.

Interleukin-17A-Induced Human Mesenchymal Stem Cells Are Superior Modulators of Immunological Function.

Interferon‐γ (IFN‐γ)‐preactivated mesenchymal stem cells (MSC‐γ) are highly immunosuppressive but immunogenic in vivo due to their inherent expression of major histocompatibility (MHC) molecules. Here, we present an improved approach where we modified human bone marrow‐derived MSC with interleukin‐17A (MSC‐17) to enhance T cell immunosuppression but not their immunogenicity. MSC‐17, unlike MSC‐γ, showed no induction or upregulation of MHC class I, MHC class II, and T cell costimulatory molecule CD40, but maintained normal MSC morphology and phenotypic marker expression. When cocultured with phytohemagglutinin (PHA)‐activated human T cells, MSCs‐17 were potent suppressors of T cell proliferation. Furthermore, MSC‐17 inhibited surface CD25 expression and suppressed the elaboration of Th1 cytokines, IFN‐γ, tumor necrosis factor‐α (TNF‐α), and IL‐2 when compared with untreated MSCs (UT‐MSCs). T cell suppression by MSC‐17 correlated with increased IL‐6 but not with indoleamine 2,3‐dioxygenase 1, cyclooxygenase 1, and transforming growth factor β‐1. MSC‐17 but not MSC‐γ consistently induced CD4+CD25highCD127lowFoxP3+ regulatory T cells (iTregs) from PHA‐activated CD4+CD25− T cells. MSC‐induced iTregs expressed CD39, CD73, CD69, OX40, cytotoxic T‐lymphocyte associated antigen‐4 (CTLA‐4), and glucocorticoid‐induced TNFR‐related protein (GITR). These suppressive MSCs‐17 can engender Tregs to potently suppress T cell activation with minimal immunogenicity and thus represent a superior T cell immunomodulator for clinical application. Stem Cells 2015;33:2850–2863

Endothelial progenitor cells enhance islet engraftment, influence β-cell function, and modulate islet connexin 36 expression.

The success of pancreatic islet transplantation is limited by delayed engraftment and suboptimal function in the longer term. Endothelial progenitor cells (EPCs) represent a potential cellular therapy that may improve the engraftment of transplanted pancreatic islets. In addition, EPCs may directly affect the function of pancreatic β-cells. The objective of this study was to examine the ability of EPCs to enhance pancreatic islet transplantation in a murine syngeneic marginal mass transplant model and to examine the mechanisms through which this occurs. We found that cotransplanted EPCs improved the cure rate and initial glycemic control of transplanted islets. Gene expression data indicate that EPCs, or their soluble products, modulate the expression of the β-cell surface molecule connexin 36 and affect glucose-stimulated insulin release in vitro. In conclusion, EPCs are a promising candidate for improving outcomes in islet transplantation, and their mechanisms of action warrant further study.

IGF2: an endocrine hormone to improve islet transplant survival

In the week following pancreatic islet transplantation, up to 50% of transplanted islets are lost due to apoptotic cell death triggered by hypoxic and pro-inflammatory cytokine-mediated cell stress. Thus, therapeutic approaches designed to protect islet cells from apoptosis could significantly improve islet transplant success. IGF2 is an anti-apoptotic endocrine protein that inhibits apoptotic cell death through the mitochondrial (intrinsic pathway) or via antagonising activation of pro-inflammatory cytokine signalling (extrinsic pathway), in doing so IGF2 has emerged as a promising therapeutic molecule to improve islet survival in the immediate post-transplant period. The development of novel biomaterials coated with IGF2 is a promising strategy to achieve this. This review examines the mechanisms mediating islet cell apoptosis in the peri- and post-transplant period and aims to identify the utility of IGF2 to promote islet survival and enhance long-term insulin independence rates within the setting of clinical islet transplantation.

Transcriptome Profiling of IL-17A Preactivated Mesenchymal Stem Cells: A Comparative Study to Unmodified and IFN-γ Modified Mesenchymal Stem Cells

Human mesenchymal stem cells pretreatment with IL-17A (MSC-17) potently enhances T cell immunosuppression but not their immunogenicity, in addition to avidly promoting the induction of suppressive regulatory T cells. The aim of this study was to identify potential mechanisms by which human MSC-17 mediate their superior immunomodulatory function. Untreated-MSC (UT-MSC), IFN-γ treated MSC (MSC-γ), and MSC-17 were assessed for their gene expression profile by microarray. Significantly regulated genes were identified for their biological functions (Database for Annotation, Visualisation and Integrated Discovery, DAVID). Microarray analyses identified 1278 differentially regulated genes between MSC-γ and UT-MSC and 67 genes between MSC-17 and UT-MSC. MSC-γ were enriched for genes involved in immune response, antigen processing and presentation, humoral response, and complement activation, consistent with increased MSC-γ immunogenicity. MSC-17 genes were associated with chemotaxis response, which may be involved in T cell recruitment for MSC-17 immunosuppression. MMP1, MMP13, and CXCL6 were highly and specifically expressed in MSC-17, which was further validated by real-time PCR. Thus, MMPs and chemokines may play a key role in mediating MSC-17 superior immunomodulatory function. MSC-17 represent a potential cellular therapy to suppress immunological T cell responses mediated by expression of an array of immunoregulatory molecules.

Manipulating human dendritic cell phenotype and function with targeted porous silicon nanoparticles

Dendritic cells (DC) are the most potent antigen-presenting cells and are fundamental for the establishment of transplant tolerance. The Dendritic Cell-Specific Intracellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN; CD209) receptor provides a target for dendritic cell therapy. Biodegradable and high-surface area porous silicon (pSi) nanoparticles displaying anti-DC-SIGN antibodies and loaded with the immunosuppressant rapamycin (Sirolimus) serve as a fit-for-purpose platform to target and modify DC. Here, we describe the fabrication of rapamycin-loaded DC-SIGN displaying pSi nanoparticles, the uptake efficiency into DC and the extent of nanoparticle-induced modulation of phenotype and function. DC-SIGN antibody displaying pSi nanoparticles favourably targeted and were phagocytosed by monocyte-derived and myeloid DC in whole human blood in a time- and dose-dependent manner. DC preconditioning with rapamycin-loaded nanoparticles, resulted in a maturation resistant phenotype and significantly suppressed allogeneic T-cell proliferation.

Immunodepletion and Hypoxia Preconditioning of Mouse Compact Bone Cells as a Novel Protocol to Isolate Highly Immunosuppressive Mesenchymal Stem Cells

Compact bones (CB) are major reservoirs of mouse mesenchymal stem cells (mMSC). Here, we established a protocol to isolate MSC from CB and tested their immunosuppressive potential. Collagenase type II digestion of BM-flushed CB from C57B/6 mice was performed to liberate mMSC precursors from bone surfaces to establish nondepleted mMSC. CB cells were also immunodepleted based on the expression of CD45 (leukocytes) and TER119 (erythroid cells) to eliminate hematopoietic cells. CD45-TER119- CB cells were subsequently used to generate depleted mMSC. CB nondepleted and depleted mMSC progenitors were cultured under hypoxic conditions to establish primary mMSC cultures. CB depleted mMSC compared to nondepleted mMSC showed greater cell numbers at subculturing and had increased functional ability to differentiate into adipocytes and osteoblasts. CB depleted mMSC had high purity and expressed key mMSC markers (>85% Sca-1, CD29, CD90) with no mature hematopoietic contaminating cells (<5% CD45, CD11b) when subcultured to passage 5 (P5). Nondepleted mMSC cultures, however, were less pure and heterogenous with <72% Sca-1+, CD29+, and CD90+ cells at early passages (P1 or P2), along with high percentages of contaminating CD11b+ (35.6%) and CD45+ (39.2%) cells that persisted in culture long term. Depleted and nondepleted mMSC nevertheless exhibited similar potency to suppress total (CD3+), CD4+ and CD8+ T cell proliferation, in a dendritic cell allostimulatory one-way mixed lymphocyte reaction. CB depleted mMSC, pretreated with proinflammatory cytokines IFN-γ, TNF-α, and IL-17A, showed superior suppression of CD8+ T cell, but not CD4+ T cell proliferation, relative to untreated-mMSC. In conclusion, CB depleted mMSC established under hypoxic conditions and treated with selective cytokines represent a novel source of potent immunosuppressive MSC. As these cells have enhanced immune modulatory function, they may represent a superior product for use in clinical allotransplantation.

Local Sphingosine Kinase 1 Activity Improves Islet Transplantation

Pancreatic islet transplantation is a promising clinical treatment for type 1 diabetes, but success is limited by extensive β-cell death in the immediate posttransplant period and impaired islet function in the longer term. Following transplantation, appropriate vascular remodeling is crucial to ensure the survival and function of engrafted islets. The sphingosine kinase (SK) pathway is an important regulator of vascular beds, but its role in the survival and function of transplanted islets is unknown. We observed that donor islets from mice deficient in SK1 (Sphk1 knockout) contain a reduced number of resident intraislet vascular endothelial cells. Furthermore, we demonstrate that the main product of SK1, sphingosine-1-phosphate, controls the migration of intraislet endothelial cells in vitro. We reveal in vivo that Sphk1 knockout islets have an impaired ability to cure diabetes compared with wild-type controls. Thus, SK1-deficient islets not only contain fewer resident vascular cells that participate in revascularization, but likely also a reduced ability to recruit new vessels into the transplanted islet. Together, our data suggest that SK1 is important for islet revascularization following transplantation and represents a novel clinical target for improving transplant outcomes.

A Combinatorial Protein Microarray for Probing Materials Interaction with Pancreatic Islet Cell Populations

Pancreatic islet transplantation has become a recognized therapy for insulin-dependent diabetes mellitus. During isolation from pancreatic tissue, the islet microenvironment is disrupted. The extracellular matrix (ECM) within this space not only provides structural support, but also actively signals to regulate islet survival and function. In addition, the ECM is responsible for growth factor presentation and sequestration. By designing biomaterials that recapture elements of the native islet environment, losses in islet function and number can potentially be reduced. Cell microarrays are a high throughput screening tool able to recreate a multitude of cellular niches on a single chip. Here, we present a screening methodology for identifying components that might promote islet survival. Automated fluorescence microscopy is used to rapidly identify islet derived cell interaction with ECM proteins and immobilized growth factors printed on arrays. MIN6 mouse insulinoma cells, mouse islets and, finally, human islets are progressively screened. We demonstrate the capability of the platform to identify ECM and growth factor protein candidates that support islet viability and function and reveal synergies in cell response.

Modification of Polyurethane Scaffolds for Localised Immunosuppression of Subcutaneous Islet Transplantation.

Introduction Hepatic islet transplantation, as a curative therapy for Type 1 Diabetes, is suboptimal for islet function and viability. Subcutaneous engraftment presents an attractive alternative, yet, is currently incapable of supporting islet survival without modification. The use of a polyurethane (PU) scaffold has previously been shown to generate a hyper-vascularised dermal layer, and co-opting this may overcome inherent dermal hypoxia and hypovascularity. However, mitigating the cutaneous immune response is crucial. The aim of this study was to evaluate scaffold loading of the immunosuppressant rapamycin (Rapa), generating a localised immunosuppressive microenvironment supporting islet engraftment. Methods PU disks (8mmx2mm) were loaded topically with 2nM of Rapa. Structural changes to the PU were evaluated using SEM. UV-VIS spectroscopy was utilised to detail Rapa absorption (278nm) over 7 days evaluating drug release kinetics. In vitro biocompatibility of the Rapa-PU was investigated through co-culture with BTC-6 murine &bgr;-cell line, murine and human islets. Islet viability was evaluated using fluorescein diacetate/propidium iodide (FDA/PI) staining. T-cell inhibitory properties were evaluated in murine and human cells using a MLR and anti-CD3/28 antibody stimulation assay. Murine in vivo biocompatibility of subcutaneously implanted Rapa-PU was assessed with immunohistochemistry. Results/Discussion SEM of Rapa-PU showed increased pore density with surface integrity disruption, due to methanol treatment, and crystal deposition. Rapa-PU release kinetics demonstrated steady release of up to 70% (60nM) in 7 days following the Higuchi and Ritger-Peppas model, indicating a time dependent release caused by scaffold degradation. In vitro no phenotypically, acute, negative effects were observed upon BTC-6 cell, murine and human islet viability with exposure to PU alone and Rapa-PU. T cell proliferation was potently suppressed by the PU alone, however, immunosuppression was also observed from Rapa released from the scaffold through conditioned media. In vivo scaffolds were well tolerated, and blood vessel formation was detected in unloaded and Rapa-PU scaffolds evaluated by CD31 immunofluorescence staining, indicating that Rapa loading does not abrogate angiogenesis and is comparable to control scaffolds. Conclusion PU scaffolds provide a stable platform for the slow elution of rapamycin, neovascularisation and graft survival. Both the scaffold and rapamycin provides local immunosuppression. In doing so, this model could allow for initial local immunosuppression to prevent early immune infiltration of the graft. Additionally, this model may reduce the requirement for initial systemic immunosuppression, and deliver a higher, less toxic, immunosuppression to the site of transplantation.