Teresa Pescara

In vitro immunomodulatory effects of microencapsulated umbilical cord Wharton jelly-derived mesenchymal stem cells in primary Sjögren’s syndrome

OBJECTIVE Human umbilical cord Wharton jelly-derived mesenchymal stem cells (hUCMS) are easy to retrieve in bulk. They may interact with immune cells by either cell contact or soluble factors. Little evidence is currently available on potential therapeutic application of hUCMS to systemic autoimmune disorders such as primary SS (pSS). We have recently developed an endotoxin-free alginate gel that can be used to microencapsulate different cell types for graft into non-immunosuppressed hosts. We aimed to assess the in vitro effects of IFN-γ-pretreated microencapsulated (CpS)-hUCMS on T cells of pSS. METHODS Ten pSS patients and 10 healthy donors were selected. Peripheral blood mononuclear cells (PBMCs) were obtained from venous blood to establish co-cultures with CpS-hUCMS. Lymphocyte proliferation and phenotypic analysis was performed by flow cytometry and real-time PCR on IFN-γ-pretreated hUCMS was performed before PBMCs co-culture. RESULTS We found that CpS-hUCMS suppress pSS T cell proliferation and restore the Treg/Th17 ratio, thereby possibly positively impacting the pSS disease process. CONCLUSION We have developed a new biohybrid drug delivery system that now waits for clinical application in autoimmune diseases, including pSS.

Restoration of t cell substes of patients with type 1 diabetes mellitus by microencapsulated human umbilical cord Wharton jelly-derived mesenchymal stem cells: An in vitro study

Human umbilical cord Wharton jelly-derived mesenchymal stem cells (hUCMS) might apply to treating chronic autoimmune disorders, as already shown for Sjögrens syndrome, including type 1 diabetes mellitus (T1D). Since naked hUCMS grafts encountered restraints, we enveloped hUCMS, within immunoisolatory microcapsules (CpS-hUCMS), made of our endotoxin-free, clinical grade alginate. We then examined the vitro effects of interferon (IFN)-γ-pretreated CpS-hUCMS on Th17 and Treg of T1D patients (n=15) and healthy controls (n=10). Peripheral blood mononuclear cells (PBMCs) were co-cultured with PBMC/CpS-hUCMS: lymphocyte proliferation was assessed by carboxyfluorescein succinimidyl esther (CFSE) dilution assay, and phenotypic analysis of regulatory and effector Tc was also performed. Cytokine expression was performed by bead array and qPCR on IFN-γ-pretreated hUCMS before PBMCs co-culture. CpS-hUCMS restored a correct Treg/Th17 ratio, relevant to the T1D disease process. In summary, we have preliminarily developed a new biohybrid system, associated with immunoregulatory properties, that is ready for in vivo application.

Functional Profiles of Human Umbilical Cord-Derived Adult Mesenchymal Stem Cells in Obese/Diabetic Versus Healthy Women

BACKGROUND Adult human mesenchymal stem cells retrieved, from the post-partum human umbilical cord Wharton jelly (hUCMS), have recently gained growing interest due to their morphological and functional properties. OBJECTIVE The main purpose of our work was to examine morphology and functional properties of hUCMS retrieved from healthy women as compared to those with obesity, or gestational or type 2 diabetes mellitus, under fair metabolic control. Possible differences between groups could shed light into the potential use of these cells for the cell therapy of a variety of diseases, regardless of the obesity/diabetes status of the donor mothers. Additionally, information on how the maternal disease may affect the cord-derived stem cells, hence possibly newborn children would be important. METHOD We have studied obese/diabetic or normal donor post-partum umbilical cord-derived hUCMS, either in basal or during differentiation protocols into several cell phenotypes and the definitive endoderm. Immunomodulatory properties of these cells, in terms of inhibition of activated lymphocyte proliferation, also was examined. RESULTS According to our preliminary results, there are functional differences, as assessed by cell and molecular assays, in terms of both, differentiation and immunomodulatory potential, between the cells derived from normal as compared to obese/diabetic mothers. CONCLUSION The findings seemingly indicate that the uterine environment of obese/diabetic mothers is quite distant from normal, regardless of metabolic control. Hence hUCMS extracted from obese/diabetic mothers do not appear to be suitable for cell therapy clinical protocols but more studies are required.

Human Umbilical Cord Wharton Jelly-Derived Adult Mesenchymal Stem Cells, in Biohybrid Scaffolds, for Experimental Skin Regeneration

The ultimate goal for skin tissue engineering is to regenerate skin lesions to allow the full restoration of morphological and functional properties as what they were before injury. To this end, we have assembled a new prototype of a biomimetic human umbilical cord adult mesenchymal stem cell (hUCMS)/fibrin-based scaffold. We have fully characterized the proposed dermal equivalent (DE) in vitro, to assess morphological, functional, and biological properties of the encased cells. We transplanted DE subcutaneously into immunocompetent rodents, to verify its full biocompatibility. Finally, we studied DE graft effects on full-thickness wounds, in immunocompetent mice to demonstrate its capability to drive the healing process in the absence of significant scarring tissue. The excellent outcome of these in vivo studies fuels hope that this new approach, based on a biohybrid DE, may be applied to the operative treatment of skin lesions (i.e., diabetic foot ulcers and burns) in man.

A8.6 A novel therapeutic approach in systemic rheumatic autoimmune disorders: encapsulated human umbilical cord wharton jelly-derived mesenchymal stem cells

Background and objectives hUCMS are adult stem cells able to exert immune-modulatory either via cell-cell contact or by the secretion of soluble mediators including indoleamine 2–3 dioxygenase (IDO) and HLA-G. We recently developed an endotoxin-free alginate matrix which can be used to microencapsulate (CpS) different cell types and grafted into a non-immunosuppressed host. We previously performed a phase I trial grafting encapsulated pancreatic islets in patients with type 1 diabetes (T1D). To date, data concerning therapeutic application of hUCMS in systemic and organ-specific autoimmune disorders are rather scarce and controversial. In addition, grafting procedures of free hUCMS are limited by safety issues. In this study we aimed to assess the in vitro immune-modulatory effects of CpS-hUCMS on T cells isolated from patients with systemic lupus erythematosus (SLE) and Sjögren’s syndrome (pSS). Methods Peripheral blood mononuclear cells (PBMCs) were isolated from SLE and pSS patients and from healthy donors (HD) to establish co-cultures with CpS-hUCMS at different ratios. Lymphocyte proliferation and phenotypic analysis before and after culture was performed by flow cytometry and real-time PCR. Phenotypic analysis of hUCMS was performed by real time PCR and western blotting. Results CpS-hUCMS were able to inhibit pSS and HD PBMC cell proliferation but not SLE PBMC cell proliferation. Of interest, in pSS and HD such inhibition was even more evident by progressively reducing hUCMS in culture. In pSS CpS-hUCMS inhibited Th1 and Th17 arm of effector T cells as well as potentiated the T regulatory cell counterpart. The evaluation of soluble molecules produced by hUCMS in such a system revealed a peculiar milieu responsible for the phenotypic and functional effects exerted on pSS PBMCs. Additional studies to understand the lack of efficacy in SLE and design different in vitro systems are currently ongoing. Conclusion Since CpS-hUCMS appear to rebalance Treg/Th17 ratio in pSS, this may suggest possible therapeutic effects by using this technology in vivo.

SAT0187 Immunomodulatory effects of human umbilical cord wharton jelly-derived mesenchymal stem cells (HUCMS) on circulating T-cell subsets in patients with sjögren’s syndrome

Background hUCMS are adult stem cells easy to retrieve in bulk. Their immunomodulatory and pro-differentiation properties has been demonstrated. Indeed they may suppress lymphocyte activation, thereby prolonging skin graft survival time. hUCMS may also promote differentiation of tolerizing dendritic cells which favors development of Treg. These properties seem to derive both from hUCMS-secreted soluble factors such as TGFβ and on contact with the target cell. Very few evidences are currently available regarding potential therapeutic application of hUCMS in systemic autoimmune disorders and in particular no data have been published in Sjogren’s syndrome (SS) to date. Objectives We aimed to assess hUCMS immunomodulatory in vitro effects on circulating SS-T cells. Methods hUCMS were isolated from umbilical cords according to the method that we recently developed and published (1). Five SS patients and 5 healthy donors (HD) were enrolled. Peripheral blood mononuclear cells (PBMC) were obtained by density gradient from SS and HD heparinized venous blood. In selected experiments CD3+ cells were isolated from PBMC by magnetic sorting prior to arrange cultures. Fluorescein diacetate and ethidium bromide and trypan blue dye exclusion assays were performed to assess cell viability. Lymphocytes proliferation was assessed by CFSE diluition assay and culture supernatants were tested for IL-17 with commercial ELISA kit. Intracellular IL-17 was performed by flow cytometry following 6 hours stimulation with PMA, ionomycin and brefeldin. Results hUCMS and PBMC were successfully co-cultured in CMRL 1066 medium with 10% fetal bovine serum (FBS). When PBMC were used, cell proliferation was dramatically inhibited by hUCMS when co-culture was performed in contact. Interestingly, the higher was PBMC:hUCMS ratio the stronger was such inhibition. IL-17 concentration in supernatants and the percentage of CD4+IL17+ cells were significantly reduced by hUCMS. When magnetic sorted CD3+ cells were used, the proliferation rate was not affected and the effect on IL-17 concentration in supernatants and the percentage of CD4+IL17+ cells was partially lost. Co-cultures in transwell and with microencapsulated hUCMS are currently ongoing. Conclusions Our preliminary results suggest possible therapeutic applications of hUCMS by selectively targeting Th17 pathogenic cells in SS. References Montanucci P et al. Tissue Eng Part A 2011 Montanucci P. et al. Biomaterials 2011;32:9254 Disclosure of Interest None Declared

A9.18 Potential Therapeutic Application of Human Umbilical Cord wharton Jelly Derived Mesenchymal Stem Cells in Primary Sjögren’s Syndrome

Background and Objectives hUCMS are adult stem cells easy to retrieve in bulk, under acceptable ethical conditions. Their immune-modulatory and pro-differentiation properties have been widely demonstrated. Modulation of the immune system is mediated by both cell contact and soluble factors such as interferon (IFN)-γ produced by hUCMS. Very few evidence is currently available with respect to potential therapeutic application of hUCMS in systemic autoimmune disorders. In particular, no data have been published in primary Sjögren’s syndrome (pSS) to date. Furthermore, we have recently developed an endotoxin-free alginate matrix which can be used to microencapsulate (CpS) different cell types and transfer them into a non-immunosuppressed host. These microcapsules containing pancreatic islets, upon approval by the Italian Ministry of Health for in vivo use, have been grafted into a cohort of patients with type 1 diabetes mellitus with no adverse effects, while proving to be immunoprotective. On this background, we aimed to assess the in vitro immune system modulation by IFN-γ pretreated CpS-hUCMS on T cells from pSS, with special regard to Th17- and Treg-cell subsets. Materials and Methods Ten pSS patients and 5 healthy donors (HD) were enrolled. Peripheral blood mononuclear cells (PBMC) were obtained by density gradient from heparinised venous blood. Co-cultures of CpS-hUCMS and PBMCs were arranged at different ratios. Lymphocyte proliferation was assessed by CFSE dilution assay. Phenotypic analysis by flow cytometry for regulatory and effector T cell subpopulations was performed after culture. Real time PCR analysis and the evaluation of culture supernatants for cytokine expression are currently ongoing. Results CpS-hUCMS were able to inhibit HD and pSS PBMC cell proliferation and this effect was inversely correlated to hUCMS number. Phenotypic analysis revealed a predominant Th17-cell response in SS which was promptly hampered by CpS-hUCMS Moreover, Th1-cell proliferation was fair and reduced Cps-hUCMS FoxP3 and IL-17 expression among CD4+ T cells was also modulated by CpS-hUCMS, thereby suggesting that a Treg/Th17 rebalance. Conclusions This is the first study evaluating the effects of hUCMSS on T cells in pSS. It appeared that CpS-hUCMS rebalance Treg/Th17 ratio and therefore may exert therapeutic effects in such disease. Furthermore, it is the first study that employs a new technology of drug delivery which may be applied in vivo in pSS patients.