Human Amniotic Epithelial Cells (hAEC)- a cellular therapy for inflammatory diseases?

Abstract

s / Placenta 32 (2011) S326–S340 S330 PLX-PAD cells are derived from the decidua of human placenta and are expanded using the company's 3D proprietary technology. The cells are characterized as CD73, CD90 CD29, CD105 positive and CD34, CD45, CD19 and CD14 negative. Analyses of the expression of stimulatory and costimulatory molecules on the surface of the PLX-PAD cells demonstrate the absence of HLA class II (HLA-DR), CD80, CD86 and CD40, supporting our findings that these cells are immunoprivileged. Accumulating data strongly suggest that PLX-PAD anti inflammatory and angiogenic properties are largely mediated via paracrine effects. PLX-PAD based cell therapy for CLI offers an opportunity for patients that have exhausted all currently available interventions. Two phase I, openlabel, dose-escalation studies, intended for the treatment of CLI were initiated in parallel in the EU and U.S. Enrollment was completed in both trials. All together 27 patients were administered by 30-50 intramuscular injections in the effected limb. Summary obtained from the 3 months clinical follow up period demonstrates that trials have currentlymet their primary safety endpoints defined as absence of adverse events as well as safe laboratory values, ECG findings and no adverse immunological reaction. Immunological data, presents no evidence for PLX-specific humoral or T-cell allosensitization, confirming the low immunogenicity of HLA unmatched allogeneic PLX cells. Analysis of efficacy parameters revealed a statistically significant improvement from base line in the Ankle-Brachial Index (ABI) (P1⁄40.033) in patients who received the intermediate dose, and in Transcutaneous Oxygen Tension (TcPO2) (P1⁄40.05) in the European study, as well as in patients’ quality of life Quality of Life (QoL) (P< 0.001) across all doses. Our data offers extensive immunological evaluation of mesenchymal-like cell therapy in CLI clinical trials. Moreover, such data may have broad implications on allogeneic cell therapy in other disorders. doi:10.1016/j.placenta.2011.07.042 HUMAN AMNIOTIC EPITHELIAL CELLS (HAEC)A CELLULAR THERAPY FOR INFLAMMATORY DISEASES? V. Vaghjiani , J. Tee , D. Arasaratnam , T. Lui , D. Lourenz , J. Tchongue , J. Chan , W. Sievert , Y. Moodley , U. Manuelpillai a Monash Institute of Medical Research, Monash University, Clayton, Australia; b Dept. of Medicine, Monash University, Clayton, Australia; c Lung Research Institute, University of Western Australia, Perth, Australia Chronic inflammation in organs such as the lung and liver arise fromdiverse stimuli including pathogens, environmental, lifestyle and genetic factors. Ongoing inflammation leads to cell death and reduced organ function and to enhanced collagen deposition (fibrosis) arising from aberrant repair mechanisms. Liver cirrhosis and lung fibrosis are amongst the leading causes of morbidity and mortality and are major burdens on healthcare systems worldwide. Due to drawbacks in conventional therapies, stem cell based approaches targeting cell replacement, tissue regeneration and inflammation and fibrosis are being widely tested in animal models mimicking these diseases. Using bleomycin and carbon tetrachloride (CCl4) to induce lung and liver inflammation/fibrosis, respectively, we investigated the effects of hAEC transplantation in mice. hAEC were isolated from amnion membranes of term delivered placentae. Mice with lung injury were injectedwith 1x106 hAEC, whilemicewith liver injury received 2x106 cells, intravenously. hAECwere found to engraft in higher numbers in lungs compared with mesenchymal stromal cells (MSC) from Wharton's jelly of the umbilical cord and were retained for longer periods in mice given bleomycin. The engrafted hAEC were recovered from the murine lung cell suspensions using CD29 FACS. The recovered hAEC were found to produce Surfactant Proteins A-D suggesting that hAEC had transdifferentiated into type II alveolar epithelial cells in the mouse lung. Mice treated with hAEC showed reduced apoptosis, decreased levels of pro-inflammatory and profibrogenic cytokines in association with elevated levels of matrix metalloproteinases and significant remodelling in the damaged lungs and livers. In mice with CCl4 induced injury, we explored whether immune cells numbers and hence inflammationwas reduced with hAEC transplantation. Human Inner Mitochondrial Membrane+ hAEC were localized in close proximity to CD3+ T-cells without evidence of host immune response in immune competent C57/Bl6 mice exposed chronically to CCl4. T-cell numbers in livers remained unaltered between mice given CCl4+hAEC and CCl4 alone, however FoxP3+ regulatory T-cells were elevated in mice receiving cells. hAEC may escape detection by T-cells due to the absence of MHC Class II, CD80/86 and low levels of MHC Class IA and CD40 expression. In vitro studies showed that hAEC secreted the immunosuppressive factors TGFb, HLA-G, HGF and showed IDO activity. These secreted factors are known to modulate T-cell and also dendritic and NK cell activity. The numbers of F4/80+ resident liver macrophages, the Kupffer cells (KC), were significantly lower in CCl4+hAEC treated mice compared to CCl4 controls. MIF-1 secreted by hAECmay inhibit KC recruitment. Factors released by KC have been shown to activate hepatic stellate cells (HSC) into pro-fibrogenic, collagendepositingmyofibroblasts. ActivatedHSCare also amajor source of inflammatory cytokines and MCP-1 that serves to recruit KC, perpetuate inflammation and lead to fibrosis. In vitro studies showed however that factors secreted by hAEC stimulated MCP-1 release by HSC, but reduced collagen levels secreted by the HSC. Collectively, these preliminary findings suggest that hAEC transplantation may be useful for targeting tissue inflammation, fibrosis and repair and that in some organs such as lungmay also contribute to cell replacement. doi:10.1016/j.placenta.2011.07.043 PHYSIOLOGICAL COMPARISON OF AUTOLOGOUS AND ALLOGENEIC CELL IMPLANTATION IN THE CENTRAL NERVOUS SYSTEM: DEFINING AND REGULATING IMMUNE CELL ACTIVITY AGAINST MESENCHYMAL AND NEURAL STEM CELL GRAFTS P. Ponsaerts Laboratory of Experimental Hematology, Vaccine and Infectious Disease Institute (Vaxinfectio), University of Antwerp, Belgium Initial cell therapy studies by our group focussed on the direct intra-spinal implantation of human mesenchymal stem cells (MSC) genetically engineered to secrete neurotrophic factors as a therapeutic tool to treat spinal cord injury in rats. Although in these studies we were unable to demonstrate any therapeutic benefit for the chosen therapy, it became clear to us that – before further cell therapy studies could be initiated – several intermediate steps needed to be optimised, among them: (i) in vitro culture, characterisation and genetic modification of different stem cell types, (ii) pre-mortem bioluminescence (BLI) and magnetic resonance imaging techniques for real-timemonitoring of cell graft localisation and survival in vivo, and (iii) post-mortem multi-colour histological analysis for unambiguous characterisation of grafted cells and surrounding tissue. Studies performed during the past 3 years therefore mainly focussed on determining the survival, differentiation and immunogenicity of autologous and allogeneic cellular implants in the CNS of immune-competent mice. Murine bone marrow-derived MSC and embryonic (E14) brain-derived neural stem cells (NSC) were cultured from FVB-Luciferase transgenic mice and further genetically engineered with the eGFP reporter gene. Following transplantation in non-injured CNS of syngeneic FVB mice and allogeneic BALB/c or C57BL/6 mice, cell survival was monitored pre-mortem using in vivo BLI (luciferase reporter gene) and post-mortem by histological analysis (eGFP reporter gene). Additional histological analysis: (i) for MSC and NSCspecific markers (respectively Sca1 and GFAP in combination with eGFP) confirmedcell graft identity, (ii) fordifferent immunecellmarkers confirmed the presence or absence of Iba1+CD11b+/microglia, CD4+ or CD8+ T-cells and NK-cells surrounding or infiltrating cell grafts, and (iii) for GFAP+ astrocytes confirmed the presence or absence of endogenous astrocytic scar tissue surrounding or infiltrating cell grafts. Finally, ex vivo ELISPOT assay was performed to identify allograft-specific T-cell responses in the CNS. Autologous transplantation of FVB-derived MSC resulted in graft survival of at least 4 weeks as demonstrated by in vivo BLI and histological analysis. However, MSC grafts became highly infiltrated by Iba1+CD11bmicroglia and encapsulated by GFAP+ astrocytic scar tissue. Allogeneic transplantation of FVB-derived MSC in BALB/c or C57BL/6 mice resulted in graft rejection from 2 weeks post-transplantation as demonstrated by in vivo BLI and histological analysis. The immune-based rejection of MSC allografts was solely mediated by highly activated Iba1+CD11b+ microglia