Human Amniotic Mesenchymal Cell Secretome for Neurodegeneration and Neuroinflammation
NCT Number: NCT06551649
Acronym: CONTRASTOME
Status: NOT_YET_RECRUITING
Study Type: INTERVENTIONAL
Phases: NA
Conditions: Amyotrophic Lateral Sclerosis|Multiple Sclerosis
Interventions: OTHER: Venous blood draw and skin biopsy
Sponsor: Fondazione Policlinico Universitario Agostino Gemelli IRCCS
Sex: ALL
Age: ADULT
Enrollment: 60
Study Design: Allocation: NON_RANDOMIZED|Intervention Model: PARALLEL|Masking: NONE|Primary Purpose: BASIC_SCIENCE
Start Date: 2024-09-01
Completion Date: 2026-09-30
Locations: Fondazione Policlinico Universitario A. Gemelli IRCCS, Roma, 00168, Italy

Summary:
Neurodegenerative diseases are debilitating conditions characterized by chronic inflammation, leading to dysfunction of both the non-neuronal cellular components of the central nervous system and peripheral blood immune cells. Thus, it is crucial to develop an innovative therapeutic strategy that not only effectively contrast neurodegeneration but also aims to reduce inflammation.

The overall aim of the study is to provide a preclinical in vitro demonstration of the immunomodulatory and pro-regenerative potential of the human amniotic mesenchymal stromal cell (hAMSC) secretome in counteracting neurodegeneration.

This potential will be evaluated in three-dimensional in vitro models of neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and chronic demyelinating disease (multiple sclerosis - MS). To this end, the study includes sample collection from patients without pharmacological treatment and without medical devices. Patients diagnosed with ALS, patients diagnosed with MS, and healthy volunteers will be recruited to collect blood samples and skin biopsies. Patient-specific and control organoid platforms, mimicking cellular heterogeneity and tridimensional interactions within the central nervous system including the inflammatory compartment, will be developed to be used as a valuable tool to investigate the in vitro efficacy of the hAMSC secretome.

Primary Outcome Measures: Neurite length measurement in a three-dimensional cellular model of amyotrophic lateral sclerosis., Organoids from induced pluripotent stem cells (iPSCs) isolated from skin biopsies of 6 amyotrophic lateral sclerosis (ALS) patients and 6 healthy controls will be generated. Neurite degeneration will be measured in fluorescence microscopy as total βIII-TUBULIN-positive fiber length divided by the number of βIII-TUBULIN-positive cells in micrometers. Measurements will be taken from at least 5 ALS and 5 control organoids to analyze correlations and differences between groups. The significance level for statistical tests and models is set at 0.05., 18 months|Percentage of apoptotic cells in a three-dimensional cellular model of amyotrophic lateral sclerosis., Organoids from induced pluripotent stem cells (iPSCs) isolated from skin biopsies of 6 amyotrophic lateral sclerosis (ALS) patients and 6 healthy controls will be generated. Apoptosis will be assessed by the percentage of propidium iodide-positive cells, representing the proportion of dead cells relative to the total number of cells. Measurements will be taken from at least 5 ALS and 5 control organoids to analyze correlations and differences between groups. The significance level for statistical tests and models is set at 0.05., 18 months|Area measurement of damaged myelin in a three-dimensional cellular model of multiple sclerosis., Organoids from induced pluripotent stem cells (iPSCs) isolated from skin biopsies of 6 multiple sclerosis (MS) patients and 6 healthy controls will be generated. Myelin sheath damage will be assessed via immunofluorescence as the reduction in myelin marker-stained area versus total section area. Measurements will be taken from at least 5 MS and 5 control organoids to analyze correlations and differences between groups. The significance level for statistical tests and models is set at 0.05., 18 months|Number of inflamed microglial cells in a three-dimensional cellular model of multiple sclerosis., Organoids from induced pluripotent stem cells (iPSCs) isolated from skin biopsies of 6 multiple sclerosis (MS) patients and 6 healthy controls will be generated. Microglial activation will be assessed by morphological variations, classifying cells as homeostatic, ramified, hyper-ramified, or amoeboid. Inflammatory microglia increase will be defined as the proportion of activated morphology cells relative to total cell count. Inflammatory markers HLA-DR and iNOS will be evaluated in terms of CD68 and IBA1 positive cells and fluorescence intensity. Measurements will be taken from at least 5 MS and 5 control organoids to analyze correlations and differences between groups. The significance level for statistical tests and models is set at 0.05., 18 months|Neurite length measurement in amyotrophic lateral sclerosis organoids after treatment with human amniotic mesenchymal stromal cell secretome., Neurite length in amyotrophic lateral sclerosis (ALS)-derived organoids treated with human amniotic mesenchymal stromal cell (hAMSC) secretome will be quantified using fluorescence microscopy as the total βIII-TUBULIN-positive fiber length divided by the number of βIII-TUBULIN-positive cells. The ability of the hAMSC secretome to restore ALS organoids (at least 5) to values close to healthy-donors organoids (at least 5) will be evaluated. The significance level is set at 0.05., 24 months|Percentage of apoptotic cells in amyotrophic lateral sclerosis organoids after treatment with human amniotic mesenchymal stromal cell secretome., Apoptosis will be measured by the reduction in propidium iodide-positive cells, expressed as a percentage of the total cells, in amyotrophic lateral sclerosis (ALS)-derived organoids treated with human amniotic mesenchymal stromal cell (hAMSC) secretome. The ability of the hAMSC secretome to restore ALS organoids (at least 5) to values close to healthy-donors organoids (at least 5) will be evaluated. The significance level is set at 0.05., 24 months|Area measurement of damaged myelin in multiple sclerosis organoids after treatment with human amniotic mesenchymal stromal cell secretome., Reduction of demyelinated areas in multiple sclerosis (MS)-derived organoids treated with human amniotic mesenchymal stromal cell (hAMSC) secretome will be measured by the increase in mature oligodendrocyte markers relative to total section area. The ability of the hAMSC secretome to restore MS organoids (at least 5) to values close to healthy-donors organoids (at least 5) will be evaluated. The significance level is set at 0.05., 24 months|Number of immunoregulatory microglial cells in multiple sclerosis organoids after treatment with human amniotic mesenchymal stromal cell secretome., The acquisition of an immunoregulatory phenotype will be determined in multiple sclerosis (MS)-derived organoids after treatment with human amniotic mesenchymal stromal cell (hAMSC) secretome by analyzing cells that are iNOS-negative, Arginase-positive, and HLA-DR low. Immunofluorescence analysis will count the number of cells positive for inflammatory/immunoregulatory markers out of total microglial IBA1+ cells. Fluorescence intensity changes will also be evaluated. The ability of the hAMSC secretome to restore MS organoids (at least 5) to values close to healthy-donors organoids (at least 5) will be evaluated. The significance level is set at 0.05., 24 months
Secondary Outcome Measures: Percentage of altered immune phenotypic markers in amyotrophic lateral sclerosis and multiple sclerosis patients, The altered immune phenotype of peripheral blood mononuclear cells (PBMC) from patients with amyotrophic lateral sclerosis (ALS, n=15) and multiple sclerosis (MS, n=15) will be compared to age- and gender-matched healthy controls PBMC (n=30). Flow cytometry will be used to identify alteration in T and B lymphocytes, and monocytes and their subsets, including naïve and memory subsets, senescence status, expression of activation markers, and costimulatory molecules. The percentage of more than 40 markers will be considered and positive expression for each marker will be recorded. Immunophenotypic variation between patient PBMC and healthy control PBMC will be considered statistically significant with a p value \< 0,05., 18 months