Phenotypical and Functional Characterization of in Vitro Expanded Bone Marrow-Derived Mesenchymal Stromal Cells from Patients with Crohn’s Disease
Authors: Bernardo Maria Ester , Avanzini Maria Antonietta , Ciccocioppo Rachele , Perotti Cesare , Cometa Angela Maria , Moretta Antonia , Valli Monica , Novara Francesca , Maccario Rita , Corazza Gino Roberto , Locatelli Franco 
Published: April 2020

Abstract
Mesenchymal stromal cells (MSCs) are multipotent progenitors, capable to differentiate into several mesenchymal lineages, such as osteoblasts, adipocytes, and chondrocytes. Thanks to their capacity to modulate immune responses and promote tissue repair, MSCs are considered a potential novel treatment for autoimmune and inflammatory diseases, including Crohn’s disease (CD). So far, the biological and functional properties of MSCs derived from bone marrow (BM) of CD patients have not been characterized. Aim of this study was to evaluate the feasibility of isolating and expanding ex vivo MSCs from BM of 7 patients with active CD (CD-MSCs; 5 males, median age 32 years, range 18–59), as well as to characterize these cells for their clonogenic efficiency, proliferative capacity, morphology, immunophenotype by flow cytometry, differentiation potential into osteoblasts and adipocytes, biosafety and ability to suppress in vitro the proliferation of autologous and allogeneic peripheral blood mononuclear cells (PBMCs). The properties of CD-MSCs were compared with those of BM-MSCs isolated from 4 healthy donors (HD-MSCs; 2 males, median age 33 years, range 16–47). Platelet lysate (PL, 5%) was employed as culture supplement to stimulate MSC growth. MSCs were successfully isolated and expanded ex vivo from BM of all 7 CD patients. The colony-forming unit-fibroblast (CFU-F) frequency after 10-day culture and the proliferative capacity were comparable in CD- and HD-MSCs. CD-MSCs showed the typical spindle-shaped morphology and ability to differentiate into osteoblasts and adipocytes. As regards the immunophenotype, CD-MSCs displayed the characteristic panel of surface markers (positivity for CD90, CD73, CD105, HLA A-B-C and negativity for CD34, CD45, CD14, CD80, CD31 molecules), with the exception of the expression of variable levels of HLA-DR at early passages (P1–P3) in culture. MSCs of 5 CD patients were propagated in long-term in vitro culture. CD-MSCs ceased their growth at variable passages (from P7 to P24) and entered a senescence phase. Senescent CD-MSCs were monitored for up to 8 weeks, without showing any change in morphology and/or proliferation rate. Results of array-Comparative Genomic Hybridization (array-CGH) demonstrated that CD-MSCs expanded in vitro do not show imbalanced chromosomal rearrangements. CD-MSCs were able to reduce in vitro PHA- and OKT3-stimulated proliferation of autologous PBMCs by up to 40% and 35%, respectively. The same degree of inhibition was observed when HD-MSCs were tested against both HD-PBMCs and CD-PBMCs. MSCs can be easily isolated and expanded ex vivo from BM of CD patients in the presence of a PL-auditioned medium; these cells exhibit similar morphological, phenotypic, and functional properties to those of HD-MSCs. These cells maintain a normal genetic asset after extensive ex vivo culture, as demonstrated by array-CGH experiments. The variable expression of HLA-DR in CD-MSCs at early passages might be related to the state of systemic inflammation of CD patients with active disease. In view of these results, autologous CD-MSCs can be considered as innovative, potentially anti-inflammatory, and reparative strategy for cell-therapy approaches in CD patients’ refractory to conventional treatments.