Phenotypical/functional characterization of in vitro-expanded mesenchymal stromal cells from patients with Crohn's disease

Background aims

Because of their capacity to modulate the immune response and promote tissue repair, mesenchymal stromal cells (MSC) represent a potential novel treatment for autoimmune/inflammatory diseases, including Crohn's disease (CD). The aim of the study was in vitro characterization of MSC from active CD patients for future clinical application.

Methods

MSC from the bone marrow (BM) of seven CD patients (median age 32 years) were expanded ex vivo in the presence of 5% platelet lysate; cells were investigated for clonogenic efficiency, proliferative capacity, morphology, immunophenotype, differentiation potential, genetic stability and ability to suppress in vitro proliferation of both autologous and allogeneic lymphocytes to polyclonal mitogens. Results were compared with those of BM MSC of four healthy donors (HD).

Results

MSC were successfully expanded from all patients. Colony-forming unit–fibroblast (CFU-F) frequency and proliferative capacity were comparable in CD and HD MSC. CD MSC showed typical spindle-shaped morphology and differentiated into osteoblasts, adipocytes and chondrocytes. Surface immunologic markers did not differ between CD and HD MSC, with the only exception of sizeable levels of HLA-DR at early culture passages [12–84% at passage (P)1] in the former. CD MSC ceased their growth at variable passages (from P8 to P25) and entered senescence without any change in morphology/proliferation rate. Array-comparative genomic hybridization demonstrated that CD MSC do not show imbalanced chromosomal rearrangements. Both CD and HD MSC inhibited in vitro proliferation of lymphocytes to mitogens.

Conclusions

CD MSC show biologic characteristics similar to HD MSC and can be considered for anti-inflammatory and reparative cell therapy approaches in patients with refractory disease.

Introduction

Within the bone marrow (BM) microenvironment, multipotent stromal cells, also referred to as mesenchymal stromal cells (MSC), are known to be precursor cells for stromal tissues that support hematopoiesis [1,2]. For many years, MSC have been considered to be mainly a component of marrow stroma, without any special function, and endowed only with structural support properties. It is now clear that MSC provide a substantial contribution to the creation of the hematopoietic stem cell (HSC) niche and play a crucial role in the development and differentiation of the lymphohematopoietic system by secreting a number of growth factors and regulatory cytokines, and by promoting cell-to-cell interactions [3., 4., 5.].

After the initial identification in post-natal BM [1], MSC have been isolated from a variety of other human tissues, including muscle connective tissue, adipose tissue, fetal tissue, placenta and umbilical cord blood (UCB) [6., 7., 8., 9., 10.]. The isolation and characterization of MSC rely on their preference to proliferate while attached to a substrate, ability to differentiate into the various mesenchymal differentiation lineages and expression or absence of a number of surface molecules on culture-expanded cells [11]. MSC possess unique immunologic properties that are displayed on all cells involved in the immune response, including T and B lymphocytes, dendritic cells and natural killer (NK) cells, as demonstrated by several independent groups both in vitro and in vivo [12., 13., 14., 15., 16.]. Based on these biologic and functional properties, MSC have already been employed successfully in the clinical setting, to either enhance hematopoietic stem cell engraftment [17,18] or treat the most severe form of acute graft versus host disease (GvHD) refractory to conventional treatments [19,20].

Moreover, MSC have been demonstrated to display chemotactic ability, migrate to sites of inflammation and injury [21] and secrete paracrine mediators able to reverse acute organ failure [22]. Indeed, MSC infusions have been used successfully in repairing tissue injury secondary to allogeneic hematopoietic stem cell transplantation (HSCT) [23]. Based on these findings, MSC can be considered a novel treatment for inflammatory diseases, where cell loss is accompanied by local and systemic inflammatory response.

Crohn's disease (CD) is a chronic inflammatory entero-pathy, the prevalence of which in western countries has dramatically increased in the last decade [24] and in which a dysregulation of the immune response toward intestinal bacteria in genetically susceptible individuals plays a pathogenetic role [25]. Despite the large number of therapeutic options available, i.e. anti-inflammatory drugs, antibiotics, immunosuppressant drugs, biologic agents and surgical strategies [26], there is a growing number of CD patients with refractory/recurrent disease. In view of this consideration and the serious side-effects of more aggressive therapies, alternative strategies are needed to both increase the proportion of patients achieving remission and improve their quality of life.

Very recently, the topical implantation of BM-derived MSC has been demonstrated to be beneficial in the healing process of experimental colitis in rats, confirming the ability of MSC to modulate immune responses and promote tissue repair through their trophic activity [27]. Moreover, a phase I clinical trial for the treatment of perianal fistulas in four CD patients with autologous, adipose tissue-derived MSC has been reported with promising results [28].

We are currently investigating the potential role of autologous BM-derived MSC as novel, anti-inflammatory cellular therapy to stimulate tissue repair in CD patients. However, so far no experimental data have been obtained on the biologic and functional characterization of BM-derived MSC from these patients. The aim of this study, therefore, was to evaluate the feasibility of isolating and expanding ex vivo MSC from BM of CD patients with active disease, and carry out a phenotypical and functional characterization of these cells in comparison with BM MSC isolated from healthy subjects. With future clinical use in mind, in order to avoid any risk associated with the use of fetal calf serum (FCS), platelet lysate (PL; 5%) was employed as culture supplement to stimulate MSC growth [29].