J Mol Neurosci. 2013;51(2):282-97. doi: 10.1007/s12031-013-9992-9. Epub 2013 Mar 27.

Characterization of in vitro expanded bone marrow-derived mesenchymal stem cells isolated from experimental autoimmune encephalomyelitis mice.

Zacharaki D, Lagoudaki R, Touloumi O, Kotta K, Voultsiadou A, Poulatsidou KN, Lourbopoulos A, Hadjigeorgiou G, Dardiotis E, Karacostas D, Grigoriadis N.

Laboratory of Experimental Neurology and Neuroimmunology, 2nd Department of Neurology, AHEPA University Hospital, Aristotle University of Thessaloniki, 1 Stilp Kyriakidi str, 54636, Thessaloniki, Macedonia, Greece.

 

Abstract

Extensive experimental studies indicate that autologous bone marrow mesenchymal stem cells (BMSCs) are able to ameliorate experimental autoimmune encephalomyelitis (EAE) and potentially multiple sclerosis. However, the impact that the inflammatory environment present in EAE may have on the biological properties of BMSCs expanded in vitro for transplantation is yet to be clarified. It was investigated whether BMSCs isolated from EAE-induced C57bl6/J mice and expanded in vitro preserve the properties of BMSCs isolated from healthy donors (BMSCs-control). The mesenchymal origin, the differentiation potential, and the transcriptional expression profile of six histone-modifying genes were studied in both groups of BMSCs. BMSCs-EAE exhibited distinct morphology and larger size compared to BMSCs-control, higher degree of proliferation and apoptosis, differences in the adipogenesis and the osteogenesis induction, and differential expression of stromal markers and markers of progenitor and mature neuronal/glial cells. Moreover, BMSCs-EAE exhibited different expression patterns on a number of histone-modifying genes compared to controls. We recorded manifold differences, both phenotypical and functional, of in vitro expanded BMSCs-EAE in comparison to their healthy donor-derived counterparts that may be attributed to the inflammatory environment they originated from. Whether our findings may be of any clinical relevance needs to be clarified in future studies, in vivo.

 

Supplements:

Multiple sclerosis (MS) is a chronic inflammatory multifocal demyelinating disease of the central nervous system (CNS). MS affects mainly young adults and to date the treatment with immunosuppressive and immunomodulatory drugs only partly delays the progress of the disease [1]. In addition, during the last decade there has been a considerable focus on promising stem cell-based therapies. The bone marrow mesenchymal stem cells (BM-MSC) represent one such source considered for transplantation in MS.

BM-MSC are distinct non-hematopoietic cells forming part of the bone marrow (BM) microenvironment (representing 0.01–0.001% of total bone marrow cells). BM-MSCs display a high differentiation potential towards cells of mesenchymal origin such as bone, cartilage, and muscle as well as they were earlier reported to transdifferentiate into almost any cell type of the three lineages. BM-MSC’s strong candidacy as therapeutic agent arises from the fact that not only can they be relatively easily isolated from the adult BM and expanded in vitro for several passages; they are also considered poorly immunogenic and able to possess immunomodulatory properties.

The therapeutic potential of murine BM-MSC in autoimmune demyelination has been examined following autologous cell transplantation [2, 3-4] or with cells originating from other species such as humans [5] in experimental autoimmune encephalomyelitis (EAE), the animal model of MS. It has already been shown that BM-MSC have a beneficial effect in the clinical course of EAE by means of immunomodulation and/or engraftment in the CNS [2, 3-5]. Briefly, they exert immunomodulatory functions, i.e. suppression of lymphocyte proliferation, modulation of B- and T-cell, dendritic, NK cells’ function through paracrine mechanisms or direct cell-to-cell contact.

Autologous transplantation of BM-MSC has been proposed as the method of choice for MS [6]. Mean doses (single or multiple) reported so far vary from 2 x 106-30 x 106/kg. In this concept, BM-MSC derived from the patient destined to be transplanted require multiple passages to reach adequate numbers for transplantation. Moreover, small clinical trials where autologous BM-MSC were either intrathecally [7-10] or intravenously [7, 11-12] administered in MS patients indicated the safety of these procedures. However, when experimental studies are considered, the donors of the cells were either naïve inbred animals [2, 3-4] or non-MS patients [5]. Under this view, there still may be a missing point over the beneficial effect of BM-MSC if autologous transplantation to patients with MS, is considered. Evidently, the impact that the inflammatory environment present either in MS or in EAE may have on the biological properties of BMSCs destined to be transplanted needs to be elucidated.

The properties of ex vivo expanded BM-MSC from healthy human individuals or other species, such as mice, have been well documented. However, there is a clear need to understand the original microenvironment of BM-MSC in demyelinating and/or autoimmune diseases of the CNS when an autologous setting of treatment is concerned to rule out any possibility of modified properties of BM-MSC originating from a diseased environment (though not directly affected by the disease) and subsequent ex vivo manipulation for long periods of time. To address this issue we isolated BM-MSC from EAE-induced mice and healthy syngeneic mice, expanded them for an equal number of passages and assayed their properties in vitro, both morphological and functional.

We reported that there are significant differences between EAE- and naïve- animal derived BM-MSC namely with regard to their size and shape, proliferation rate, and differentiation capacities [13]. Both EAE- and naïve- animal derived BM-MSC exhibited MSC properties of osteogenic and adipocytic differentiation though to a significantly different degree. Moreover, BM-MSC originating from EAE-induced mice exhibited different expression patterns on a number of histone modifying genes compared to controls, thereby indicating potential epigenetic changes in these cells [13]. No cytogenetic abnormalities were observed for either cell group.

Interestingly enough, some controversies exist on the potential changes of human BM-MSC isolated from MS patients when compared to those originating from healthy controls [14],[15] Moreover, no deleterious effect on the functional/biological properties of the BM-MSC isolated from EAE donors has recently been reported. Importantly enough, these cells were able to control EAE similarly as the naïve animal isolated BM-MSC did [16], thus indicating a rather controversial finding compared to ours [13].

However, careful study of the already published reports indicate that variability in BM-MSC isolation and culture protocols, the inflammatory status of MS or EAE donors when human or animal studies, respectively, are considered, may have an impact on the examined cells. In our study [13], BM-MSCs were isolated during the peak of the acute phase of EAE (20-25 days post-induction) whereas in the study of Kassis et al [16] the same cells originated from 6-9 week-old-EAE mice, ie at various time points after the induction of the disease (6-7 week-old mice), thus indicating a broad spectrum of the underlying inflammatory component of the disease. Moreover, it is of notice that the animals transplanted with naïve BM-MSCs exhibited an almost 3 day delay of onset of the disease compared to either saline or EAE-isolated – BM-MSCs. Therefore, some difference at least partial, is evident when the clinical efficacy of the transplanted cells, is considered.

Currently available data deem it necessary to further evaluate whether in vitro expanded BM-MSC from diseased individuals/animals are equivalent to healthy donors/animals-derived BM-MSC via properly designed in vivo transplantations with suitable animal models. Functional assays should focus on the BM-MSC in vivo behavior (homing, engraftment, proliferation and differentiation status) after transplantation in diseased and naïve animals. Our results together with those more recently published [16], clearly indicate the timing and consequently the level of the underlying disease activity may be crucial for the BM-MSCs biologic behavior when considered for autologous transplantation in EAE and potentially in MS.

Nikolaos Grigoriadis-fig1

Figure 1: In vitro morphological differences between control (A) and EAE (B) – isolated BMSCs with homogenous population (A) and spindle cells with long processes and multiradial cells with extensive spherical nuclei (B), respectively.

 

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