Dana Foudah

Monitoring the genomic stability of in vitro cultured rat bone-marrow-derived mesenchymal stem cells

Bone-marrow-derived mesenchymal stem cells (MSCs) are multipotent cells capable of self-renewal and differentiation into multiple cell types. Accumulating preclinical and clinical evidence indicates that MSCs are good candidates to use as cell therapy in many degenerative diseases. For MSC clinical applications, an adequate number of cells are necessary so an extensive expansion is required. However, spontaneous immortalization and malignant transformation of MSCs after culture expansion have been reported in human and mouse, while very few data are present for rat MSCs (rMSCs). In this study, we monitored the chromosomal status of rMSCs at several passages in vitro, also testing the influence of four different cell culture conditions. We first used the conventional traditional cytogenetic techniques, in order to have the opportunity to observe even minor structural abnormalities and to identify low-degree mosaic conditions. Then, a more detailed genomic analysis was conducted by array comparative genomic hybridization. We demonstrated that, irrespective of culture conditions, rMSCs manifested a markedly aneuploid karyotype and a progressive chromosomal instability in all the passages we analyzed and that they are anything but stable during in vitro culture. Despite the fact that the risk of neoplastic transformation associated with this genomic instability needs to be further addressed and considering the apparent genomic stability reported for in vitro cultured human MSCs (hMSCs), our findings underline the fact that rMSCs may not in fact be a good model for effectively exploring the full clinical therapeutic potential of hMSCs.

From cytogenomic to epigenomic profiles: monitoring the biologic behavior of in vitro cultured human bone marrow mesenchymal stem cells

IntroductionBone marrow mesenchymal stem cells (BM-MSCs) are multipotent cells that can differentiate into different cell lineages and have emerged as a promising tool for cell-targeted therapies and tissue engineering. Their use in a therapeutic context requires large-scale in vitro expansion, increasing the probability of genetic and epigenetic instabilities. Some evidence shows that an organized program of replicative senescence is triggered in human BM-MSCs (hBM-MSCs) on prolonged in vitro expansion that includes alterations in phenotype, differentiation potential, telomere length, proliferation rates, global gene-expression patterns, and DNA methylation profiles.MethodsIn this study, we monitored the chromosomal status, the biologic behavior, and the senescence state of hBM-MSCs derived from eight healthy donors at different passages during in vitro propagation. For a more complete picture, the telomere length was also monitored in five of eight donors, whereas the genomic profile was evaluated in three of eight donors by array-comparative genomic hybridization (array-CGH). Finally, an epigenomic profile was delineated and compared between early and late passages, by pooling DNA of hBM-MSCs from four donors.ResultsOur data indicate that long-term culture severely affects the characteristics of hBM-MSCs. All the observed changes (that is, enlarged morphology, decreased number of cell divisions, random loss of genomic regions, telomere shortening) might be regulated by epigenetic modifications. Gene Ontology analysis revealed that specific biologic processes of hBM-MSCs are affected by variations in DNA methylation from early to late passages.ConclusionsBecause we revealed a significant decrease in DNA methylation levels in hBM-MSCs during long-term culture, it is very important to unravel how these modifications can influence the biologic features of hBM-MSCs to keep track of this organized program and also to clarify the conflicting observations on hBM-MSC malignant transformation in the literature.

Mesenchymal Stem Cells Neuronal Differentiation Ability: A Real Perspective for Nervous System Repair?

Mesenchymal Stem Cells (MSCs) are a bone marrow-derived population present in adult tissues that possess the important property of dividing when called upon and of differentiating into specialized cells. The evidence that MSCs were able to transdifferentiate into specialized cells of tissues different from bone marrow, in particular into nervous cells, opened up the possibility of using MSCs to substitute damaged neurons, that are normally not replaced but lost, in order to repair the Nervous System. The first neuronal differentiation protocols were based on the use of a mixture of toxic drugs which induced MSCs to rapidly acquire a neuronal-like morphology with the expression of specific neuronal markers. However, many subsequent studies demonstrated that the morphological and molecular modifications of MSCs were probably due to a stress response, rather than to a real differentiation into neuronal cells, thus throwing into question the possible use of MSCs to repair the nervous system. Currently, some papers are suggesting again that it may be possible to induce neuronal differentiation of MSCs by using several differentiation protocols, and by accompanying the morphological evidence of differentiation with functional evidence, thus demonstrating that MSC-derived cells not only seem to be neurons, but that they also function like neurons. In this review, we have attempted to shed light on the capacity of MSCs to genuinely differentiate into nervous cells, and to identify the most reliable protocols for obtaining neurons from MSCs for nervous system repair.

DNA Methylation Changes during In Vitro Propagation of Human Mesenchymal Stem Cells: Implications for Their Genomic Stability?

Mesenchymal stem cells (MSCs) hold great promise for the treatment of numerous diseases. A major problem for MSC therapeutic use is represented by the very low amount of MSCs which can be isolated from different tissues; thus ex vivo expansion is indispensable. Long-term culture, however, is associated with extensive morphological and functional changes of MSCs. In addition, the concern that they may accumulate stochastic mutations which lead the risk of malignant transformation still remains. Overall, the genome of human MSCs (hMSCs) appears to be apparently stable throughout culture, though transient clonal aneuploidies have been detected. Particular attention should be given to the use of low-oxygen environment in order to increase the proliferative capacity of hMSCs, since data on the effect of hypoxic culture conditions on genomic stability are few and contradictory. Furthermore, specific and reproducible epigenetic changes were acquired by hMSCs during ex vivo expansion, which may be connected and trigger all the biological changes observed. In this review we address current issues on long-term culture of hMSCs with a 360-degree view, starting from the genomic profiles and back, looking for an epigenetic interpretation of their genetic stability.

Evaluation of tubulin polymerization and chronic inhibition of proteasome as citotoxicity mechanisms in bortezomib-induced peripheral neuropathy.

Bortezomib (BTZ) is the first proteasome inhibitor entered in clinical practice. Peripheral neuropathy is likely to be a class side effect of these drugs, although its severity is largely variable, and it deserves to be further investigated, since the mechanisms of BTZ-induced peripheral neurotoxicity (BiPN) are still unknown. In our study, we investigated in vivo and in vitro possible pathogenic events relevant to BiPN using a well-established rat model, with particular reference to the extent of proteasome inhibition and the effects on α-tubulin polymerization in sciatic nerves and dorsal root ganglia specimens obtained from animals treated with chronic regimens at a dose of 0.2 mg/kg intravenously. The same assessments were also performed after a single injection. Moreover, these studies were replicated in vitro using embryonic DRG neurons exposed to 100 nM BTZ and adult DRG neurons exposed to 10–50 nM BTZ for 24 h and 48 h. A significant increase in the polymerized fraction of α-tubulin and prolonged proteasome inhibition were observed after the chronic BTZ treatment in vivo. Recovery to physiological levels was observed after a 4-week follow-up post-treatment period. Proteasome inhibition and increased α-tubulin polymerization were also observed following BTZ treatment of both embryonic and adult DRG neurons in vitro. Our in vivo results suggest that proteasome inhibition and alteration of tubulin dynamics contribute to BiPN. The in vitro systems here described reliably replicate the in vivo results, and might therefore be used for further mechanistic studies on the effects of proteasome inhibitors on neurons.

Human mesenchymal stem cells express neuronal markers after osteogenic and adipogenic differentiation

Mesenchymal stem cells (MSCs) are multipotent cells that are able to differentiate into mesodermal lineages (osteogenic, adipogenic, chondrogenic), but also towards non-mesodermal derivatives (e.g. neural cells). Recent in vitro studies revealed that, in the absence of any kind of differentiation stimuli, undifferentiated MSCs express neural differentiation markers, but the literature data do not all concur. Considering their promising therapeutic potential for neurodegenerative diseases, it is very important to expand our knowledge about this particular biological property of MSCs. In this study, we confirmed the spontaneous expression of neural markers (neuronal, glial and progenitor markers) by undifferentiated human MSCs (hMSCs) and in particular, we demonstrated that the neuronal markers βIII-tubulin and NeuN are expressed by a very high percentage of hMSCs, regardless of the number of culture passages and the culture conditions. Moreover, the neuronal markers βIII-tubulin and NeuN are still expressed by hMSCs after in vitro osteogenic and adipogenic differentiation. On the other hand, chondrogenically differentiated hMSCs are negative for these markers. Our findings suggest that the expression of neuronal markers could be common to a wide range of cellular types and not exclusive for neuronal lineages. Therefore, the expression of neuronal markers alone is not sufficient to demonstrate the differentiation of MSCs towards the neuronal phenotype. Functional properties analysis is also required.

Mesenchymal stem cells enhance GABAergic transmission in co-cultured hippocampal neurons

Bone marrow-derived mesenchymal stem cells (MSCs) are multipotent stem cells endowed with neurotrophic potential combined with immunological properties, making them a promising therapeutic tool for neurodegenerative disorders. However, the mechanisms through which MSCs promote the neurological recovery following injury or inflammation are still largely unknown, although cell replacement and paracrine mechanisms have been hypothesized. In order to find out what are the mechanisms of the trophic action of MSCs, as compared to glial cells, on CNS neurons, we set up a co-culture system where rat MSCs (or cortical astrocytes) were used as a feeding layer for hippocampal neurons without any direct contact between the two cell types. The analysis of hippocampal synaptogenesis, synaptic vesicle recycling and electrical activity show that MSCs were capable to support morphological and functional neuronal differentiation. The proliferation of hippocampal glial cells induced by the release of bioactive substance(s) from MSCs was necessary for neuronal survival. Furthermore, MSCs selectively increased hippocampal GABAergic pre-synapses. This effect was paralleled with a higher expression of the potassium/chloride KCC2 co-transporter and increased frequency and amplitude of mIPSCs and sIPSCs. The enhancement of GABA synapses was impaired by the treatment with K252a, a Trk/neurotrophin receptor blocker, and by TrkB receptor bodies hence suggesting the involvement of BDNF as a mediator of such effects. The results obtained here indicate that MSC-secreted factors induce glial-dependent neuronal survival and trigger an augmented GABAergic transmission in hippocampal cultures, highlighting a new effect by which MSCs could promote CNS repair. Our results suggest that MSCs may be useful in those neurological disorders characterized by an impairment of excitation versus inhibition balance.

Mesengenic differentiation: comparison of human and rat bone marrow mesenchymal stem cells.

Background and Objectives: Cellular therapies using Mesenchymal Stem Cells (MSCs) represent a promising approach for the treatment of degenerative diseases, in particular for mesengenic tissue regeneration. However, before the approval of clinical trials in humans, in vitro studies must be performed aimed at investigating MSCs’ biology and the mechanisms regulating their proliferation and differentiation abilities. Besides studies on human MSCs (hMSCs), MSCs derived from rodents have been the most used cellular type for in vitro studies. Nevertheless, the transfer of the results obtained using animal MSCs to hMSCs has been hindered by the limited knowledge regarding the similarities existing between cells of different origins. Aim of this paper is to highlight similarities and differences and to clarify the sometimes reported different results obtained using these cells. Methods and Results: We compare the differentiation ability into mesengenic lineages of rat and human MSCs cultured in their standard conditions. Our results describe in which way the source from which MSCs are derived affects their differentiation potential, depending on the mesengenic lineage considered. For osteogenic and chondrogenic lineages, the main difference between human and rat MSCs is represented by differentiation time, while for adipogenesis hMSCs have a greater differentiation potential. Conclusions: These results on the one hand suggest to carefully evaluate the transfer of results obtained with animal MSCs, on the other hand they offer a clue to better apply MSCs into clinical practice.

Anti-proliferative and anti-migratory effects of baicalin on cholangiocarcinoma cell line egi-1

Cholangiocarcinoma (CCA) is the second most frequent primary liver neoplasia. It mainly arises from the malignant transformation of biliary epithelial cells, although it might originate from either hepatic progenitor cells at the Hering canals or transformed hepatocytes. CCA is a highly aggressive tumor with extremely poor prognosis and limited therapeutic approaches. Baicalin (BA) is one of the main bioactive flavonoids identified in the Scutellaria Baicalensis Georgi root dried extract which is extensively used in the Chinese traditional medicine. Together with the anti-inflammatory effect, the anti-neoplastic action is the most relevant BA property demonstrated on cancer cells of different origin. Being aware of the need of new therapeutic weapons for CCA treatment, we investigated whether Baicalin could exert anti-proliferative and anti-migratory effect on EGI-1 cells, a highly metastatic CCA cell line derived from bile duct carcinoma. We first tested different BA concentrations (from 5 to 200μM) in limiting EGI-1 viability using MTT assay. After 24h and 48h treatment, 5 and 10μM BA had no effect while rising from 25μM to 200μM (i.e. 25, 50, 100 and 200μM) BA exerted a significant cell viability reduction already at 24h and increased after 48h BA exposure. This reduction well correlated with the adherent absolute cell number decrease and it cannot be due to BA induced cell cycle impairment after neither 24 nor 48h treatment. We also evaluated the anti-migratory BA potential by a wound healing assay adding different BA concentrations (5, 25, 50,100 and 200μM) to the culture medium immediately after performing a wound on confluent cell cultures. All BA concentrations but 5μM induced a significant reduction in the EGI-1 migration rate after 24h treatment. Moreover 25, 50 and 10μM BA showed similar migration inhibition extent at 24 and 48h whilst 200μM BA exerted a stronger inhibitory effect already after 24h exposure which increased with time in a significant way. Taken together our preliminary results demonstrate that BA impairs CCA cell viability and migration suggesting a promising adjuvant therapeutic use for BA as antitumoral agent.

Antitumoral effects of Hibiscus Sabdarifa on human oral squamous cell carcinoma and multiple myeloma cells

Epidemiological data consistently demonstrate a reduced cancer risk associated with a polyphenols rich diet. Hibiscus sabdarifa (HS), a polyphenols rich plant widely consumed worldwide as beverage and used in folk medicine, has recently gained interest thanks to its antioxidant, anti-inflammatory and chemopreventive properties. In the present study we investigated the antitumoral potential of HS extract in two different human tumor cell lines: Multiple Myeloma cells (RPMI 8226) and Oral Squamous Cell Carcinoma cells (SCC-25). MTT assays showed that HS extract induced a dose-dependent viability reduction in both the cells lines. For the subsequent experiments we used HS at the concentration of 5 mg/ml that was the most effective in inducing cell viability reduction after 48h of treatment. Viable cell count using trypan blue staining demonstrated that the HS extract induced decrease in cell growth of both the cell lines and this was due to a reversible cytostatic rather than a cytotoxic effect. Wound-healing and cell invasion assays, respectively performed by a scratch of cell monolayer and Boyden Chamber transwell test, demonstrated that HS extract was able to reduce motility and invasiveness in both RPMI 8226 and SCC-25 cells. The chemical inhibition of ERK1/ERK2 and PI3K, with U0126 and wortmannin respectively, reduces proliferation and migration of both SSC-25 and RPMI cells and HB extract treatment played an additive action with the inhibitors. In conclusion, our results suggest that HS extract have antitumoral properties, since it proved to inhibit tumoral cell growth and cell migration and invasiveness. It is interesting to note that HS extract is effective against two very different tumor cell lines. In fact, RPMI 8226 cells are of hematopoietic origin and grow in suspension, whereas SCC-25 cells derive from epithelium and are characterized by adherent cell growth. Therefore, although further studies are needed to clarify the molecular mechanisms involved in its action, we proposed HS as a potential chemopreventive agent.