Differentiation of single cell derived human mesenchymal stem cells into cells with a neuronal phenotype: RNA and microRNA expression profile.

The adult bone marrow contains a subset of non-haematopoietic cells referred to as bone marrow mesenchymal stem cells (BMSCs). Mesenchymal stem cells (MSCs) have attracted immense research interest in the field of regenerative medicine due to their ability to be cultured for successive passages and multi-lineage differentiation. The molecular mechanisms governing the self-renewal and differentiation of MSCs remain largely unknown. In a previous paper we demonstrated the ability to induce human clonal MSCs to differentiate into cells with a neuronal phenotype (DMSCs). In the present study we evaluated gene expression profiles by Sequential Analysis of Gene Expression (SAGE) and microRNA expression profiles before and after the neuronal differentiation process. Various tissue-specific genes were weakly expressed in MSCs, including those of non-mesodermal origin, suggesting multiple potential tissue-specific differentiation, as well as stemness markers. Expression of OCT4, KLF4 and c-Myc cell reprogramming factors, which are modulated during the differentiation process, was also observed. Many peculiar nervous tissue genes were expressed at a high level in DMSCs, along with genes related to apoptosis. MicroRNA profiling and correlation with mRNA expression profiles allowed us to identify putative important genes and microRNAs involved in the differentiation of MSCs into neuronal-like cells. The profound difference in gene and microRNA expression patterns between MSCs and DMSCs indicates a real functional change during differentiation from MSCs to DMSCs.

Human astrocytes can be induced to differentiate into cells with neuronal phenotype.

Several recent studies have proposed that astrocytes may contribute to neurogenesis, not only as a source of trophic substances regulating it, but also as stem cells themselves. In order to better understand these mechanisms, primary astrocyte cultures were established from human fetal brain. After 3-4 weeks in culture, astrocytes (about 95% GFAP+; neurofilament, NF-; neuro-specific enolase, NSE-) were treated with a cocktail of protein kinase activators and FGF-1. After 5 h of treatment, most cells showed morphological changes that increased progressively up to 24-48 h, exhibiting a round cell body with long processes. Immunocytochemistry showed that treatment-induced NF and NSE expression in about 40% of cells. Nestin expression increased after treatment, whereas GFAP immunostaining was not significantly modified. Western blot and RT-PCR confirmed the results. No neuronal electrophysiological properties were observed after treatment, suggesting an incomplete maturation under these experimental conditions. Understanding the regenerative capability and neurogenic potential of astrocytes might be useful in devising therapeutic approaches for a variety of neurological disorders.

Differentiation of human bone marrow stem cells into cells with a neural phenotype: diverse effects of two specific treatments.

BACKGROUND: It has recently been demonstrated that the fate of adult cells is not restricted to their tissues of origin. In particular, it has been shown that bone marrow stem cells can give rise to cells of different tissues, including neural cells, hepatocytes and myocytes, expanding their differentiation potential. RESULTS: In order to identify factors able to lead differentiation of stem cells towards cells of neural lineage, we isolated stromal cells from human adult bone marrow (BMSC). Cells were treated with: (1) TPA, forskolin, IBMX, FGF-1 or (2) retinoic acid and 2-mercaptoethanol (BME). Treatment (1) induced differentiation into neuron-like cells within 24 hours, while a longer treatment was required when using retinoic acid and BME. Morphological modifications were more dramatic after treatment (1) compared with treatment (2). In BMSC both treatments induced the expression of neural markers such as NF, GFAP, TUJ-1 and neuron-specific enolase. Moreover, the transcription factor Hes1 increased after both treatments. CONCLUSION: Our study may contribute towards the identification of mechanisms involved in the differentiation of stem cells towards cells of neural lineage.

Differentiation of human adult CD34+ stem cells into cells with a neural phenotype: role of astrocytes.

It has recently been reported that adult hematopoietic stem cells can differentiate into neural cells, opening new frontiers in therapy for neurodegenerative diseases. In this study, adult human hematopoietic stem cells (HSCs) were isolated via magnetic bead sorting, using a specific CD34 antibody and cultured with human astrocyte culture conditioned medium (ACM). In order to evaluate their differentiation into neurons and/or astrocytes, ACM-treated cultures were probed for the expression of several neural markers. We observed morphological modifications and, after 20 days of treatment, cell morphology displayed extending processes. Immunocytochemistry, Western blotting and RT-PCR showed the expression of neuronal markers such as neurofilaments, neuron specific enolase (NSE) and NeuN in ACM-treated HSCs cultured in poly-L-lysine-coated dishes. On the contrary, when the same ACM-treated cells were grown on a plastic substrate, they expressed high levels of glial fibrillary acidic protein (GFAP), with only weak expression of neuronal markers. Nestin, a neural progenitor cell marker, was present in treated cells, regardless of the substrate. These results demonstrate that astrocytes can generate a suitable microenvironment for inducing HSCs to differentiate into neural cells. Therefore, adult bone marrow may represent a readily accessible source of cells for treating neurodegenerative diseases.

S100b counteracts effects of the neurotoxicant trimethyltin on astrocytes and microglia.

Central nervous system degenerative diseases are often characterized by an early, strong reaction of astrocytes and microglia. Both these cell types can play a double role, protecting neurons against degeneration through the synthesis and secretion of trophic factors or inducing degeneration through the secretion of toxic molecules. Therefore, we studied the effects of S100B and trimethyltin (TMT) on human astrocytes and microglia with two glial models, primary cultures of human fetal astrocytes and a microglia cell line. After treatment with 10(-5) M TMT, astrocytes showed morphological alterations associated with an increase in glial fibrillary acidic protein (GFAP) expression and changes in GFAP filament organization. Administration of S100B before TMT treatment prevented TMT-induced changes in morphology and GFAP expression. A decrease in inducible nitric oxide synthase expression was observed in astrocytes treated with TMT, whereas the same treatment induced iNOS expression in microglia. In both cases, S100B prevented TMT-induced changes. Tumor necrosis factor-alpha mRNA expression in astrocytes was not modified by TMT treatment, whereas it was increased in microglia cells. S100B pretreatment blocked the TMT-induced increase in TNF-alpha expression in microglia. To trace the mechanisms involved in S100B activity, the effect of BAY 11-7082, an inhibitor of nuclear factor-kappaB (NF-kappaB) activation, and of PD98059, an inhibitor of MEK-ERK1/2, were investigated. Results showed that the protective effects of S100B against TMT toxicity in astrocytes depend on NF-kappaB, but not on ERK1/2 activation. These results might help in understanding the role played by glial cells in brain injury after exposure to chemical neurotoxicants and support the view that S100B may protect brain cells in case of injury. (c) 2005 Wiley-Liss, Inc.

Inhibition of cytokines expression in human microglia infected by virulent and non-virulent mycobacteria.

The pathogenesis of tuberculosis (TBC) meningitis is still unknown. As shown by previous studies, human microglia can be the target of mycobacteria, but no data are available about their cellular response to infection. Consequently, we studied the expression of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1) and IL-10 in human microglia pure cultures infected with the two variants of Mycobacterium avium (domed-opaque (SmD) and transparent (SmT)) and with Mycobacterium tuberculosis. Results showed that microglia was productively infected by mycobacteria which could grow inside the cells. Mycobacteria internalization was more rapid for M. avium, but M. tuberculosis infection turned out to be more efficient due to the incorporation of densely packed bacteria. TNF-alpha expression was not affected by M. avium, whereas an increase followed by a decrease was observed in M. tuberculosis. Both IL-1 and IL-10 cytokine expression was rapidly inhibited by infection with the more virulent bacteria, whereas the non-pathogenic one had almost no effect. Also, the expression of the co-stimulatory molecule CD137, a member of tumor necrosis factor receptor family, was affected by infection with virulent mycobacteria. Our results show that microglia response to mycobacterial infection is modulated in correlation with virulence, mainly toward inhibition of inflammatory response. This observation might be one of the mechanisms by which non-pathogenic mycobacteria are quickly eliminated, explaining one of the bases of virulence.

Expression of CD137 and its ligand in human neurons, astrocytes, and microglia: modulation by FGF-2.

CD137 (ILA, 4-1BB), a member of the tumor necrosis factor receptor family, and its ligand CD137-L were assayed by RT-PCR and immunocytochemistry in cultured human brain cells. Results demonstrated that both neurons and astrocytes expressed specific RNA for CD137 and its protein, which was found both on the plasma membrane and in the cytoplasm. Surprisingly, microglia, which also expressed CD137 mRNA, showed negative immunostaining. CD137-L-specific RNA was detected only in astrocytes and neurons. When brain cells were treated with fibroblast growth factor-2 (FGF-2), upregulation of CD137 but not of its ligand was observed in neurons and astrocytes. Protein localization was also affected. In microglia, an inhibition of RNA expression was induced by treatment, whereas CD137-L remained negative. Our data are the first demonstration that human brain cells express a protein found thus far in activated immunocompetent cells and epithelia. Moreover, they suggest not only that CD137 and CD137-L might play a role in interaction among human brain cells, but also that FGF-2 might have an immunoregulatory function in brain, modulating interaction of the central nervous system with peripheral immunocompetent cells.