Coaxing bone marrow stromal mesenchymal stem cells towards neuronal differentiation: progress and uncertainties

Multipotent adult stem cells capable of developing into particular neuronal cell types have great potential for autologous cell replacement therapy for central nervous system neurodegenerative disorders and traumatic injury. Bone marrow-derived stromal mesenchymal stem cells (BMSCs) appear to be attractive starting materials. One question is whether BMSCs could be coaxed to differentiate in vitro along neuronal or glial lineages that would aid their functional integration post-transplantation, while reducing the risk of malignant transformation. Recent works suggest that BMSCs could indeed be differentiated in vitro to exhibit some cellular and physiological characteristics of neural cell lineages, but it is not likely to be achievable with simple chemical treatments. We discussed recent findings pertaining to efforts in neuronal differentiation of BMSCs in vitro, and results obtained when these were transplanted in vivo. Received 19 January 2006; received after revision 24 February 2006; accepted 12 April 2006     

Resistance of in vivo-selected spontaneously transformed cells and Rous sarcoma virus-transformed cells to macrophage-mediated cytotoxicity.

The cytotoxic activity (CTA) of activated peritoneal macrophages (MP) on variant lines of Syrian hamster embryo (HE) cells of differing malignant characteristics was studied. The target cells were a line of low-malignant cells resulting from spontaneous transformation of HE cells in vitro (STHE strain), and malignant variants selected from them in vivo (STHE-LM-4, STHE-LM-8, and STHE-75/18 strains). In addition, we used cells of the HET-SR-1 strain; these are HE cells transformed in vitro by a tumorigenic Rous sarcoma virus (Schmidt-Ruppin strain, RSV-SR), or the TU-SR strain induced by RSV-SR in vivo. Thioglycollate-elicited peritoneal MP from Syrian hamsters were activated in vitro with bacterial levan, LPS or MDP and used as effector cells. MP-mediated cytolysis was determined by means of a 42-h radioactivity release assay with 3H-thymidine-labeled target cells. We found that only the parental STHE cells were susceptible towards fully-activated MP-mediated CTA. All three of the in vivo-selected malignant variants of the STHE cell sublines, as well as the tumorigenic RSV-SR transformants, were resistant to cytolysis by activated MP. Non-activated thioglycollate-elicited MP did not lyse any of the tumor cells studied.     

Resistance of in vivo-selected spontaneously transformed cells and Rous sarcoma virus-transformed cells to macrophage-mediated cytotoxicity

The cytotoxic activity (CTA) of activated peritoneal macrophages (MP) on variant lines of Syrian hamster embryo (HE) cells of differing malignant characteristics was studied. The target cells were a line of low-malignant cells resulting from spontaneous transformation of HE cells in vitro (STHE strain), and malignant variants selected from them in vivo (STHE-LM-4, STHE-LM-8, and STHE-75/18 strains). In addition, we used cells of the HET-SR-1 strain; these are HE cells transformed in vitro by a tumorigenic Rous sarcoma virus (Schmidt-Ruppin strain, RSV-SR), or the TU-SR strain induced by RSV-SR in vivo. Thioglycollate-elicited peritoneal MP from Syrian hamsters were activated in vitro with bacterial levan, LPS or MDP and used as effector cells. MP-mediated cytolysis was determined by means of a 42-h radioactivity release assay with³H-thymidine-labeled target cells. We found that only the parental STHE cells were susceptible towards fully-activated MP-mediated CTA. All three of the in vivo-selected malignant variants of the STHE cell sublines, as well as the tumorigenic RSV-SR transformants, were resistant to cytolysis by activated MP. Non-activated thioglycollate-elicited MP did not lyse any of the tumor cells studied.     

Inhibition of proliferation by 1-8U in interferon-α-responsive and non-responsive cell lines

Interferon (IFN)-inducible proteins of the 1-8 gene family mediate homotypic adhesion and transduction of antiproliferative signals. Their induction correlates with inhibition of cell growth while they are often repressed in the course of malignant transformation and tumor development. Ras-mediated transformation of mouse mast cells is associated with downregulation of 1-8U expression and interferon-α (IFN-α) treatment reverts the proliferation rate to normal levels together with induction of 1-8U. Conversely, the antiproliferative responses of IFN-α in sensitive human melanoma cells are accompanied by 1-8U induction. Here we provide direct evidence that recombinant expression of 1-8U in human cell lines is sufficient to block cell proliferation. Based on the abundant expression and subcellular localization to the plasma membrane and exosome-like structures, we propose a model capable of explaining the pleiotropic functions of 1-8 family proteins in tumor cells and during normal development. Received 15 January 2003; received after revision 21 March 2003; accepted 25 March 2003 RID="*" ID="*"Corresponding author.     

Disappearance of the gliofibrillar protein (GFA) during the cultivation of glioblastoma cells]

Absence of GFA in established cell lines from glioblastomas promped us to look for this protein from the first replication of malignant cells in vitro and in following subcultrues. Our results show that GFA disappeared during the first twelve replications, often before the fifth replication. No correlation was observed with a morphological transformation of the cells.     

Effect of prostaglandins and dibutyryl cyclic AMP on the morphology of cells in primary astroglial cultures and on metabolic enzymes of GABA and glutamate metabolism

Prostaglandins (PGE1) and dibutyryl cyclic AMP (dBc AMP) induce similar morphological changes in astrocytes obtained in primary cultures. PGE1 and dBc AMP increased 2 enzymes of GABA and glutamate metabolism, GABA-T and AAT, but did not modify GDH and GLN-S. Prostaglandins probably affect the cAMP content of glial cells and act in the same way as dBc AMP on glial cell differentiation.     

Nitric oxide stimulates human neural progenitor cell migration via cGMP-mediated signal transduction

Neuronal migration is one of the most critical processes during early brain development. The gaseous messenger nitric oxide (NO) has been shown to modulate neuronal and glial migration in various experimental models. Here, we analyze a potential role for NO signaling in the migration of fetal human neural progenitor cells. Cells migrate out of cultured neurospheres and differentiate into both neuronal and glial cells. The neurosphere cultures express neuronal nitric oxide synthase and soluble guanylyl cyclase that produces cGMP upon activation with NO. By employing small bioactive enzyme activators and inhibitors in both gain and loss of function experiments, we show NO/cGMP signaling as a positive regulator of migration in neurosphere cultures of early developing human brain cells. Since NO signaling regulates cell movements from developing insects to mammalian nervous systems, this transduction pathway may have evolutionary conserved functions.     

The contribution of neuronal–glial–endothelial–epithelial interactions to colon carcinogenesis

Several different cell types constitute the intestinal wall and interact in different manners to maintain tissue homeostasis. Elegant reports have explored these physiological cellular interactions revealing that glial cells and neurons not only modulate peristalsis and mechanical stimulus in the intestines but also control epithelial proliferation and sub-epithelial angiogenesis. Although colon carcinoma arises from epithelial cells, different sub-epithelial cell phenotypes are known to support the manifestation and development of tumors from their early steps on. Therefore, new perspectives in cancer research have been proposed, in which neurons and glial cells not only lead to higher cancer cell proliferation at the tumor invasion front but also further enhance angiogenesis and neurogenesis in tumors. Transformation of physiological neural activity into a pro-cancer event is thus discussed for colon carcinogenesis herein.     

Mechanisms of glial-guided neuronal migration in vitro and in vivo

Our laboratory has developed an in vitro model system in which glial-guided neuronal migration can be observed in real time. Cerebellar granule neurons migrate on astroglial fibers by apposing their cell soma against the glial arm, forming a specialized migration junction, and extending a motile leading process in the direction of migration. In vitro assays indicate that the neuronal antigen astrotactin functions as a neuron-glia ligand, and is likely to play a role in the movement of neurons along glial fibers. In heterotypic recombinations of neurons and glia from mouse cerebellum and rat hippocampus, neurons migrate on heterotypic glial processes with a cytology, speed and mode of movement identical to that of neuronal migration on homotypic glial fibers, suggesting that glial fibers provide a permissive pathway for neuronal migration in developing brain. In vivo analyses of developing cerebellum demonstrate a close coordination of afferent axon ingrowth relative to target cell migration. These studies indicate that climbing fibers contact immature Purkinje neurons during the migration and settling of Purkinje cells, implicating a role for afferents in the termination of migration.     

Mechanisms of glial-guided neuronal migration in vitro and in vivo

Summary Our laboratory has developed an in vitro model system in which glial-guided neuronal migration can be observed in real time. Cerebellar granule neurons migrate on astroglial fibers by apposing their cell soma against the glial arm, forming a specialized migration junction, and extending a motile leading process in the direction of migration. In vitro assays indicate that the neuronal antigen astrotactin functions as a neuron-glia ligand, and is likely to play a role in the movement of neurons along glial fibers. In heterotypic recombinations of neurons and glia from mouse cerebellum and rat hippocampus, neurons migrate on heterotypic glial processes with a cytology, speed and mode of movement identical to that of neuronal migration on homotypic glial fibers, suggesting that glial fibers provide a permissive pathway for neuronal migration in developing brain. In vivo analyses of developing cerebellum demonstrate a close coordination of afferent axon ingrowth relative to target cell migration. These studies indicate that climbing fibers contact immature Purkinje neurons during the migration and settling of Purkinje cells, implicating a role for afferents in the termination of migration.     

Effect of prostaglandins and dibutyryl cyclic AMP on the morphology of cells in primary astroglial cultures and on metabolic enzymes of GABA and glutamate metabolism

Summary Prostaglandins (PGE₁) and dibutyryl cyclic AMP (dBc AMP) induce similar morphological changes in astrocytes obtained in primary cultures. PGE₁ and dBc AMP increased 2 enzymes of GABA and glutamate metabolism, GABA-T and AAT, but did not modify GDH and GLN-S. Prostaglandins probably affect the cAMP content of glial cells and act in the same way as dBc AMP on glial cell differentiation.     

In vitro malignant transformation of hamster lung cells by 2-chlorobutadiene]

Normal Hamster lung cells from an established line were treated with 1-500 microgram . ml-1 2-chlorobutadiene. Those treated with 1 microgram . ml-1 of the compound showed malignant transformations 14 weeks after treatment. The treatment with higher concentrations did not accelerate the transformation process.     

Loss of virulence of canine distemper virus is associated with a structural change recognized by a monoclonal antibody.

The monoclonal antibody (mAB) L1, which binds to the nucleocapsid protein of canine distemper virus (CDV), was shown to bind to avirulent CDV obtained after serial passages in Vero cells, but not to two different virulent demyelinating CDV-strains propagated in dog glial cell cultures. However, when both virulent CDV-strains were passaged through Vero cells they expressed, after a number of passages, an epitope recognized by mAB L1. The occurrence of the L1 epitope appeared to coincide with loss of virulence in animal inoculation experiments.     

Loss of virulence of canine distemper virus is associated with a structural change recognized by a monoclonal antibody

The monoclonal antibody (mAB) L1, which binds to the nucleocapsid protein of canine distemper virus (CDV), was shown to bind to avirulent CDV obtained after serial passages in Vero cells, but not to two different virulent demyelinating CDV-strains propagated in dog glial cell cultures. However, when both virulent CDV-strains were passaged through Vero cells they expressed, after a number of passages, an epitope recognized by mAB L1. The occurrence of the L1 epitope appeared to coincide with loss of virulence in animal inoculation experiments.     

In vitro neurogenesis: development and functional implications of iPSC technology

Neurogenesis is the developmental process regulating cell proliferation of neural stem cells, determining their differentiation into glial and neuronal cells, and orchestrating their organization into finely regulated functional networks. Can this complex process be recapitulated in vitro using induced pluripotent stem cell (iPSC) technology? Can neurodevelopmental and neurodegenerative diseases be modeled using iPSCs? What is the potential of iPSC technology in neurobiology? What are the recent advances in the field of neurological diseases? Since the applications of iPSCs in neurobiology are based on the capacity to regulate in vitro differentiation of human iPSCs into different neuronal subtypes and glial cells, and the possibility of obtaining iPSC-derived neurons and glial cells is based on and hindered by our poor understanding of human embryonic development, we reviewed current knowledge on in vitro neural differentiation from a developmental and cellular biology perspective. We highlight the importance to further advance our understanding on the mechanisms controlling in vivo neurogenesis in order to efficiently guide neurogenesis in vitro for cell modeling and therapeutical applications of iPSCs technology.     

Enteric glial cells are susceptible to <Emphasis Type="Italic">Clostridium difficile</Emphasis> toxin B

Clostridium difficile causes nosocomial/antibiotic-associated diarrhoea and pseudomembranous colitis. The major virulence factors are toxin A and toxin B (TcdB), which inactivate GTPases by monoglucosylation, leading to cytopathic (cytoskeleton alteration, cell rounding) and cytotoxic effects (cell-cycle arrest, apoptosis). C. difficile toxins breaching the intestinal epithelial barrier can act on underlying cells, enterocytes, colonocytes, and enteric neurons, as described in vitro and in vivo, but until now no data have been available on enteric glial cell (EGC) susceptibility. EGCs are crucial for regulating the enteric nervous system, gut homeostasis, the immune and inflammatory responses, and digestive and extradigestive diseases. Therefore, we evaluated the effects of C. difficile TcdB in EGCs. Rat-transformed EGCs were treated with TcdB at 0.1–10 ng/ml for 1.5–48 h, and several parameters were analysed. TcdB induces the following in EGCs: (1) early cell rounding with Rac1 glucosylation; (2) early G2/M cell-cycle arrest by cyclin B1/Cdc2 complex inactivation caused by p27 upregulation, the downregulation of cyclin B1 and Cdc2 phosphorylated at Thr161 and Tyr15; and (3) apoptosis by a caspase-dependent but mitochondria-independent pathway. Most importantly, the stimulation of EGCs with TNF-α plus IFN-γ before, concomitantly or after TcdB treatment strongly increased TcdB-induced apoptosis. Furthermore, EGCs that survived the cytotoxic effect of TcdB did not recover completely and showed not only persistent Rac1 glucosylation, cell-cycle arrest and low apoptosis but also increased production of glial cell-derived neurotrophic factor, suggesting self-rescuing mechanisms. In conclusion, the high susceptibility of EGCs to TcdB in vitro, the increased sensitivity to inflammatory cytokines related to apoptosis and the persistence of altered functions in surviving cells suggest an important in vivo role of EGCs in the pathogenesis of C. difficile infection.     

Mesenchymal stem cells secretome: a new paradigm for central nervous system regeneration?

The low regeneration potential of the central nervous system (CNS) represents a challenge for the development of new therapeutic strategies. Mesenchymal stem cells (MSCs) have been proposed as a possible therapeutic tool for CNS disorders. In addition to their differentiation potential, it is well accepted nowadays that their beneficial actions can also be mediated by their secretome. Indeed, it was already demonstrated, both in vitro and in vivo, that MSCs are able to secrete a broad range of neuroregulatory factors that promote an increase in neurogenesis, inhibition of apoptosis and glial scar formation, immunomodulation, angiogenesis, neuronal and glial cell survival, as well as relevant neuroprotective actions on different pathophysiological contexts. Considering their protective action in lesioned sites, MSCs’ secretome might also improve the integration of local progenitor cells in neuroregeneration processes, opening a door for their future use as therapeutical strategies in human clinical trials. Thus, in this review we analyze the current understanding of MSCs secretome as a new paradigm for the treatment of CNS neurodegenerative diseases.     

The multifaceted role of glial cells in amyotrophic lateral sclerosis

Despite indisputable progress in the molecular and genetic aspects of amyotrophic lateral sclerosis (ALS), a mechanistic comprehension of the neurodegenerative processes typical of this disorder is still missing and no effective cures to halt the progression of this pathology have yet been developed. Therefore, it seems that a substantial improvement of the outcome of ALS treatments may depend on a better understanding of the molecular mechanisms underlying neuronal pathology and survival as well as on the establishment of novel etiological therapeutic strategies. Noteworthy, a convergence of recent data from multiple studies suggests that, in cellular and animal models of ALS, a complex pathological interplay subsists between motor neurons and their non-neuronal neighbours, particularly glial cells. These observations not only have drawn attention to the physiopathological changes glial cells undergo during ALS progression, but they have moved the focus of the investigations from intrinsic defects and weakening of motor neurons to glia–neuron interactions. In this review, we summarize the growing body of evidence supporting the concept that different glial populations are critically involved in the dreadful chain of events leading to motor neuron sufferance and death in various forms of ALS. The outlined observations strongly suggest that glial cells can be the targets for novel therapeutic interventions in ALS.     

Molecular and cellular pathogenesis of melanoma initiation and progression

Melanoma is a malignant tumor of melanocytes that can spread to other organs of the body, resulting in severe and/or lethal malignancies. Melanocytes are pigment-producing cells found in the deep layer of the epidermis and are originated from melanocytes stem cells through a cellular process called melanogenesis. Several genes and epigenetic and micro-environmental factors are involved in this process via the regulation and maintenance of the balance between melanocytes stem cells proliferation and their differentiation into melanocytes. Dysregulation of this balance through gain or loss of function of key genes implicated in the control and regulation of cell cycle progression and/or differentiation results in melanoma initiation and progression. This review aims to provide a comprehensive overview about the origin of melanocytes, the oncogenic events involved in melanocytes stem cells transformation, and the mechanisms implicated in the perpetuation of melanoma malignant phenotype.