Human dental pulp stem cells cultured in serum-free supplemented medium.

UNLABELLED: Growing evidence show that human dental pulp stem cells (DPSCs) could provide a source of adult stem cells for the treatment of neurodegenerative pathologies. In this study, DPSCs were expanded and cultured with a protocol generally used for the culture of neural stem/progenitor cells. METHODOLOGY: DPSC cultures were established from third molars. The pulp tissue was enzymatically digested and cultured in serum-supplemented basal medium for 12 h. Adherent (ADH) and non-adherent (non-ADH) cell populations were separated according to their differential adhesion to plastic and then cultured in serum-free defined N2 medium with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). Both ADH and non-ADH populations were analyzed by FACS and/or PCR. RESULTS: FACS analysis of ADH-DPSCs revealed the expression of the mesenchymal cell marker CD90, the neuronal marker CD56, the transferrin receptor CD71, and the chemokine receptor CXCR3, whereas hematopoietic stem cells markers CD45, CD133, and CD34 were not expressed. ADH-DPSCs expressed transcripts coding for the Nestin gene, whereas expression levels of genes coding for the neuronal markers ß-III tubulin and NF-M, and the oligodendrocyte marker PLP-1 were donor dependent. ADH-DPSCs did not express the transcripts for GFAP, an astrocyte marker. Cells of the non-ADH population that grew as spheroids expressed Nestin, ß-III tubulin, NF-M and PLP-1 transcripts. DPSCs that migrated out of the spheroids exhibited an odontoblast-like morphology and expressed a higher level of DSPP and osteocalcin transcripts than ADH-DPSCs. CONCLUSION: Collectively, these data indicate that human DPSCs can be expanded and cultured in serum-free supplemented medium with EGF and bFGF. ADH-DPSCs and non-ADH populations contained neuronal and/or oligodendrocyte progenitors at different stages of commitment and, interestingly, cells from spheroid structures seem to be more engaged into the odontoblastic lineage than the ADH-DPSCs.

Expression of heme oxygenase-1 in neural stem/progenitor cells as a potential mechanism to evade host immune response.

Besides their therapeutic benefit as cell source, neural stem/progenitor cells (NSPCs) exhibit immunosuppressive properties of great interest for modulating immune response in the central nervous system. To decipher the mechanisms of NSPC-mediated immunosuppression, activated T cells were exposed to NSPCs isolated from fetal rat brains. Analyses revealed that NSPCs inhibited T-cell proliferation and interferon-gamma production in a dose-dependent manner. A higher proportion of helper T cells (CD4+ T cells) was found in the presence of NSPCs, but analyses of FoxP3 population indicated that T-cell suppression was not secondary to an induction of suppressive regulatory T cells (FoxP3+ CD4+ CD25+). Conversely, induction of the high affinity interleukin-2 (IL-2) receptor (CD25) and the inability of IL-2 to rescue T-cell proliferation suggest that NSPCs display immunosuppressive activity without affecting T-cell activation. Cultures in Transwell chambers or addition of NSPC-conditioned medium to activated T cells indicated that part of the suppressive activity was not contact dependent. We therefore searched for soluble factors that mediate NSPC immunosuppression. We found that NSPCs express several immunosuppressive molecules, but the ability of these cells to inhibit T-cell proliferation was only counteracted by heme oxygenase (HO) inhibitors in association or not with nitric oxide synthase inhibitors. Taken together, our findings highlight a dynamic crosstalk between NSPCs and T lymphocytes and provide the first evidence of an implication of HO-1 in mediating the immunosuppressive effects of the NSPCs.

Comparison of spheroids formed by rat glioma stem cells and neural stem cells reveals differences in glucose metabolism and promising therapeutic applications.

Cancer stem cells (CSCs) are thought to be partially responsible for cancer resistance to current therapies and tumor recurrence. Dichloroacetate (DCA), a compound capable of shifting metabolism from glycolysis to glucose oxidation, via an inhibition of pyruvate dehydrogenase kinase was used. We show that DCA is able to shift the pyruvate metabolism in rat glioma CSCs but has no effect in rat neural stem cells. DCA forces CSCs into oxidative phosphorylation but does not trigger the production of reactive oxygen species and consecutive anti-cancer apoptosis. However, DCA, associated with etoposide or irradiation, induced a Bax-dependent apoptosis in CSCs in vitro and decreased their proliferation in vivo. The former phenomenon is related to DCA-induced Foxo3 and p53 expression, resulting in the overexpression of BH3-only proteins (Bad, Noxa, and Puma), which in turn facilitates Bax-dependent apoptosis. Our results demonstrate that a small drug available for clinical studies potentiates the induction of apoptosis in glioma CSCs.

Assessing the potential clinical utility of transplantations of neural and mesenchymal stem cells for treating neurodegenerative diseases.

Treatments for neurodegenerative diseases have little impact on the long-term patient health. However, cellular transplants of neuroblasts derived from the aborted embryonic brain tissue in animal models of neurodegenerative disorders and in patients have demonstrated survival and functionality in the brain. However, ethical and functional problems due to the use of this fetal tissue stopped most of the clinical trials. Therefore, new cell sources were needed, and scientists focused on neural (NSCs) and mesenchymal stem cells (MSCs). When transplanted in the brain of animals with Parkinson's or Huntington's disease, NSCs and MSCs were able to induce partial functional recovery by promoting neuroprotection and immunomodulation. MSCs are more readily accessible than NSCs due to sources such as the bone marrow. However, MSCs are not capable of differentiating into neurons in vivo where NSCs are. Thus, transplantation of NSCs and MSCs is interesting for brain regenerative medicine. In this chapter, we detail the methods for NSCs and MSCs isolation as well as the transplantation procedures used to treat rodent models of neurodegenerative damage.

Neural stem/progenitor cells as a promising candidate for regenerative therapy of the central nervous system.

Neural transplantation is a promising therapeutic strategy for neurodegenerative diseases and other disorders of the central nervous system (CNS) such as Parkinson and Huntington diseases, multiple sclerosis or stroke. Although cell replacement therapy already went through clinical trials for some of these diseases using fetal human neuroblasts, several significant limitations led to the search for alternative cell sources that would be more suitable for intracerebral transplantation.Taking into account logistical and ethical issues linked to the use of tissue derived from human fetuses, and the immunologically special status of the CNS allowing the occurrence of deleterious immune reactions, neural stem/progenitor cells (NSPCs) appear to be an interesting cell source candidate. In addition to their ability for replacing cell populations lost during the pathological events, NSPCs also display surprising therapeutic effects of neuroprotection and immunomodulation. A better knowledge of the mechanisms involved in these specific characteristics will hopefully lead in the future to a successful use of NSPCs in regenerative medicine for CNS disorders.

Ectopic expression of the immune adaptor protein CD3zeta in neural stem/progenitor cells disrupts cell-fate specification.

Immune signaling and neuroinflammatory mediators have recently emerged as influential variables that regulate neural precursor/stem cell (NPC) behavior and function. In this study, we investigated whether the signaling adaptor protein CD3ζ, a transmembrane protein involved in T cell differentiation and function and recently shown to regulate neuronal development in the central nervous system (CNS), may have a role in NPC differentiation. We analyzed the expression profile of CD3ζ in embryonic rat brain during neurogenic periods and in neurosphere-derived neural cells, and we investigated the action of CD3ζ on cell differentiation. We found that CD3ζ expression coincided with neuronal commitment, but its forced expression in NPCs prevented the production of neurons and oligodendrocytes, but not astroglial cells. This blockade of neuronal differentiation was operated through an ITAM-independent mechanism, but required the Asp36 of the CD3ζ transmembrane domain involved in membrane receptor interaction. Together, our findings show that ectopic CD3ζ expression in NPCs impaired their normal cell-fate specification and suggest that variations of CD3ζ expression in the developing CNS might result in neurodevelopmental anomalies.

The immune molecule CD3zeta and its downstream effectors ZAP-70/Syk mediate ephrin signaling in neurons to regulate early neuritogenesis.

Recent studies have highlighted the key role of the immune protein CD3ζ in the maturation of neuronal circuits in the CNS. Yet, the upstream signals that might recruit and activate CD3ζ in neurons are still unknown. In this study, we show that CD3ζ functions early in neuronal development and we identify ephrinA1-dependent EphA4 receptor activation as an upstream regulator of CD3ζ. When newly born neurons are still spherical, before neurite extension, we found a transient CD3ζ aggregation at the cell periphery matching the initiation site of the future neurite. This accumulation of CD3ζ correlated with a stimulatory effect on filopodia extension via a Rho-GEF Vav2 pathway and a repression of neurite outgrowth. Conversely, cultured neurons lacking CD3ζ isolated from CD3ζ(-/-) mice showed a decreased number of filopodia and an enhanced neurite number. Stimulation with ephrinA1 induces the translocation of both CD3ζ and its activated effector molecules, ZAP-70/Syk tyrosine kinases, to EphA4 receptor clusters. EphrinA1-induced growth cone collapse was abrogated in CD3ζ(-/-) neurons and was markedly reduced by ZAP-70/Syk inhibition. Moreover, ephrinA1-induced ZAP-70/Syk activation was inhibited in CD3ζ(-/-) neurons. Altogether, our data suggest that CD3ζ mediates the ZAP-70/Syk kinase activation triggered by ephrinA-activated pathway to regulate early neuronal morphogenesis.

Proapoptotic and antiapoptotic actions of Stat1 versus Stat3 underlie neuroprotective and immunoregulatory functions of IL-11.

Current therapies for multiple sclerosis target inflammation but do not directly address oligodendrocyte protection or myelin repair. The gp130 family cytokines ciliary neurotrophic factor, leukemia inhibitory factor, and IL-11 have been identified as oligodendrocyte growth factors, and IL-11 is also strongly immunoregulatory, but their underlying mechanisms of action are incompletely characterized. In this study, we demonstrate that these effects of IL-11 are mediated via differential regulation of apoptosis in oligodendrocytes versus Ag-presenting dendritic cells (DCs), and are dependent on lineage-specific activity of the transcription factors Stat1 versus Stat3. Focal demyelinating lesions induced in cerebral cortices of IL-11Rα(-/-) mice using stereotactic microinjection of lysolecithin were larger than in controls, and remyelination was delayed. In IL-11Rα(-/-) mice, lesions displayed extensive oligodendrocyte loss and axonal transection, and increased infiltration by inflammatory cells including CD11c(+) DCs, CD3(+) lymphocytes, and CD11b(+) phagocytes. In oligodendrocyte progenitor cell (OPC) cultures, IL-11 restricted caspase 9 activation and apoptosis, and it increased myelination in OPC-neuron cocultures. Importantly, siRNA inhibition of Stat1 enhanced the antiapoptotic effects of IL-11 on OPCs, but IL-11 induced apoptosis in the presence of Stat3 silencing. In contrast, IL-11 augmented caspase activation and apoptosis in cultures of CD11c(+) DCs, but not in CD11b(+) or CD3(+) cells. Inhibition of Stat3 exacerbated the proapoptotic effects of IL-11 on DCs, whereas they were ablated in Stat1(-/-) cultures. Collectively, these findings reveal novel mechanisms underlying the actions of a neuroprotective and immunoregulatory member of the gp130 cytokine family, suggesting avenues to enhance oligodendrocyte viability and restrict CNS inflammation in multiple sclerosis.

Targeting oligodendrocyte protection and remyelination in multiple sclerosis.

Multiple sclerosis is an inflammatory demyelinating disease of the brain and spinal cord with a presumed autoimmune etiology. Conduction block in demyelinated axons underlies early neurological symptoms, whereas axonal transection is believed responsible for more permanent later deficits. Approved treatments for the disease are immunoregulatory and reduce the rate of lesion formation and clinical exacerbation, but are only partially effective in preventing the onset of disability in multiple sclerosis patients. Approaches that directly protect myelin-producing oligodendrocytes and enhance remyelination may improve long-term outcomes and reduce the rate of axonal transection. Studies in genetically modified animals have improved our understanding of mechanisms underlying central nervous system pathology in multiple sclerosis models, and have identified pathways that regulate oligodendrocyte viability and myelin repair. However, although clinical trials are ongoing, many have been unsuccessful, and no treatments are yet approved that target these areas in multiple sclerosis. In this review, we examine avenues for oligodendrocyte protection and endogenous myelin repair in animal models of demyelination and remyelination, and their relevance as therapeutics in human patients.

Trophic and immunoregulatory properties of neural precursor cells: benefit for intracerebral transplantation.

Intracerebral xenotransplantation of porcine fetal neuroblasts (pNB) is considered as an alternative to human neuroblasts for the treatment of neurodegenerative diseases. However, pNB are systematically rejected, even in an immunoprivileged site such as the brain. Within this context, neural stem/precursor cells (NSPC), which were suggested as exhibiting low immunogenicity, appeared as a useful source of xenogeneic cells. To determine the advantage of using porcine NSPC (pNSPC) in xenotransplantation, pNB and pNSPC were grafted into the striatum of rats without immunosuppression. At day 63, all the pNB were rejected while 40% of the rats transplanted with pNSPC exhibited large and healthy grafts with numerous pNF70-positive cells. The absence of inflammation at day 63 and the occasional presence of T cells in pNSPC grafts evoked a weak host immune response which might be partly due to the immunosuppressive properties of the transplanted cells. T cell proliferation assays confirmed such a hypothesis by revealing an inhibitory effect of pNSPC on T cells through a soluble factor. In addition to their immunosuppressive effect, in contrast to pNB, very few pNSPC differentiated into tyrosine hydroxylase-positive neurons but the cells triggered an intense innervation of the striatum by rat dopaminergic fibers coming from the substantia nigra. Further experiments will be required to optimize the use of pNSPC in regenerative medicine but here we show that their immunomodulatory and trophic activities might be of great interest for restorative strategies. This article is part of a Special Issue entitled "Interaction between repair, disease, & inflammation."

In vitro analyses of the immunosuppressive properties of neural stem/progenitor cells using anti-CD3/CD28-activated T cells.

Neural stem/progenitor cells (NSPCs) are multi-potent cells defined by their ability to self-renew and differentiate into cells of glial and neuronal lineage. Because of these properties, NSPCs have been proposed as therapeutic tools to replace lost neurons. Recent observations in animal models of immune-related diseases indicate that NSPCs display immunomodulatory properties that might be a great interest for cell therapy. In particular, transplantation of NSPCs might be very useful as local immunosuppressive agent to promote the long-term survival of neuronal xenotransplant in the brain. To study this possibility, we have analysed the impact of NSPCs on anti-CD3/CD28-activated T cells. In vitro analyses clearly show that porcine, rat, and mouse NSPCs inhibit the proliferation of activated T cells. This result raises new perspectives concerning the use of NSPCs in cell therapy.

AUF1 and Hu proteins in the developing rat brain: implication in the proliferation and differentiation of neural progenitors.

Posttranscriptional events such as RNA stabilization are important for cell differentiation, but little is known about the impact of AU-rich binding proteins (AUBPs) on the fate of neural cells. Expression of destabilizing AUBPs such as AUF1 and neuronal-specific stabilizing proteins such as HuB, HuC and HuD was therefore analyzed in the developing central nervous system. Real-time RT-PCR indicated a specific developmental pattern in the postnatal cerebellum, with a progressive down-regulation of AUF1 from P1, whereas HuB was strongly up-regulated at about P7. These changes were accompanied by a progressive increase in AUF1p45 and the disappearance of one HuB isoform from P15, suggesting particular roles for these AUBPs in the developing cerebellum. AUF1 was detected in the three main cerebellar layers, whereas Hu proteins were found only in postmitotic neurons. A role for Hu proteins in the early stages of neuronal differentiation is further supported by arrest of cell proliferation following induction of HuB or HuD expression in a neural stem cell line. The decrease in nestin expression suggest that HuD, but not HuB, favors the transition of neural progenitors into early neuroblasts, but other factors are most probably required for their full differentiation into neurons, insofar as GAP-43 was not detected in HuD-transfected cells. These data suggest critical roles for HuB at the very earliest stages of neuronal differentiation, such as cell cycle exit, and HuD might also be involved in the transition of neural progenitors into early neuroblasts. Taken together, the present results strengthen the importance of AUBPs in brain ontogenesis.

The signaling adaptor protein CD3zeta is a negative regulator of dendrite development in young neurons.

A novel idea is emergxsing that a large molecular repertoire is common to the nervous and immune systems, which might reflect the existence of novel neuronal functions for immune molecules in the brain. Here, we show that the transmembrane adaptor signaling protein CD3zeta, first described in the immune system, has a previously uncharacterized role in regulating neuronal development. Biochemical and immunohistochemical analyses of the rat brain and cultured neurons showed that CD3zeta is mainly expressed in neurons. Distribution of CD3zeta in developing cultured hippocampal neurons, as determined by immunofluorescence, indicates that CD3zeta is preferentially associated with the somatodendritic compartment as soon as the dendrites initiate their differentiation. At this stage, CD3zeta was selectively concentrated at dendritic filopodia and growth cones, actin-rich structures involved in neurite growth and patterning. siRNA-mediated knockdown of CD3zeta in cultured neurons or overexpression of a loss-of-function CD3zeta mutant lacking the tyrosine phosphorylation sites in the immunoreceptor tyrosine-based activation motifs (ITAMs) increased dendritic arborization. Conversely, activation of endogenous CD3zeta by a CD3zeta antibody reduced the size of the dendritic arbor. Altogether, our findings reveal a novel role for CD3zeta in the nervous system, suggesting its contribution to dendrite development through ITAM-based mechanisms.

Cell surface antigens on rat neural progenitors and characterization of the CD3 (+)/CD3 (-) cell populations.

While the hematopoietic lineage has been extensively studied using cluster of differentiation (CD) antibodies, very few data are available on the extracellular epitopes expressed by rat neural progenitors (rNPC) and their derivatives. In the present study, we used flow cytometry to screen 47 cell surface antigens, initially known as immune markers. The quantitative analyses were performed on rat neurospheres and compared with primary cultures of astroglial cells or cerebellar neurons. Several antigens such as CD80 or CD86 were clearly undetectable while others, like CD26 or CD161, showed a weak expression. Interestingly, 10% and 15% of the cells were immunopositive for CD172a and CD200, two immunoglobulin superfamily members preferentially expressed by glial or neuronal cells, respectively. Over 40% of the cells were immunopositive for CD3, CD71, or MHCI. The biological significance of the latter markers in rNPC remains to be determined but analyses of the CD3(-)/CD3(+) populations isolated by magnetic cell separation revealed differences in their cell fate. Indeed, CD3(+) cells did not establish neurospheres and differentiated mostly into GFAP(+) cells while CD3(-) cells were able to generate neurospheres upon mitogen treatment and gave rise to GFAP(+), A2B5(+), Tuj-1(+), and RIP(+) cells under differentiating conditions. In contrast, CD71(-)/CD71(+) cells did not show any significant difference in their proliferating and differentiating potentials. Finally, it is worth noting that an subpopulation of cells in rat neurospheres exhibit an immunoreactivity against anti-CD25 (IL2 receptor) and anti-CD62L (L-selectin) antibodies. The results reveal particular surface antigen profiles, giving new perspectives on the properties of rat brain-derived cells.