Carlos Bueno

Mesenchymal stem cells derived from dental tissues

Regeneration of tissues occurs naturally due to the existence of stem cells with the capacity to self-regenerate and differentiate; however, regenerative capacity decreases with age, and in many cases, regeneration is not sufficient to repair the damage produced by degenerative, ischaemic, inflammatory, or tumour-based diseases. In the last decade, advances have been made in the understanding of stem cells, the genes that control the alternative fates of quiescence and differentiation, and the niches that provide specific signals that modulate cell fate decisions. Embryonic stem-cell research is shedding light on the secrets of development. Adult stem cells (AS cells) are available from several sources. Bone marrow and connective tissue have been used in preliminary clinical trials for regenerative therapy. Recently, several types of AS cells have been isolated from teeth, including dental pulp stem cells, stem cells from human exfoliated deciduous teeth, periodontal ligament stem cells, dental follicle progenitor stem cells and stem cells from apical papilla. Preliminary data suggest that these cells have the capacity to differentiate into osteoblasts, adipocytes, chondrocytes and neural cells. If confirmed, these data would support the use of these cells, which are easily obtained from extracted teeth, in dental therapies, including in regenerative endodontics, providing a new therapeutic modality.

Mesenchymal stem cells rescue Purkinje cells and improve motor functions in a mouse model of cerebellar ataxia

Mesenchymal stem cells have been proven to be potentially effective in the treatment of a large variety of diseases, including neurodegenerative disorders. Of these, cerebellar ataxia is a group of disorders characterized by the degeneration of the cerebellum, particularly the Purkinje cells, responsible for motor coordination and control of the motor functions. To analyze the possibility of using bone marrow-derived mesenchymal stem cells in treating ataxia, we transplanted these cells in the cerebellum of newborn Lurcher mutant mice, a very aggressive mouse model characterized by the selective early post-natal death of Purkinje cells in the cerebellum. Two months after the surgical procedure, the treated mice presented significant improvements in the motor behavior tests performed. Histological analysis of the cerebellum indicated that the donor cells had migrated throughout the cerebellum, as well as a significant increase in the number of Purkinje cells. Many grafted stem cells were located adjacent to the Purkinje cell layer, and expressed BDNF, NT-3 or GDNF, neurotrophic factors implicated in Purkinje cell survival. Also, a small percentage of the grafted stem cells had fused with Purkinje cells. Thus, we have shown that mesenchymal stem cells are capable of integrating into the central nervous system, migrate towards the areas where neurodegenerative processes are occurring, and rescue the degenerating cells through cell trophic effects. This is an adequate and feasible model that could be translated into a therapeutic approach for clinical assays in neurodegenerative diseases.

Human adult periodontal ligament-derived cells integrate and differentiate after implantation into the adult mammalian brain.

Previous studies suggest that neural crest (NC)-derived stem cells may reside in NC derivatives including the human periodontal ligament (hPDL). The isolation and manipulation of autologous NC-derived cells could be an accessible source of adult neural stem cells for their use in cell replacement and gene transfer to the diseased central nervous system. In this study, we examined the expression of NC markers and neural differentiation potential of hPDL-derived cells both in vitro and in vivo. In vitro we found that hPDL-derived cells expressed stem cell markers (Oct3/4, Nestin, Sox2, and Musashi-1) and a subset of NC cell markers (Slug, p75NTR, Twist, and Sox9). hPDL-derived cells differentiated into neural-like cells based on cellular morphology and neural marker expression (TUJ1, MAP2, MAP1b, GAD65/67, GABA, NeuN, ChAT, GAT1, synaptophysin, GFAP, NG2, and O4). In vivo, hPDL-derived cells survive, migrate, and give rise to DCX+, NF-M+, GABA+, GFAP+, and NG2+ cells after grafting the adult mouse brain. Some of the grafted hPDL-derived cells were located in stem cell niches such as the ventricular epithelium and the subventricular zone of the anterolateral ventricle wall as well as in the subgranular zone of the hippocampal dentate gyrus. Thus, the hPDL contains stem cells that originate from the NC and can differentiate into neural cells. The engraftment and differentiation properties of hPDL-derived cells in the adult brain indicate that they are a potential stem cell source to be used in neuroregenerative and/or neurotrophic medicine.

Wnt signal specifies the intrathalamic limit and its organizer properties by regulating Shh induction in the alar plate.

The structural complexity of the brain depends on precise molecular and cellular regulatory mechanisms orchestrated by regional morphogenetic organizers. The thalamic organizer is the zona limitans intrathalamica (ZLI), a transverse linear neuroepithelial domain in the alar plate of the diencephalon. Because of its production of Sonic hedgehog, ZLI acts as a morphogenetic signaling center. Shh is expressed early on in the prosencephalic basal plate and is then gradually activated dorsally within the ZLI. The anteroposterior positioning and the mechanism inducing Shh expression in ZLI cells are still partly unknown, being a subject of controversial interpretations. For instance, separate experimental results have suggested that juxtaposition of prechordal (rostral) and epichordal (caudal) neuroepithelium, anteroposterior encroachment of alar lunatic fringe (L-fng) expression, and/or basal Shh signaling is required for ZLI specification. Here we investigated a key role of Wnt signaling in the molecular regulation of ZLI positioning and Shh expression, using experimental embryology in ovo in the chick. Early Wnt expression in the ZLI regulates Gli3 and L-fng to generate a permissive territory in which Shh is progressively induced by planar signals of the basal plate.

Human Adipose Stem Cell–Conditioned Medium Increases Survival of Friedreich's Ataxia Cells Submitted to Oxidative Stress

Friedreichs ataxia (FA) is a multisystemic disorder characterized by progressive gait, ataxia, and cardiomyopathy. There are few treatments for this disease; thus, we analyzed in vitro the possible beneficial effect of adult stem cells in FA. To this end, human adipose stem cells from healthy individuals and periodontal ligament cells from FA patients were isolated and cultured. FA cells are especially vulnerable to oxidative stress; thus, they were submitted to this condition and cultured in adipose stem cell-conditioned medium. This resulted in increased cell survival and upregulation of oxidative-stress-related genes as well as frataxin, among other genes. A number of trophic factors were shown to be expressed by the adipose stem cells, especially brain-derived neurotrophic factor (BDNF), which was also identified in the conditioned medium. The culture of the ataxic cells under oxidative stress and in the presence of this trophic factor confirmed its protective effect. Thus, this work demonstrates that adipose stem cell-conditioned medium from healthy individuals is capable of changing the transcription levels of oxidative-stress-related genes in cells that are particularly susceptible to this condition, avoiding cellular degeneration. Also, this work shows how neurotrophic factors, particularly BDNF, are capable of increasing cell survival in response to oxidative stress, which occurs in many neurodegenerative diseases.

Pallial origin of basal forebrain cholinergic neurons in the nucleus basalis of Meynert and horizontal limb of the diagonal band nucleus

The majority of the cortical cholinergic innervation implicated in attention and memory originates in the nucleus basalis of Meynert and in the horizontal limb of the diagonal band nucleus of the basal prosencephalon. Functional alterations in this system give rise to neuropsychiatric disorders as well as to the cognitive alterations described in Parkinson and Alzheimers diseases. Despite the functional importance of these basal forebrain cholinergic neurons very little is known about their origin and development. Previous studies suggest that they originate in the medial ganglionic eminence of the telencephalic subpallium; however, our results identified Tbr1-expressing, reelin-positive neurons migrating from the ventral pallium to the subpallium that differentiate into cholinergic neurons in the basal forebrain nuclei projecting to the cortex. Experiments with Tbr1 knockout mice, which lack ventropallial structures, confirmed the pallial origin of cholinergic neurons in Meynert and horizontal diagonal band nuclei. Also, we demonstrate that Fgf8 signaling in the telencephalic midline attracts these neurons from the pallium to follow a tangential migratory route towards the basal forebrain.

Glioblastoma progression is assisted by induction of immunosuppressive function of pericytes through interaction with tumor cells

The establishment of immune tolerance during Glioblastoma Multiforme (GBM) progression, is characterized by high levels expression of anti-inflammatory cytokines, which suppress the function of tumor assocciated myeloid cells, and the activation and expansion of tumor antigen specific T cells. However, the mechanisms underlying the failed anti-tumor immune response around the blood vessels during GBM, are poorly understood. The consequences of possible interactions between cancer cells and the perivascular compartment might affect the tumor growth. In this work we show for the first time that GBM cells induce immunomodulatory changes in pericytes in a cell interaction-dependent manner, acquiring an immunosuppresive function that possibly assists the evasion of the anti-tumor immune response and consequently participates in tumor growth promotion. Expression of high levels of anti-inflammatory cytokines was detected in vitro and in vivo in brain pericytes that interacted with GBM cells (GBC-PC). Furthermore, reduction of surface expression of co-stimulatory molecules and major histocompatibility complex molecules in GBC-PC correlated with a failure of antigen presentation to T cells and the acquisition of the ability to supress T cell responses. In vivo, orthotopic xenotransplant of human glioblastoma in an immunocompetent mouse model showed significant GBM cell proliferation and tumor growth after the establishment of interspecific immunotolerance that followed GMB interaction with pericytes.

Nuclear polymorphism and non-proliferative adult neurogenesis in human neural crest-derived cells.

Abstract Self-renewal and lineage regulation of neural stem cells in the adult mammalian brain (aNSCs) are still far from been understood. Although previous studies have reported that some aNSCs in neurogenic niches showed irregular nuclei, their functional significance remains elusive. We used neural crest-derived human periodontal ligament stem cells (hPDLSCs) as an in vitro cell model of neurogenesis to investigate the functional significance of nuclear polymorphisms. Here, we show that hPDLSCs-derived neurons are not directly generated through cell division from stem cells. In fact, the cell shape of neural precursors is reset and start their neuronal development as round spheres. The hPDLSCs-derived neurons gradually adopted a complex morphology by forming several processes, that grew and arborized, adquiring dendritic-like and axonal-like identities, giving rise to a variety of neuron-like morphologies. To our knowledge, this article provides the first observation of these morphological events during in vitro neurogenesis and neuron polarization in human aNCSCs, and we have discovered a transient cell nuclei lobulation coincident to in vitro neurogenesis, without being related to cell proliferation. We observed that small DNA containing structures move within the cell to specific directions and temporarily form lobed nuclei. Morphological analysis also reveals that neurogenic niches in the adult mouse brain contains cells with nuclear shapes highly similar to those observed during in vitro neurogenesis from hPDLSCs. Our results provide strong evidence that neuronal differentiation from aNSCs may also occur during in vivo adult mammalian neurogenesis without being related to cell proliferation. In addition, we demonstrate that hPDLSCs-derived neurons display a sequence of morphologic development highly similar to those observed in primary neuronal cultures derived from rodent brains during neurogenesis, providing strong evidence that it is possible to reproduce neurogenic processes and obtain neurons from hPDLSCs. Beyond the central nervous system, the presence of lobed nuclei has been reported in most blood and immune cells, but the functional significance of multilobed nuclear structures is not yet stablished. Our results suggest that multilobed nuclear structures is associated to nuclear movement within the cell.Self-renewal and lineage regulation of neural stem cells in the adult mammalian brain (aNSCs) are still far from been understood. Although previous studies have reported that some aNSCs in neurogenic niches showed irregular nuclei, their functional significance remains elusive. We used neural crest-derived human periodontal ligament stem cells (hPDLSCs) as an in vitro cell-model of neurogenesis to investigate the functional significance of nuclear polymorphisms. Here, we show that hPDLSCs-derived neurons are not directly generated through cell division from stem cells. In fact, the cell shape of neural precursors is reset and start their neuronal development as round spheres. the hPDLSCs-derived neurons gradually adopted a complex morphology by forming several processes, that grew and arborized, adquiring dendritic-like and axonal-like identities, giving rise to a variety of neuron-like morphologies. To our knowledge, this article provides the first observation of these morphological events during in vitro neurogenesis and neuron polarization in human aNCSCs, and we have discovered a transient cell nuclei lobulation coincident to in vitro neurogenesis, without being related to cell proliferation. Morphological analysis also reveals that the V-SVZ of the anterolateral ventricle wall and the SGZ of the hippocampal dentate gyrus in the adult mouse brain contains cells with nuclear shapes highly similar to those observed during in vitro neurogenesis from hPDLSCs. Our results provide strong evidence that neuronal differentiation from aNSCs may also occur during in vivo adult mammalian neurogenesis without being related to cell proliferation. In addition, we demonstrate that hPDLSC-derived neurons and primary neuronal cultures derived from rodent brains show similar polarity formation patterns during neurogenesis, providing strong evidence that it is possible to reproduce neurogenic processes and obtain human neurons from hPDLSCs. Thus, hPDLSCs could be used as an in vitro human cell-based model for neurogenesis and neuronal polarization.

Clinical Phenotypes Associated to Engrailed 2 Gene Alterations in a Series of Neuropediatric Patients

The engrailed homeobox protein (EN) plays an important role in the regionalization of the neural tube. EN distribution regulates the cerebellum and midbrain morphogenesis, as well as retinotectal synaptogenesis. In humans, the EN1 and EN2 genes code for the EN family of transcription factors. Genetic alterations in the expression of EN2 have been related to different neurologic conditions and more particularly to autism spectrum disorders (ASD). We aimed to study and compare the phenotypes of three series of patients: (1) patients with encephalic structural anomalies (ESA) and abnormalities in the genomic (DNA) and/or transcriptomic (RNAm) of EN2 (EN2-g), (2) ESA patients having other gene mutations (OG-g), and (3) ESA patients free of these mutations (NM-g). Subjects and Methods: We have performed a descriptive study on 109 patients who suffer from mental retardation (MR), cerebral palsy (CP), epilepsy (EP), and behavioral disorders (BD), showing also ESA in their encephalic MRI. We studied genomic DNA and transcriptional analysis (cDNA) on EN2 gene (EN2), and in other genes (OG): LIS1, PTAFR, PAFAH1B2, PAFAH1B3, FGF8, PAX2, D17S379, D17S1866, and SMG6 (D17S5), as a routine genetic diagnosis in ESA patients. Results: From 109 patients, fifteen meet the exclusion criteria. From the remaining 94 patients, 12 (12.8%) showed mutations in EN2 (EN2-g), 20 showed mutations in other studied genes (OG-g), and 62 did not showed any mutation (NM-g). All EN2-g patients, suffered from MR, nine EP, seven BD and four CP. The proportions of these phenotypes in EN2-g did not differ from those in the OG-g, but it was significantly higher when comparing EN2-g with NM-g (MR: p = 0.013; EP: p = 0.001; BD: p = 0.0001; CP: p = 0.07, ns). Groups EN2-g and OG-g showed a 100 and a 70% of comorbidity, respectively, being significantly (p = 0.04) greater than NM-group (62.9%). Conclusion: Our series reflects a significant effect of EN2 gene alterations in neurodevelopmental abnormalities associated to ESA. Conversely, although these EN2 related anomalies might represent a predisposition to develop brain diseases, our results did not support direct relationship between EN2 mutations and specific clinical phenotypes.

03-P085 Molecular characteristics of FGF8b mouse protein during its morphogenetic activity in the mouse mid-hind brain boundary (MHB)

ated nerves and leads to corneal abnormalities. Pax6 expression was observed in a subset of cells in the mouse maxillomandibular trigeminal placode and the ganglion. This expression has never been described previously. A possible specific role of Pax6 in trigeminal ganglion development was hypothesised. The trigeminal ganglion is formed in Pax6 / mice, and analysis of the trigeminal nerve major divisions shows that the ophthalmic branch (opV) is present. However, in the absence of any eyes in the homozygous mutants, it projects solely to the eyelids. On the other hand, the maxillary branch (Mx) is abnormal, with reduced arborisation. In particular, the whisker follicles, the major target of the Mx division, seem to be abnormally innervated in Pax6 / mice with defects in the encirclement of the axons around the follicles. These findings confirmed the association between changes in Pax6 gene dosage and axonal pathfinding, as has already been described for other discrete neuronal populations. Moreover, the downregulated expression in Pax6 / mice of two neuronal markers, TrkC and Ngn2, suggests defects in the specification of neurones, confirming the role of Pax6 in trigeminal precursor specification.