Stem cell transplantation in brain tumors: a new field for molecular imaging?

Neural stem cells have been proposed as a new and promising treatment modality in various pathologies of the central nervous system, including malignant brain tumors. However, the underlying mechanism by which neural stem cells target tumor areas remains elusive. Monitoring of these cells is currently done by use of various modes of molecular imaging, such as optical imaging, magnetic resonance imaging and positron emission tomography, which is a novel technology for visualizing metabolism and signal transduction to gene expression. In this new context, the microenvironment of (malignant) brain tumors and the blood-brain barrier gains increased interest. The authors of this review give a unique overview of the current molecular-imaging techniques used in different therapeutic experimental brain tumor models in relation to neural stem cells. Such methods for molecular imaging of gene-engineered neural stem/progenitor cells are currently used to trace the location and temporal level of expression of therapeutic and endogenous genes in malignant brain tumors, closing the gap between in vitro and in vivo integrative biology of disease in neural stem cell transplantation.

Taurine increases hippocampal neurogenesis in aging mice.

Aging is associated with increased inflammation and reduced hippocampal neurogenesis, which may in turn contribute to cognitive impairment. Taurine is a free amino acid found in numerous diets, with anti-inflammatory properties. Although abundant in the young brain, the decrease in taurine concentration with age may underlie reduced neurogenesis. Here, we assessed the effect of taurine on hippocampal neurogenesis in middle-aged mice. We found that taurine increased cell proliferation in the dentate gyrus through the activation of quiescent stem cells, resulting in increased number of stem cells and intermediate neural progenitors. Taurine had a direct effect on stem/progenitor cells proliferation, as observed in vitro, and also reduced activated microglia. Furthermore, taurine increased the survival of newborn neurons, resulting in a net increase in adult neurogenesis. Together, these results show that taurine increases several steps of adult neurogenesis and support a beneficial role of taurine on hippocampal neurogenesis in the context of brain aging.

L’effet du vieillissement sur les cellules souches neurales adultes

La neurogenèse persiste à l’âge adulte dans deux régions du système nerveux central (SNC) des mammifères : la zone sous-ventriculaire (SVZ) du cerveau antérieur et la zone sous-granulaire (SGZ) de l’hippocampe. Cette neurogenèse est possible grâce à la capacité de prolifération des cellules souches présentes dans les niches de la SVZ et la SGZ, mais en vieillissant, le cerveau subit une diminution dramatique du nombre de cellules souches neurales adultes (CSNa), une diminution de la prolifération cellulaire et une altération des niches de neurogenèse. Cependant, une importante question reste sans réponse : comment la perte tardive des CSNa est temporellement reliée aux changements de l’activité de prolifération et de la structure de la principale niche de neurogenèse (la SVZ)? Afin d’avoir un aperçu sur les événements initiaux, nous avons examiné les changements des CSNa et de leur niche dans la SVZ entre le jeune âge et l’âge moyen. La niche de la SVZ des souris d’âge moyen (12 mois) subit une réduction de l’expression des marqueurs de plusieurs sous-populations de précurseurs neuraux en comparaison avec les souris jeunes adultes (2 mois). Anatomiquement, cela est associé avec des anomalies cytologiques, incluant une atrophie générale de la SVZ, une perte de la couche de cellules sousépendymaires par endroit et l’accumulation de gouttelettes lipidiques de grande taille dans l’épendyme. Fonctionnellement, ces changements sont corrélés avec une diminution de l’activité de la SVZ et une réduction du nombre de nouveaux neurones arrivant aux bulbes olfactifs. Pour déterminer si les CSNa de la SVZ ont subi des changements visibles, nous avons évalué les paramètres clés des CSNa in vivo et in vitro. La culture cellulaire montre qu’un nombre équivalent de CSNa ayant la capacité de former des neurosphères peut être isolé du cerveau du jeune adulte et d’âge moyen. Cependant, à l’âge moyen, les précurseurs neuraux semblent moins sensibles aux facteurs de croissance durant leur différenciation in vitro. Les CSNa donnent des signes de latence in vivo puisque leur capacité d’incorporation et de rétention du BrdU diminue. Ensemble, ces données démontrent que, tôt dans le processus du vieillissement, les CSNa et leur niche dans la SVZ subissent des changements significatifs, et suggèrent que la perte de CSNa liée au vieillissement est secondaire à ces événements.

Les cytokines inflammatoires modulent la prolifération et la différenciation in vitro des cellules souches/progénitrices de la moelle épinière.

La moelle épinière (MÉ) est essentielle pour relier les informations motrices et sensorielles entre le cerveau et la périphérie. Malheureusement, elle peut facilement être endommagée suite à un traumatisme médullaire (TM) ou des pathologies comme la sclérose en plaques. Chez les vertébrés inférieurs, tels les amphibiens, la MÉ lésée se régénère via ses cellules souches endogènes, alors que celle des mammifères démontre une très faible habileté régénératrice post-traumatique. Des travaux récents ont démontré que la MÉ des mammifères contient des cellules souches neurales latentes correspondant aux cellules épendymaires du canal central. D’autres études ont prouvé qu’à la suite d’un TM, les cellules souches épendymaires (cSÉ) prolifèrent, migrent vers le site de la lésion et se différencient principalement en cellules gliales. Promouvoir la régénération de la MÉ endommagée via la modulation des cellules souches endogènes devient donc une voie thérapeutique intéressante. Isolant des cellules souches/progénitrices de la MÉ via la culture de neurosphères (NS), nos études in vitro, en présence de cytokines inflammatoires ou de milieu conditionné auxmacrophages, suggèrent que la réponse inflammatoire influence fortement la prolifération et la différentiation des cSÉ. Dans l’objectif de définir le programme génétique relié à l’activation des cSÉ de la MÉ, nous avons débuté l’élaboration d’un protocole d’isolement des cSÉ à l’aide d’un modèle de souris transgénique.

L'expression de nestine est associée à l'entrée des cardiomyocytes de rats néonataux dans le cycle cellulaire.

L’infarctus du myocarde est une des conséquences possibles de l’ischémie cardiaque; il se traduit par la mort des cardiomyocytes se situant en aval du blocus coronaire, puis par la formation d’une cicatrice formée essentiellement de dépôts de matrices extracellulaires sécrétées par les myofibroblastes. Nestine est une protéine filamenteuse intermédiaire de classe VI couramment associée à la prolifération et à la migration cellulaire. Chez l’homme et les rongeurs, à la suite d’un infarctus du myocarde, une sous-population de cardiomyocytes localisée à la zone infarcie/péri-infarcie exprimait la forme striée de nestine. Le but principal de cette étude était de déterminer la source cellulaire des cardiomyocytes nestine (+) observée dans le cœur infarci ainsi que le mécanisme de signalisation cellulaire sous-jacent impliqué dans l’expression de nestine. L’utilisation de souris transgénique a révélé que l’augmentation des cardiomyocytes nestine (+) dans le cœur infarci des souris n’était pas attribuable à la différenciation de cellules souches/progénitrices nestine (+) en cardiomyocytes nestine (+). Le traitement des cardiomyocytes ventriculaires de rats néonataux avec l’activateur des protéines kinases C PDBu et l’inhibition concomitante des voies p38 MAPK a mené à l’augmentation du nombre de ces cellules exprimant nestine. De plus, une population importante de cardiomyocytes ventriculaires de rats néonataux a incorporé la bromodéxoxyuridine, signe d’une capacité à réentrer dans le cycle cellulaire et à synthétiser de l’ADN. Sur la base de ces observations, l’apparition de cardiomyocytes nestine (+) dans le cœur infarci des rongeurs et des hommes pourrait possiblement refléter une sous-population de cardiomyocytes en prolifération tentant de régénérer le cœur infarci.

Implications physiopathologiques de la Nestine lors du remodelage pulmonaire et cardiaque à la suite de l’infarctus du myocarde, du diabète et de l’hypertension pulmonaire

Il est reconnu que la protéine filamenteuse intermédiaire Nestine est exprimée lors du processus de cicatrisation et du remodelage fibrotique. De plus, nous avons identifié l’expression de la Nestine au sein de deux populations distinctes qui sont directement impliquées dans les réponses de fibroses réparative et réactive. Ainsi, une population de cellules souches neurales progénitrices résidentes du coeur de rat adulte exprime la Nestine et a été identifiée à titre de substrat de l’angiogenèse et de la neurogenèse cardiaque. Également, la Nestine est exprimée par les myofibroblastes cicatriciels cardiaques et il a été établi que la protéine filamenteuse intermédiaire joue un rôle dans la prolifération de ces cellules. Ainsi, l’objectif général de cette thèse était de mieux comprendre les évènements cellulaires impliqués dans la réponse neurogénique des cellules souches neurales progénitrices résidentes cardiaques Nestine(+) (CSNPRCN(+)) lors de la fibrose réparative cardiaque et d’explorer si l’apparition de fibroblastes Nestine(+) est associée avec la réponse de fibrose réactive secondaire du remodelage pulmonaire. Une première publication nous a permis d’établir qu’il existe une régulation à la hausse de l’expression de la GAP43 (growth associated protein 43) et que cet événement transitoire précède l’acquisition d’un phénotype neuronal par les CSNPRCN(+) lors du processus de cicatrisation cardiaque chez le rat ayant subi un infarctus du myocarde. De plus, la surimposition de la condition diabétique de type 1, via l’injection unique de Streptozotocine chez le rat, abolit la réponse neurogénique des CSNPRCN(+), qui est normalement induite à la suite de l’ischémie cardiaque ou de l’administration de 6-hydroxydopamine. Le second article a démontré que le développement aigu de la fibrose pulmonaire secondaire de l’infarctus du myocarde chez le rat est associé avec une augmentation de l’expression protéique de la Nestine et de l’apparition de myofibroblastes pulmonaires Nestine(+). Également, le traitement de fibroblastes pulmonaires avec des facteurs de croissances peptidiques pro-fibrotiques a augmenté l’expression de la Nestine par ces cellules. Enfin, le développement initial de la condition diabétique de type 1 chez le rat est associé avec une absence de fibrose réactive pulmonaire et à une réduction significative des niveaux protéiques et d’ARN messager de la Nestine pulmonaire. Finalement, la troisième étude représentait quant à elle un prolongement de la deuxième étude et a alors examiné le remodelage pulmonaire chronique chez un modèle établi d’hypertension pulmonaire. Ainsi, les poumons de rats adultes mâles soumis à l’hypoxie hypobarique durant 3 semaines présentent un remodelage vasculaire, une fibrose réactive et une augmentation des niveaux d’ARN messager et de la protéine Nestine. De plus, nos résultats ont démontré que la Nestine, plutôt que l’alpha-actine du muscle lisse, est un marqueur plus approprié des diverses populations de fibroblastes pulmonaires activés. Également, nos données suggèrent que les fibroblastes pulmonaires activés proviendraient en partie de fibroblastes résidents, ainsi que des processus de transition épithélio-mésenchymateuse et de transition endothélio-mésenchymateuse. Collectivement, ces études ont démontré que des populations distinctes de cellules Nestine(+) jouent un rôle majeur dans la fibrose réparative cardiaque et la fibrose réactive pulmonaire.

Protochordate Zic genes define primitive somite compartments and highlight molecular changes underlying neural crest evolution

The vertebrate Zic gene family encodes C2H2 zinc finger transcription factors closely related to the Gli proteins. Zic genes are expressed in multiple areas of developing vertebrate embryos, including the dorsal neural tube where they act as potent neural crest inducers. Here we describe the characterization of a Zic ortholog from the amphioxus Branchiostoma floridae and further describe the expression of a Zic ortholog from the ascidian Ciona intestinalis. Molecular phylogenetic analysis and sequence comparisons suggest the gene duplications that formed the vertebrate Zic family were specific to the vertebrate lineage. In Ciona maternal CiZic/Ci-macho1 transcripts are localized during cleavage stages by asymmetric cell division, whereas zygotic expression by neural plate cells commences during neurulation. The amphioxus Zic ortholog AmphiZic is expressed in dorsal mesoderm and ectoderm during gastrulation, before being eliminated first from midline cells and then from all neurectoderm during neurulation. After neurulation, expression is reactivated in the dorsal neural tube and dorsolateral somite. Comparison of CiZic and AmphiZic expression with vertebrate Zic expression leads to two main conclusions. First, Zic expression allows us to define homologous compartments between vertebrate and amphioxus somites, showing primitive subdivision of vertebrate segmented mesoderm. Second, we show that neural Zic expression is a chordate synapomorphy, whereas the precise pattern of neural expression has evolved differently on the different chordate lineages. Based on these observations we suggest that a change in Zic regulation, specifically the evolution of a dorsal neural expression domain in vertebrate neurulae, was an important step in the evolution of the neural crest.

A novel alternative to cryopreservation for the short-term storage of stem cells for use in cell therapy using alginate encapsulation

Efficient transport of stem/progenitor cells without affecting their survival and function is a key factor in any practical cell-based therapy. However, the current approach using liquid nitrogen for the transfer of stem cells requires a short delivery time window is technically challenging and financially expensive. The present study aims to use semipermeable alginate hydrogels (crosslinked by strontium) to encapsulate, store, and release stem cells, to replace the conventional cryopreservation method for the transport of therapeutic cells within world-wide distribution time frame. Human mesenchymal stem cell (hMSC) and mouse embryonic stem cells (mESCs) were successfully stored inside alginate hydrogels for 5 days under ambient conditions in an air-tight environment (sealed cryovial). Cell viability, of the cells extracted from alginate gel, gave 74% (mESC) and 80% (hMSC) survival rates, which compared favorably to cryopreservation. More importantly, the subsequent proliferation rate and detection of common stem cell markers (both in mRNA and protein level) from hMSCs and mESCs retrieved from alginate hydrogels were also comparable to (if not better than) results gained following cryopreservation. In conclusion, this new and simple application of alginate hydrogel encapsulation may offer a cheap and robust alternative to cryopreservation for the transport and storage of stem cells for both clinical and research purposes.

Morphological characterization of periodontium-derived human stem cells

The aim of this study has been to characterize adult human somatic periodontium-derived stem cells (PDSCS) isolated from human periodontium and to follow their differentiation after cell culture. PDSCS were isolated from human periodontal tissue and cultured as spheres in serum-free medium. After 10 days the primary spheres were dissociated and the secondary spheres sub-cultured for another 1-2 weeks. Cells from different time points were analyzed, and immunohistochemical and electron microscopic investigations carried out. Histological analysis showed differentiation of spheres deriving from the PDSCS with central production of extracellular matrix beginning 3 days after sub-culturing. Isolated PDSCS developed pseudopodia which contained actin. Tubulin was found in the central portion of the cells. Pseudopodia between different cells anastomosed, indicating intercellular transport. Immunostaining for osteopontin demonstrated a positive reaction in primary spheres and within extracellular matrix vesicles after sub-culturing. In cell culture under serum-free conditions human PDSCS form spheres which are capable of producing extracellular matrix. Further investigations have do be carried out to investigate the capability of these cells to differentiate into osteogenic progenitor cells.

Ascl1 coordinately regulates gene expression and the chromatin landscape during neurogenesis

The proneural transcription factor Ascl1 coordinates gene expression in both proliferating and differentiating progenitors along the neuronal lineage. Here, we used a cellular model of neurogenesis to investigate how Ascl1 interacts with the chromatin landscape to regulate gene expression when promoting neuronal differentiation. We find that Ascl1 binding occurs mostly at distal enhancers and is associated with activation of gene transcription. Surprisingly, the accessibility of Ascl1 to its binding sites in neural stem/progenitor cells remains largely unchanged throughout their differentiation, as Ascl1 targets regions of both readily accessible and closed chromatin in proliferating cells. Moreover, binding of Ascl1 often precedes an increase in chromatin accessibility and the appearance of new regions of open chromatin, associated with de novo gene expression during differentiation. Our results reveal a function of Ascl1 in promoting chromatin accessibility during neurogenesis, linking the chromatin landscape at Ascl1 target regions with the temporal progression of its transcriptional program.

Controversial role of toll-like receptor 4 in adult stem cells

Adult or somatic stem cells are tissue-resident cells with the ability to proliferate, exhibit self-maintenance as well as to generate new cells with the principal phenotypes of the tissue in response to injury or disease. Due to their easy accessibility and their potential use in regenerative medicine, adult stem cells raise the hope for future personalisable therapies. After infection or during injury, they are exposed to broad range of pathogen or damage-associated molecules leading to changes in their proliferation, migration and differentiation. The sensing of such damage and infection signals is mostly achieved by Toll-Like Receptors (TLRs) with Toll-like receptor 4 being responsible for recognition of bacterial lipopolysaccharides (LPS) and endogenous danger-associated molecular patterns (DAMPs). In this review, we examine the current state of knowledge on the TLR4-mediated signalling in different adult stem cell populations. Specifically, we elaborate on the role of TLR4 and its ligands on proliferation, differentiation and migration of mesenchymal stem cells, hematopoietic stem cells as well as neural stem cells. Finally, we discuss conceptual and technical pitfalls in investigation of TLR4 signalling in stem cells.

The neurogenic potential of astrocytes is regulated by inflammatory signals

Although the adult brain contains neural stem cells (NSCs) that generate new neurons throughout life, these astrocyte-like populations are restricted to two discrete niches. Despite their terminally differentiated phenotype, adult parenchymal astrocytes can re-acquire NSC-like characteristics following injury, and as such, these 'reactive' astrocytes offer an alternative source of cells for central nervous system (CNS) repair following injury or disease. At present, the mechanisms that regulate the potential of different types of astrocytes are poorly understood. We used in vitro and ex vivo astrocytes to identify candidate pathways important for regulation of astrocyte potential. Using in vitro neural progenitor cell (NPC)-derived astrocytes, we found that exposure of more lineage-restricted astrocytes to either tumor necrosis factor alpha (TNF-α) (via nuclear factor-κB (NFκB)) or the bone morphogenetic protein (BMP) inhibitor, noggin, led to re-acquisition of NPC properties accompanied by transcriptomic and epigenetic changes consistent with a more neurogenic, NPC-like state. Comparative analyses of microarray data from in vitro-derived and ex vivo postnatal parenchymal astrocytes identified several common pathways and upstream regulators associated with inflammation (including transforming growth factor (TGF)-β1 and peroxisome proliferator-activated receptor gamma (PPARγ)) and cell cycle control (including TP53) as candidate regulators of astrocyte phenotype and potential. We propose that inflammatory signalling may control the normal, progressive restriction in potential of differentiating astrocytes as well as under reactive conditions and represent future targets for therapies to harness the latent neurogenic capacity of parenchymal astrocytes.

Human neural stem cell-derived cultures in three- dimensional substrates form spontaneously functional neuronal networks

Differentiated human neural stem cells were cultured in an inert three-dimensional (3D) scaffold and, unlike two-dimensional (2D) but otherwise comparable monolayer cultures, formed spontaneously active, functional neuronal networks that responded reproducibly and predictably to conventional pharmacological treatments to reveal functional, glutamatergic synapses. Immunocytochemical and electron microscopy analysis revealed a neuronal and glial population, where markers of neuronal maturity were observed in the former. Oligonucleotide microarray analysis revealed substantial differences in gene expression conferred by culturing in a 3D vs a 2D environment. Notable and numerous differences were seen in genes coding for neuronal function, the extracellular matrix and cytoskeleton. In addition to producing functional networks, differentiated human neural stem cells grown in inert scaffolds offer several significant advantages over conventional 2D monolayers. These advantages include cost savings and improved physiological relevance, which make them better suited for use in the pharmacological and toxicological assays required for development of stem cell-based treatments and the reduction of animal use in medical research.

In Vivo Comparison of Hard Tissue Regeneration with Human Mesenchymal Stem Cells Processed with Either the FICOLL Method or the BMAC Method

Objective: To compare new bone formation in maxillary sinus augmentation procedures using biomaterial associated with mesenchymal stem cells (MSCs) separated by two different isolation methods. Background: In regenerative medicine open cell concentration systems are only allowed for clinical application under good manufacturing practice conditions. Methods: Mononuclear cells, including MSCs, were concentrated with either the synthetic poylsaccharid (FICOLL) method (classic open system-control group, n = 6 sinus) or the bone marrow aspirate concentrate (BMAC) method (closed system-test group, n = 12 sinus) and transplanted in combination with biomaterial. A sample of the cells was characterized by their ability to differentiate. After 4.1 months (SD +/- 1.0) bone biopsies were obtained and analyzed. Results: The new bone formation in the BMAC group was 19.9% (90% confidence interval [CI], 10.9-29), and in the FICOLL group was 15.5% (90% CI, 8.6-22.4). The 4.4% difference was not significant (90% CI, -4.6-13.5; p = 0.39). MSCs could be differentiated into osteogenic, chondrogenic, and adipogenic lineages. Conclusion: MSCs harvested from bone marrow aspirate in combination with bovine bone matrix particles can form lamellar bone and provide a reliable base for dental implants. The closed BMAC system is suited to substitute the open FICOLL system in bone regeneration procedures.

Systemic delivery of adult stem cells improves cardiac function in spontaneously hypertensive rats

Heart regeneration after myocardial infarction (MI) can occur after cell therapy, but the mechanisms, cell types and delivery methods responsible for this improvement are still under investigation. In the present study, we evaluated the impact of systemic delivery of bone marrow cells (BMC) and cultivated mesenchymal stem cells (MSC) on cardiac morphology, function and mortality in spontaneously hypertensive rats (SHR) submitted to coronary occlusion. Female syngeneic adult SHR, submitted or not (control group; C) to MI, were treated with intravenous injection of MSC (MI + MSC) or BMC (MI + BM) from male rats and evaluated after 1, 15 and 30 days by echocardiography. Systolic blood pressure (SBP), functional capacity, histology, mortality rate and polymerase chain reaction for the Y chromosome were also analysed. Myocardial infarction induced a decrease in SBP and BMC, but not MSC, prevented this decrease. An improvement in functional capacity and ejection fraction (38 +/- 4, 39 +/- 3 and 58 +/- 2% for MI, MI + MSC and MI + BM, respectively; P < 0.05), as well as a reduction of the left ventricle infarcted area, were observed in rats from the MI + BM group compared with the other three groups. Treated animals had a significantly reduced lesion tissue score. The mortality rate in the C, MI + BM, MI + MSC and MI groups was 0, 0, 16.7 and 44.4%, respectively (P < 0.05 for the MI + MSC and MI groups compared with the C and MI + BM groups). The results of the present study suggest that systemic administration of BMC can improve left ventricular function, functional capacity and, consequently, reduce mortality in an animal model of MI associated with hypertension. We speculate that the cells transiently home to the myocardium, releasing paracrine factors that recruit host cells to repair the lesion.