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Summary Despite heterogeneity across the six layers of the mammalian cortex, all excitatory neurons are generated from a single founder population of neuroepithelial stem cells. However, how these progenitors alter their layer competence over time remains unknown. Here, we used human embryonic stem cell-derived cortical progenitors to examine the role of fibroblast growth factor (FGF) and Notch signaling in influencing cell fate, assessing their impact on progenitor phenotype, cell-cycle kinetics, and layer specificity. Forced early cell-cycle exit, via Notch inhibition, caused rapid, near-exclusive generation of deep-layer VI neurons. In contrast, prolonged FGF2 promoted proliferation and maintained progenitor identity, delaying laminar progression via MAPK-dependent mechanisms. Inhibiting MAPK extended cell-cycle length and led to generation of layer-V CTIP2+ neurons by repressing alternative laminar fates. Taken together, FGF/MAPK regulates the proliferative/neurogenic balance in deep-layer corticogenesis and provides a resource for generating layer-specific neurons for studying development and disease., Highlights • FGF/MAPK regulates the proliferative/neurogenic balance in deep-layer corticogenesis • FGF/MAPK signaling maintains the progenitor pool and generates layer-VI neurons • MAPK inhibition prolongs cell cycle to yield layer-V neurons, repressing other fates • Protocols to generate layer-specific cortical neurons to study development and disease, Despite heterogeneity across human cortical layers, all excitatory neurons evolve from a single founder stem cell population. How these progenitors alter layer competence over time is unknown. Using human pluripotent stem cell-derived cortical progenitors, we demonstrate that FGF/MAPK signaling regulates the proliferative/neurogenic balance in deep-layer corticogenesis. Modulating FGF/MAPK enables generation of layer-specific neurons to study development and disease.

Midbrain dopaminergic (DA) neurons include many subtypes characterized by their location, connectivity and function. Surprisingly, mechanisms underpinning the specification of A9 neurons [responsible for motor function, including within ventral midbrain (VM) grafts for treating Parkinson's disease (PD)] over adjacent A10, remains largely speculated. We assessed the impact of synaptic targeting on survival, integration, and phenotype acquisition of dopaminergic neurons within VM grafts generated from fetal tissue or human pluripotent stem cells (PSCs). VM progenitors were grafted into female mice with 6OHDA-lesions of host midbrain dopamine neurons, with some animals also receiving intrastriatal quinolinic acid (QA) injections to ablate medium spiny neurons (MSN), the A9 neuron primary target. While loss of MSNs variably affected graft survival, it significantly reduced striatal yet increased cortical innervation. Consequently, grafts showed reduced A9 and increased A10 specification, with more DA neurons failing to mature into either subtype. These findings highlight the importance of target acquisition on DA subtype specification during development and repair. SIGNIFICANCE STATEMENT Parish and colleagues highlight, in a rodent model of Parkinson's disease (PD), the importance of synaptic target acquisition in the survival, integration and phenotypic specification of grafted dopamine neurons derived from fetal tissue and human stem cells. Ablation of host striatal neurons resulted in reduced dopamine neuron survival within grafts, re-routing of dopamine fibers from striatal to alternate cortical targets and a consequential reduced specification of A9 dopamine neurons (the subpopulation critical for restoration of motor function) and increase in A10 DA neurons.

The derivation of neurotransmitter and region-specific neuronal populations from human pluripotent stem cells (PSC) provides impetus for advancing cell therapies into the clinic. At the forefront is our ability to generate ventral midbrain (VM) dopaminergic (DA) progenitors, suitable for transplantation in Parkinson’s disease (PD). Pre-clinical studies, however, have highlighted the low proportion of DA neurons within these grafts and their inferior plasticity by comparison to human fetal donor transplants. Here we sought to examine whether modification of the host environment, through viral delivery of a developmentally critical molecule, glial cell line-derived neurotrophic factor (GDNF), could improve graft survival, integration and function in Parkinsonian rodents. Utilising LMX1A- and PITX3-GFP hPSC reporter lines, we tracked the response of DA progenitors implanted into either a GDNF-rich environment, or in a second group, after a 3-week delay in onset of exposure. We found that early exposure of the graft to GDNF promoted survival of DA and non-DA cells, leading to enhanced motor recovery in PD rats. Delayed overexpression of intrastriatal GDNF also promoted motor recovery in transplanted rats, through alternate selective mechanisms including enhanced A9/A10 specification, increased DA graft plasticity, greater activation of striatal neurons and elevated DA metabolism. Lastly, transcriptional profiling of the grafts highlighted novel genes underpinning these changes. Collectively these results demonstrate the potential of targeted neurotrophic gene therapy strategies to improve human PSC graft outcomes.

Pluripotent stem cells (PSCs) are a valuable tool for interrogating development, disease modelling, drug discovery and transplantation. Despite the burgeoned capability to fate restrict human PSCs to specific neural lineages, comparative protocols for mouse PSCs have not similarly advanced. Mouse protocols fail to recapitulate neural development, consequently yielding highly heterogeneous populations, yet mouse PSCs remain a valuable scientific tool as differentiation is rapid, cost effective and an extensive repertoire of transgenic lines provides an invaluable resource for understanding biology. Here we developed protocols for neural fate restriction of mouse PSCs, using knowledge of embryonic development and recent progress with human equivalents. These methodologies rely upon naïve ground-state PSCs temporarily transitioning through LIF-responsive stage prior to neural induction and rapid exposure to regional morphogens. Neural subtypes generated included those of the dorsal forebrain, ventral forebrain, ventral midbrain and hindbrain. This rapid specification, without feeder layers or embryoid-body formation, resulted in high proportions of correctly specified progenitors and neurons with robust reproducibility. These generated neural progenitors/neurons will provide a valuable resource to further understand development, as well disorders affecting specific neuronal subpopulations.

Abnormal development of ventral midbrain (VM) dopaminergic (DA) pathways, essential for motor and cognitive function, may underpin a number of neurological disorders and thereby highlight the importance of understanding the birth and connectivity of the associated neurons. While a number of regulators of VM DA neurogenesis are known, processes involved in later developmental events, including terminal differentiation and axon morphogenesis, are less well understood. Recent transcriptional analysis studies of the developing VM identified genes expressed during these stages, including the cell adhesion molecule with homology to L1 (Chl1). Here, we map the temporal and spatial expression of CHL1 and assess functional roles of substrate-bound and soluble-forms of the protein during VM DA development. Results showed early CHL1 in the VM, corresponding with roles in DA progenitor migration and differentiation. Subsequently, we demonstrated roles for CHL1 in both axonal extension and repulsion, selectively of DA neurons, suggestive of a role in guidance towards forebrain targets and away from hindbrain nuclei. In part, CHL1 mediates these roles through homophilic CHL1-CHL1 interactions. Collectively, these findings enhance our knowledge of VM DA pathways development, and may provide new insights into understanding DA developmental conditions such as autism spectrum disorders.

PURPOSE: To evaluate microscopic behavior and viability of dental pulp stem cells under glucose and glutamine deprivation. METHODS: Human tooth tissues were minced in isolated pieces and cultured until the desired cellular proliferation for experimental phases. Cells were cultured under variations of glucose and glutamine in both serum presence and absence, and then those cells were evaluated according to number and viability by MTT assay. The confocal microscopy analyzed cytoskeleton, nucleus, and mitochondria integrity. RESULTS: A low concentration of glucose favored cellular viability and microscopic behavior; the presence of glutamine in culture medium was favorable only when associated with glucose. The cellular biological potential in culture could be preserved in serum absence if nutritional requirements are adequate. CONCLUSION: Cell microscopic behavior and viability have demonstrated better patterns on serum-free low glucose culture medium with glutamine deprivation.

Tissue engineering aims at developing the necessary technological strategies for replacement or regeneration tissues. However, the number of cells required is much greater than the number obtained from a cell source. Expanding the cells' number in cell culture for a long period is required until the necessary amount of cells is obtained. While in culture, cells often go unwanted differentiation. Little attention has been given to the use of proteolytic enzymes in cell passage. Review the importance of extracellular matrix and surface proteins for cell behavior and the possible mechanisms of cellular changes that may occur due to the use of proteolytic enzymes is an essential issue. Possible alternative to replace these enzymes in cell passage has also been developed., Tissue engineering aims at developing strategies for tissue replacement. However, the number of cells required is much greater than the number obtained from a cell source. Expanding the cells' number in cell culture for a long period is required until the necessary amount of cells is obtained but, cells often go unwanted differentiation. Proteolytic enzymes are commonly used in culture and it might lead to such cellular changes. This article aims at reviewing the possible mechanisms of cellular changes that may occur due to the use of proteolytic enzymes and possible alternatives to replace the use of those enzymes.

Introduction: Tissue Engineering (TE) aims to bio manufacture human tissues, an endeavor that requires a large number of cells, preferably autologous ones. Typically, an autologous cell source is unable to provide the required amount of cells and it is necessary that such cells be cultured during a prolonged period of time. One of the methodologies used to obtain and expand autologous cells in culture is explants; however, explants are commonly discarded at the moment of the first cell passage. Given the difficulty in obtaining those cell sources, a good alternative would be to reuse the explants and put them back into culture after cell passage. Nevertheless, various modifications have been seen in long-term cell culture, such as modifications of gene expression, and number and amount of proteins. Objective: To evaluate changes in gene expression of extracellular matrix (ECM) and adhesion molecules (AM) in human mesenchymal dental pulp progenitor cells (hMDPC) culture of reused explants. Methods: Gene expression of cells derived from second passage reused explants culture was accessed by Real Time PCR array of Human ECM and AM. Results: Twenty-nine genes showed at least a 5-fold change increase or decrease compared to the control group. Conclusion: Long-term culture frequently induces gene expression changes in ECM or AM. Re using explants is an option to expand the cell source in order to decrease culture time. However, it is necessary to understand, map and control those changes in order to ensure clinical application safety.

Foram analisados efeitos de diferentes histórias de incontrolabilidade por perda ou ganho de pontos sobre o desempenho posterior de participantes humanos na construção de frases. Inicialmente, os participantes podiam ganhar ou perder pontos independentemente de qualquer característica da frase construída. Posteriormente, recebiam pontos por construir frases iniciadas apenas pelo pronome "ele". Os resultados mostram que a exposição à incontrolabilidade pode dificultar condições posteriores de novas aprendizagens sob reforçamento positivo. Interessantemente, essas dificuldades foram menos acentuadas e, em certos casos, até mesmo superadas, no caso de uma história de exposição a ganhos incontroláveis de pontos. Em contrapartida, no caso de uma história de perdas incontroláveis de pontos, aprendizagens subsequentes sob reforço positivo tenderam a ser prejudicadas. Esses resultados contribuem para os estudos de incontrolabilidade e desamparo aprendido, em particular por apresentar alternativas metodológicas passíveis de aplicação a respostas verbais em humanos. Human participants were exposed to a task in which, by constructing sentences, the effects of different stories of uncontrollability were assessed through the loss or acquisition of points in a subsequent exposure to a new learning condition. Initially, subjects could lose or win points regardless of any specific response. Subsequently, they would receive points for constructing any sentence beginning with the pronoun "he". Results show that exposure to uncontrollability can make it difficult to acquire a subsequent learning task. In particular, the effects of the history of exposure to uncontrollable points gains were relatively short-lived, concentrated at the beginning of the learning process and tending to recover throughout the exposure to the new task. On the other hand, the effects of uncontrollability through the loss of points had a detrimental effect on the acquisition of the new task. These results contribute to the study of uncontrollability and learned helplessness, particularly by proposing alternative methods using human verbal responses.

Improved control over spatiotemporal delivery of growth factors is needed to enhance tissue repair. Current methods are limited-requiring invasive procedures, poor tissue targeting, and/or limited control over dosage and duration. Incorporation into implantable biomaterials enables stabilized delivery and avoids burst release/fluctuating doses. Here, the physical forces of fibrils formed by self-assembly of epitope-containing peptides are exploited. This biomimetic hydrogel is loaded with neurotrophic factor BDNF via a shear-induced gel-solution transition, unique to noncovalent hydrogels. This results in a biomaterial with three desirable features: a nanofibrillar scaffold, presentation of a laminin epitope, and slow release of BDNF. In a stroke-injury model, synergistic actions of this trimodal strategy on the integration of transplanted human neural progenitor cells, and protection of peri-infarct tissue are identified. These BDNF-functionalized hydrogels promote the integration of transplanted human embryonic stem cell-derived neural progenitors-resulting in larger grafts with greater cortical differentiation, appropriate for neuronal replacement. Furthermore, BDNF promotes the infiltration of host endothelial cells into the graft to augment vascularization of the graft, and adjacent penumbra tissue. These findings demonstrate the benefits of multifaceted tissue-specific hydrogels to provide biomimetics of the host tissue, while sustain protein delivery, to promote endogenous and graft-derived tissue repair.

Stereotyped behaviors have been routinely used as characters for phylogeny inference, but the same cannot be said of the plastic aspects of performance, which routinely are taken as a result of ecological processes. In this paper we examine the evolution of one of these plastic behavioral phenotypes, thus fostering a bridge between ecological and evolutionary processes. Foraging behavior in spiders is context dependent in many aspects, since it varies with prey type and size, spider nutritional and developmental state, previous experience and, in webweavers, is dependent on the structure of the web. Reeling is a predatory tactic typical of cobweb weavers (Theridiidae), in which the spider moves the prey toward her by pulling the capture thread (gumfoot) to which it is adhered. Predatory reeling is dependent on the gumfoot for its expression, and has not been previously reported in orbweavers. In order to investigate the evolution of this web dependent behavior, we built artificial, pseudogumfoot lines in orbwebs and registered parameters of the predatory tactics in this modified web. Aspects of the predatory tactics of 240 individuals (12 species in 4 families) were measured, and the resulting data were optimized on the phylogeny of Orbiculariae. All species perform predatory reeling with the pseudogumfoot lines. Thus, predatory reeling is homologous for the whole Orbiculariae group. In nature, holes made by insects in ecribellate orbs produce pseudogumfoot lines (similar to our experimentally modified webs), and thus reeling occurred naturally in ecribellates. Nevertheless, outside lab conditions, predatory reeling does not occur among cribellate orbweavers, so that this behavior could not have been selected for in the cribellate ancester of orbweavers. Cribellate spiders are flexible enough as to present novel and adaptive predatory responses (reeling) even when exposed for the first time to conditions outside their usual environment. Thus, the evolution of reeling suggests an alternative mechanism for the production of evolutionary novelties; that is, the exploration of unusual ecological conditions and of the regular effects these abnormal conditions have on phenotype expression.

A memória é considerada por muitos pesquisadores como um dos produtos mais fascinantes da evolução biológica, justamente por proporcionar aos animais a vantagem adaptativa de se utilizar de uma experiência prévia para a solução de problemas necessários para a sobrevivência. Os animais se utilizam de processos mnemônicos durante o forrageamento, a seleção de parceiros sexuais, seleção de habitat e a preferência alimentar. As aranhas são um modelo interessante em estudos de memória e há evidências do uso de experiências passadas na otimização do forrageio: a teia permite que as aranhas capturem e armazenem mais de um item alimentar. Há carência na literatura de estudos comparativos evolutivos da memória, o que dificulta identificar as possíveis pressões seletivas. O primeiro objetivo deste trabalho foi fazer um estudo comparativo de aspectos da memória predatória em aranhas do grupo Orbiculariae. Este grupo é monofilético e tem como principal sinapomorfia a construção de teia orbicular. Porém, ao longo da evolução, o padrão de teia foi se modificando desde uma estrutura plana até uma tridimensional. Tais modificações podem ter gerado diferenças comportamentais e no sistema de memória dessas aranhas. Por este motivo, o outro objetivo, foi traçar a evolução da memória dentro do grupo. Delinearam-se três grupos de estudo com sete espécies. Na condição natural, observou-se a captura de duas presas sucessivas. Na condição experimental I, ofereceu-se uma primeira presa (p1) à aranha e, em seguida, ofereceu-se uma segunda (p2); durante o deslocamento da aranha até p2, retirou-se p1 da teia. Na condição II, ao invés de se oferecer p2, a aranha foi atraída à periferia (simulando a queda de p2). No deslocamento da aranha até a fonte de vibração removeu-se p1. O indício de memória é observado quando, na supressão da presa, a aranha executa um comportamento denominado Busca. Neste, a aranha executa sacudidas/detecções dos fios no centro/refúgio da teia. A busca por p1 roubada foi observada em todas as espécies estudadas, exceto em Z. geniculata no grupo I. A presença de p2 parece interferir na expressão da memória de p1; deixar p2 na teia enquanto busca por p1, aumenta as chances de p2 também ser roubada por ação dos cleptoparasitas. Zosis apresenta algumas peculiaridades, entre elas a ausência de veneno. Dessa forma, o tempo gasto durante a imobilização de p2 parece ser suficiente para causar o esquecimento de p1. Todas as espécies buscaram por p1 quando não houve oferta de p2, inclusive Zosis. O tempo de busca, na condição I, diminui da base de Araneoidea até as famílias mais derivadas. No grupo II, há uma aparente inversão do sinal filogenético e o tempo de busca aumenta na família mais derivada. A estrutura da teia tridimensional parece ser mais complexa e, por este motivo, exigiria um sistema de navegação espacial também mais complexo quando comparado às aranhas orbitelas. A construção da teia parece ter sido crucial para a captura e armazenamento de presas excedentes, e tal comportamento, juntamente com a diversificação das teias, favoreceu a variação e evolução da memória na Ordem Araneae Memory is one of the most fascinating products of biological evolution, because it endows animals with the adaptive use a prior experiences to solve survival problems. Animals use mnemonic processes during foraging, selection of sexual partners, habitat selection, and feeding preference. Spiders are an interesting model for studies of memory, and there is evidence of their using of past experiences in optimizing foraging: the web allows the spiders to capture and store more than one food item. In literature, there are few comparative studies, and no one about the evolution of memory, making it difficult to identify the possible selective pressures on memory. The first objective here was to make a comparative study on the aspects of predatory memory in spiders of the group Orbiculariae. This group is monophyletic and its main synapomorphy is building an orbicular web. However, during evolution, the web pattern changed from a flat to a three-dimensional structure. Such changes may have caused differences in behavior and memory. For that reason, another goal was to trace the evolution of memory within the group. We used three experimental groups with seven species each. In natural conditions, there was an observation of the capture of two successive preys. In the experimental condition I, a first prey (p1) was offered to the spider and, subsequently, another prey (p2) was offered; during the locomotion of the spider towards p2, p1 was removed from the web. In condition II, instead of offering p2, the spider was attracted to the periphery (simulating the fall of p2). As the spider moved towards the source of vibration, p1 was removed. The evidence of memory is observed when, in the suppression of the prey, the spider performs a behavior called search behavior: the spider runs plucking on threads in the web hub/retreat. The search for stolen p1 was observed in all species studied, except for Zosis geniculata in group I. The presence of p2 appears to interfere with the expression of p1 memory, and also leaving p2 on the web while searching for p1, increases the chances of having p2 stolen by the action of kleptoparasites. Zosis presents some peculiarities, including the absence of poison glands, leading to a prolonged immobilization of p2, in a time lag that could then be sufficient forget p1. All species sought by p1 when there was no offer of p2, including Zosis. The search time, in condition I, decreases from the base of Araneoidea until the most derived families. In group II, there is an apparent reversal of the phylogenetic signal and the search time increases in the most derived family. The three-dimensional structure of the derived webs seems to be more complex and, therefore, it would require a spatial navigation system also more complex if compared to the flat orbweb. The construction of the web seems to have been crucial for prey capture and storage of surpluses, and such behavior, along with the diversification of the webs, favored the variation and evolution of memory in the Order Araneae

Background and Objective: Cyclosporin A-induced gingival overgrowth comprises a variety of signaling pathways (including growth factors and proteoglycans) that are still not completely understood. In the present study, gingival overgrowth was investigated in transplant patients receiving cyclosporin A (cyclosporin A group) and compared with gingival tissues never exposed to the drug (control group) by analyzing the gene expression of the cell-surface heparan sulfate proteoglycans syndecan-2, syndecan-4 and betaglycan. Material and Methods: mRNA analysis was carried out by reverse transcription–polymerase chain reaction amplification of pooled samples from nine patients of the cyclosporin A group and six control subjects. The groups were compared by the Student's t-test. Results: The expression of heparan sulfate proteoglycans was increased in the cyclosporin A group (165% for syndecan-2, 308% for syndecan-4, and 42% for betaglycan) compared with the control group. Conclusion: Our findings agree with the current concept of cyclosporin A-induced gingival overgrowth and provide new evidence that its noncollagenous extracellular matrix is overexpressed.