Caracterização e expansão in vitro de células tronco epidermais derivadas da crista neural

TCC(graduação) - Universidade Federal de Santa Catarina. Centro de Ciências Biológicas. Biologia.A implementação de tecnologias baseadas no uso de células tronco adultas requer a identificação de fontes prontamente disponíveis bem como o estabelecimento de metodologias de cultivo, expansão e diferenciação dessas células. Este trabalho teve como objetivo principal a caracterização e a expansão in vitro de células tronco epidermais da crista neural (EPI-NCSCs). Tais células residem no folículo piloso adulto e são remanescentes da crista neural - estrutura embrionária que origina o sistema nervoso periférico, melanócitos da pele, e musculatura lisa, ossos e cartilagens da região cefálica. Devido à acessibilidade e amplo potencial de diferenciação, as EPI-NCSCs são consideradas candidatas promissoras para futuras aplicações em Medicina Regenerativa e Bioengenharia Tecidual. Usando técnicas de cultivo de células em condição aderente e lançando mão das metodologias de imunofluorescência, RT-PCR, western blot e citometria de fluxo, demonstramos nesse trabalho que as EPI-NCSCs isoladas de vibrissas de camundongos adultos são caracterizadas pela expressão de genes característicos da CN (incluindo nestina, p75NTR, Pax3, Slug, Snail, Sox10 e Twist) e podem ser rotineiramente cultivadas e expandidas in vitro. Além disso, verificamos que a combinação dos fatores de crescimento EGF e FGF2 no meio de cultivo estimula a proliferação e direciona essas células a um estado de progenitor neuronal às expensas dos fenótipos glial e de músculo liso – o que foi evidenciado, respectivamente, pelo aumento na taxa de incorporação de BrdU e pela aquisição de morfologia semelhante à neuronal aliada a expressão de proteínas neurais. Em última análise, esses resultados evidenciam a possibilidade de obtenção de uma linhagem de células neuronais a partir de um órgão de fácil acesso como a pele, bem como as vantagens do uso dos fatores de crescimento EGF e FGF2 nesse processo. Tal linhagem poderia futuramente servir de base para tratamento de lesões do sistema nervoso, testes de drogas in vitro, e outras aplicações biotecnológicas

Hierarchical reactivation of transcription during mitosis-to-G1 transition by Brn2 and Ascl1 in neural stem cells

During mitosis, chromatin condensation is accompanied by a global arrest of transcription. Recent studies suggest transcriptional reactivation upon mitotic exit occurs in temporally coordinated waves, but the underlying regulatory principles have yet to be elucidated. In particular, the contribution of sequence-specific transcription factors (TFs) remains poorly understood. Here we report that Brn2, an important regulator of neural stem cell identity, associates with condensed chromatin throughout cell division, as assessed by live-cell imaging of proliferating neural stem cells. In contrast, the neuronal fate determinant Ascl1 dissociates from mitotic chromosomes. ChIP-seq analysis reveals that Brn2 mitotic chromosome binding does not result in sequence-specific interactions prior to mitotic exit, relying mostly on electrostatic forces. Nevertheless, surveying active transcription using single-molecule RNA-FISH against immature transcripts reveals differential reactivation kinetics for key targets of Brn2 and Ascl1, with transcription onset detected in early (anaphase) versus late (early G1) phases, respectively. Moreover, by using a mitotic-specific dominant-negative approach, we show that competing with Brn2 binding during mitotic exit reduces the transcription of its target gene Nestin. Our study shows an important role for differential binding of TFs to mitotic chromosomes, governed by their electrostatic properties, in defining the temporal order of transcriptional reactivation during mitosis-to-G1 transition

Genome editing as a tool to explore transcriptional and epigenetic regulation in neural stem cells and brain cancer

Mammalian neural stem cell (NSC) lines provide a useful experimental model for basic and applied research across stem cell and developmental biology, regenerative medicine and neuroscience. NSCs are clonally expandable, genetically stable, and easily transfectable - experimental attributes compatible with functional genetic analyses. However, targeted genetic manipulations have not been reported for mammalian NSC lines. Here, we deploy the CRISPR/Cas9 technology and demonstrate a variety of diverse targeted genetic modifications in both mouse and human NSC lines such as: targeted transgene insertion at safe harbour loci; biallelic knockout of neurodevelopmental genes; knock-in of epitope tags and fluorescent reporters; and engineering of glioma driver mutations at endogenous proto-oncogenes. Leveraging these new optimised methods, we explored gene editing to model the earliest stages of paediatric gliomagenesis in primary human NSCs. Our data indicate that oncogenic mutations in histone H3.3 play a role in NSC transformation and may operate through suppression of replication induced senescence. By comparing cellular responses of NSC cultures from different compartments of the developing brain, we also identify differences in susceptibility to distinct H3.3 mutations that mirror the disease etiology. Altogether, our findings indicate that CRISPR/Cas9-assisted genome editing in NSC lines is a versatile tool to explore gene function in CNS development and cancer biology. Our project resulted in the creation of valuable human cellular models of paediatric gliomagenesis, which will allow us to further our understanding of the disease and carry out cell based drug discovery projects

Mesenchymal-epithelial crosstalk shapes intestinal regionalisation via Wnt and Shh signalling

Organs are anatomically compartmentalised to cater for specialised functions. In the small intestine (SI), regionalisation enables sequential processing of food and nutrient absorption. While several studies indicate the critical importance of non-epithelial cells during development and homeostasis, the extent to which these cells contribute to regionalisation during morphogenesis remains unexplored. Here, we identify a mesenchymal-epithelial crosstalk that shapes the developing SI during late morphogenesis. We find that subepithelial mesenchymal cells are characterised by gradients of factors supporting Wnt signalling and stimulate epithelial growth in vitro. Such a gradient impacts epithelial gene expression and regional villus formation along the anterior-posterior axis of the SI. Notably, we further provide evidence that Wnt signalling directly regulates epithelial expression of Sonic Hedgehog (SHH), which, in turn, acts on mesenchymal cells to drive villi formation. Taken together our results uncover a mechanistic link between Wnt and Hedgehog signalling across different cellular compartments that is central for anterior-posterior regionalisation and correct formation of the SI

From cohorts to molecules: Adverse impacts of endocrine disrupting mixtures

Convergent evidence associates exposure to endocrine disrupting chemicals (EDCs) with major human diseases, even at regulation-compliant concentrations. This might be because humans are exposed to EDC mixtures, whereas chemical regulation is based on a risk assessment of individual compounds. Here, we developed a mixture-centered risk assessment strategy that integrates epidemiological and experimental evidence. We identified that exposure to an EDC mixture in early pregnancy is associated with language delay in offspring. At human-relevant concentrations, this mixture disrupted hormone-regulated and disease-relevant regulatory networks in human brain organoids and in the model organisms Xenopus leavis and Danio rerio, as well as behavioral responses. Reinterrogating epidemiological data, we found that up to 54% of the children had prenatal exposures above experimentally derived levels of concern, reaching, for the upper decile compared with the lowest decile of exposure, a 3.3 times higher risk of language delay.ISSN:0036-8075ISSN:1095-920

From cohorts to molecules : Adverse impacts of endocrine disrupting mixtures

Convergent evidence associates exposure to endocrine disrupting chemicals (EDCs) with major human diseases, even at regulation-compliant concentrations. This might be because humans are exposed to EDC mixtures, whereas chemical regulation is based on a risk assessment of individual compounds. Here, we developed a mixture-centered risk assessment strategy that integrates epidemiological and experimental evidence. We identified that exposure to an EDC mixture in early pregnancy is associated with language delay in offspring. At human-relevant concentrations, this mixture disrupted hormone-regulated and disease-relevant regulatory networks in human brain organoids and in the model organisms Xenopus leavis and Danio rerio, as well as behavioral responses. Reinterrogating epidemiological data, we found that up to 54% of the children had prenatal exposures above experimentally derived levels of concern, reaching, for the upper decile compared with the lowest decile of exposure, a 3.3 times higher risk of language delay