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Neural crest stem cells (NCPCs) have been shown to differentiate into various cell types and tissues during embryonic development, including sensory neurons. The few studies addressing the generation of NCPCs and peripheral sensory neurons (PSNs) from human induced pluripotent stem cells (hiPSCs), generated sensory cells without displaying robust activity. Here, we describe an efficient strategy for hiPSCs differentiation into NCPCs and functional PSNs using chemically defined media and factors to achieve efficient differentiation, confirmed by the expression of specific markers. After 10 days hiPSCs differentiated into NCPCs, cells were then maintained in neural induction medium containing defined growth factors for PSNs differentiation, followed by 10 days in neonatal human epidermal keratinocytes- (HEKn-) conditioned medium (CM). We observed a further increase in PSN markers expression and neurites length after CM treatment. The resulting neurons elicited action potentials after current injection and released substance P (SP) in response to nociceptive agents such as anandamide and resiniferatoxin. Anandamide induced substance P release via activation of TRPV1 and not CB1. Transcriptomic analysis of the PSNs revealed the main dorsal root ganglia neuronal markers and a transcriptional profile compatible with C fiber-low threshold mechanoreceptors. TRPV1 was detected by immunofluorescence and RNA-Seq in multiple experiments. In conclusion, the developed strategy generated PSNs useful for drug screening that could be applied to patient-derived hiPSCs, consisting in a powerful tool to model human diseases in vitro. Made available in DSpace on 2019-02-07T23:37:27Z (GMT). No. of bitstreams: 1 APGenerationiPSCFelipe.pdf: 7505321 bytes, checksum: 135c87796cec61c67776b2a6e5db7f0f (MD5) Previous issue date: 2019-02-07 Article 277.

SOUSA, André Filipe Santos - Generation and characterization of functional sensory neurons from hESCs and hiPSCs. Coimbra : [s.n.], 2016. Dissertação de Mestrado em Biologia Celular e Molecular. The lack of access to the neuronal tissue, limited our understanding about its development and physiology of pain in humans. Therefore, the generation of functional human sensory neurons for disease modelling, drug screening and clinical applications is an urgent and unmet need. Nociceptors are the sensory neurons related to sense the pain, characterized by the presence of transient receptor potential channels which have sensory functions linked to transduction of noxious stimuli as well as signalling within the pain system. In this thesis, we first characterized the nociceptors generated from induced pluripotent stem cell with a protocol developed on the laboratory, comparing it to a protocol described by Young et al., 2014. We used techniques as real-time reverse transcription-polymerase chain reaction and staining to check the neuronal phenotyping of the cells generated, as well as calcium imaging as a functional assay, to test about the functionality of transient receptor potential (TRP) channels, in order to characterize the neurons generated. We showed that only after 50 days of differentiation with both protocols we can have functional mature sensory neurons, with transient receptor potential cation channel sub family V, member 1 and transient receptor potential cation channel subfamily A, member 1 being activated. Moreover, the best results were achieved with the adapted protocol. Next, we focused on different approaches to improve the differentiation protocol. Changing cell line and replating dilution, did not enhance the protocol. Hence, we next hypothesized that current hurdles to speed up differentiation might be overcome by overexpression of key transcription factors involved in the sensory neurons differentiation: PR domain 12, brain-specific homeobox 3A, insulin gene enhancer and kruppel-link zinc finger transcription factor. A technique used on the laboratory, recombinase-mediated cassette exchange, was employed to generate two cell lines, one overexpressing PRDM12 and the other overexpressing ISL1-BRN3A-KLF7. We proved that in the new cell lines, the transcription factors were being correctly overexpressed, and collectively our results demonstrate that overexpressing PRDM12 improves the homogeneity of the cells generated. Further studies are required to evidence the impact of this new cell lines on the upgrading of the differentiation protocol. This work will open new opportunities for investigating in vitro disease modelling and evaluation of pharmacological responds to pain research. A falta de acesso ao tecido neuronal limita o nosso conhecimento acerca do seu desenvolvimento e da fisiologia da dor nos humanos. Portanto, a geração de neurónios sensoriais humanos funcionais de forma à criação de modelos de doenças relacionadas com a dor, triagem de drogas e aplicações clinicas é uma necessidade urgente e não atendida. Os nociceptors são os neurónios sensoriais responsáveis por receber o estímulo da dor, caracterizados pela presença dos TRP channels que têm funções sensoriais na medida em que estão ligados à transdução de estímulos nocivos assim como à sinalização dentro do sistema da dor. Nesta tese, primeiramente caracterizamos os nociceptors gerados a partir de células estaminais recorrendo a um protocolo desenvolvido no nosso laboratório, comparando-o a um protocolo descrito por Young et al., 2014. Foram usadas técnicas como qRT-PCR e staining, de forma a conferirmos o fenótipo neuronal das células geradas, assim como calcium imaging como um teste funcional acerca da atividade dos TRP channels com o objetivo de caracterizarmos os neurónios gerados. Aqui, demonstramos que apenas após 50 dias de diferenciação com ambos protocolos conseguimos obter neurónios sensoriais maduros funcionais, com a ativação de TRPV1 e TRPA1. Ainda, o protocolo adaptado no laboratório teve melhores resultados. De seguida, focamo-nos nas várias formas de melhorarmos o protocolo de diferenciação. Mudar a linha celular usada e a diluição na fase de replating não aperfeiçoou o protocolo. Sendo assim, supomos que as barreiras em conseguir uma diferenciação mais rápida podiam ser ultrapassadas pelo aumento de expressão de fatores de transcrição envolvidas na diferenciação de neurónios sensoriais: PRDM12, BRN3A, ISL1 e KLF7. Uma técnica usada no laboratório, RMCE, foi usada de forma a gerar duas linhas celulares, uma com o aumento de expressão de PRDM12, e a outra com o aumento de expressão de ISL1, BRN3A e KLF7. Neste trabalho provamos que as novas linhas celulares estavam de facto com a expressão aumentada dos fatores de transcrição inseridos, e os nossos resultados demonstram que o aumento de expressão de PRDM12 contribuiu para o melhoramento da homogeneidade das células geradas. Futuros estudos têm de ser feitos de forma a evidenciar o impacto destas duas novas linhas celulares no aprimoramento do protocolo de diferenciação. Este trabalho vai abrir novas oportunidades para a investigação in vitro de modelos de doenças e a avaliação de respostas farmacológicas na investigação da dor.