Construction of hiPSC-derived Excitatory Neural Network-like Tissue / 中山大学学报(医学科学版)

ObjectiveTo construct a neural network-like tissue with the potential of synaptic formation in vitro by seeding human induced pluripotent stem cell-derived neural precursor cells （hiPSC-NPCs） on decellularized optic nerve （DON）， so as to provide a promising approach for repair of nerve tissue injury. MethodsThrough directional induction and tissue engineering technology， human induced pluripotent stem cells （hiPSCs） and 3D DON scaffolds were combined to construct neural network-like tissues. Then the hiPSCs were directionally induced into human neural precursor cells （hNPCs） and neurons. Immunofluorescence staining was used to identify cell differentiation efficiency. 3D DON scaffolds were prepared. Morphology and cytocompatibility of scaffolds were identified by scanning electron microscopy and Tunnel staining. Induced hiPSC-NPCs were seeded on DON scaffolds. Immunofluorescence staining， scanning electron microscopy， transmission electron microscopy and patch clamp were used to observe the morphology and functional identification of constructed neural network tissues. Results①The results of immunofluorescence staining suggested that most of hiPSC-NPCs differentiated into neurons in vitro. We had successfully constructed a neural network dominated by neurons. ② The results of scanning electron microscopy and immunohistochemistry suggested that a neural network-like tissue with predominating excitatory neurons in vitro was successfully constructed. ③The results of immunohistochemical staining， transmission electron microscopy and patch clamp indicated that the neural network-like tissue had synaptic transmission function. ConclusionA neural network-like tissue mainly composed of excitatory neurons has been constructed by the combination of natural uniform-channel DON scaffold and hiPSC-NPCs， which has the function of synaptic transmission. This neural network plays a significant role in stem cell derived replacement therapy， and offers a promising prospect for repair of spinal cord injury （SCI） and other neural tissue injuries.

Kinin B2-Receptor in human iPSC differentiation into cardiomyocytes / Receptor B2 de cininas na diferenciação de iPSC humanas em cardiomiócitos

Cardiovascular diseases are responsible for almost one third of all global deaths yearly, and therefore are largely studied. Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CM) have emerged as an exciting technology for cardiac disease modelling and personalised therapy. Nevertheless, issues concerning functional and molecular maturation are still faced. In addition to this, differentiation protocols generally yield a heterogeneous mixed population comprised of nodal, atrial and ventricular-like subtypes, being unsuitable for therapeutic purposes. Bradykinin (BK) is a vasoactive peptide which exerts important physiological roles in the cardiovascular system, having been previously described as important for cellular, keratinocyte and skeletal muscle differentiation. This project performed in cooperation with PluriCell Biotech, a startup specialized in the production and differentiation of hiPSC-CM, has sought (1) characterizing gene and protein expression of molecular markers of maturation and of subtype specification throughout of differentiation; (2) Assessing the electrical functionality of hiPSC-CM through the characterization of subtype-specific action potentials (APs) and (3) Investigating whether the progress of hiPSCCM maturation is regulated by BK through kinin-B2 receptors (B2R). Our results have validated the model that proposes a developmental-dependent switch between skeletal (ssTnI) and cardiac (cTnI) isoforms of troponin I as differentiation progresses, at least to some extent. Furthermore, prolonged time in culture has resulted in higher levels of expression of the ventricular marker MLC2v and in increased rates of ventricular-like action APs. Electrophysiological analysis of hiPSC-CM reveals a mixed population with AP morphologies correspondent to nodal, atrial and ventricular subtypes, all showing pronounced automaticity as well as other features of immature cardiomyocytes, such as low amplitude and depolarization velocity. Such findings are coherent with those from other groups who have attempted to differentiate mature native-like cardiac cells from pluripotent stem cells sources, without fully succeeding. After showing that differentiating hiPSC-CM express a functional and responsive B2R, the receptor was subjected to chronic activation with 10µM BK and 1µM BK or inhibition with 5µM Firazyr+BK. Even though B2R modulation has not interfered negatively with differentiation yields nor cell morphology, analysis of gene andprotein expression of ssTnI or cTnI and of the ventricular marker MLC2v, have revealed no significant results in comparison to untreated controls. This suggests that BK does not interfere on hiPSC-CM maturation nor subtype specification, although we cannot rule out that it could be leading to other unexplored effects. We recommend a closer look into which intracellular signalling pathways become active upon B2R stimulation in hiPSC-CM, in order to narrow down cellular processes for further investigation

Doenças cardiovasculares são responsáveis por quase um terço de todas as mortes globais anualmente, e por isto o sistema cardiovascular é amplamente estudado. Cardiomiócitos derivados a partir de células-tronco pluripotentes induzidas humanas (hiPSCCM) emergiram como uma promissora tecnologia para modelagem de doenças cardíacas e terapia personalizada. No entanto, desafios acerca de sua maturação funcional e molecular ainda são enfrentados. Além disso, protocolos de diferenciação geralmente levam à obtenção de populações heterogêneas contendo células com fenótipos similares aos de cardiomiócitos nodais, atriais e ventriculares sendo, portanto, inapropriadas para fins terapêuticos. A bradicinina (BK) é um peptídio vasoativo que exerce importantes papeis fisiológicos no sistema cardiovascular, além de ter sido previamente descrita como importante para a diferenciação neuronal, de queratinócitos e de músculo esquelético. Este projeto foi realizado em colaboração com a empresa PluriCell Biotech, uma startup especializada na produção e diferenciação de hiPSC-CM, e buscou (1) caracterizar a expressão gênica e proteíca de marcadores moleculares de maturação e de especificação de subtipos cardíacos durante a diferenciação; (2) avaliar a funcionalidade elétrica de hiPSC-CM por meio da caracterização de seus potenciais de ação (PAs) e (3) Investigar se o progresso da diferenciação de hiPSCCM é regulado por bradicinina por meio do receptor B2 (B2R). Nossos resultados validaram o modelo que propõe um switch na expressão das isoformas funcionais de troponina I esquelética (ssTnI) e cardíaca (cTnI), durante o desenvolvimento e diferenciação celular, pelo menos parcialmente. Além disso, tempo prolongado em cultura resultou em maiores níveis de expressão do marcador ventricular MLC2v, assim como maiores frequências de PAs com morfologias similares a de cardiomiócitos ventriculares. Análise eletrofisiológica de hiPSCCM revelam a existência de uma população mista contendo PAs correspondentes aos subtipos nodais, atriais e ventriculares, assim como pronunciada automaticidade e outros atributos típicos de cardiomiócitos imaturos, como baixa amplitude e devagar velocidade de despolarização. Estes resultados são coerentes com os de outros grupos que ainda não foram totalmente bem-sucedidos em diferenciar células cardíacas maduras similares acardiomiócitos nativos a partir de células-troncos pluripotentes. Após mostrar que as hiPSCCM expressam receptores B2 funcionais e responsivos, submetemos o receptor a uma ativação crônica com BK 10µM e BK 1µM ou inibição crônica com Firazyr 5µM + BK. Apesar da modulação do B2R não ter interferido de forma negativa no rendimento da diferenciação ou na morfologia celular, análise de expressão gênica e proteica de ssTnI e cTnI e do marcador ventricular MLC2v não revelou resultados significativos em comparação aos controles não-tratados. Isto sugere que a BK não interfere na maturação e especificação de subtipos cardíacos em hiPSC-CM, apesar de não podermos ignorar o fato de que ela poderia estar desencadeando outros efeitos inexplorados. Nós recomendamos um estudo mais aprofundado acerca de quais vias de sinalização se tornam ativas após estimulação do receptor B2 em hiPSC-CM, com o objetivo de afunilar quais processos celulares poderiam ser investigados em uma próxima etapa deste estudo

Coupled electrophysiological recording and single cell transcriptome analyses revealed molecular mechanisms underlying neuronal maturation

The mammalian brain is heterogeneous, containing billions of neurons and trillions of synapses forming various neural circuitries, through which sense, movement, thought, and emotion arise. The cellular heterogeneity of the brain has made it difficult to study the molecular logic of neural circuitry wiring, pruning, activation, and plasticity, until recently, transcriptome analyses with single cell resolution makes decoding of gene regulatory networks underlying aforementioned circuitry properties possible. Here we report success in performing both electrophysiological and whole-genome transcriptome analyses on single human neurons in culture. Using Weighted Gene Coexpression Network Analyses (WGCNA), we identified gene clusters highly correlated with neuronal maturation judged by electrophysiological characteristics. A tight link between neuronal maturation and genes involved in ubiquitination and mitochondrial function was revealed. Moreover, we identified a list of candidate genes, which could potentially serve as biomarkers for neuronal maturation. Coupled electrophysiological recording and single cell transcriptome analysis will serve as powerful tools in the future to unveil molecular logics for neural circuitry functions.