RESUMO
Three-dimensional (3D) tissue models have gained recognition for their improved ability to mimic the native cell microenvironment compared to traditional two-dimensional models. This progress has been driven by advances in tissue-engineering technologies such as 3D bioprinting, a promising method for fabricating biomimetic living tissues. While bioprinting has succeeded in generating various tissues to date, creating neural tissue models remains challenging. In this context, we present an accelerated approach to fabricate 3D sensory neuron (SN) structures using a transgenic human pluripotent stem cell (hPSC)-line that contains an inducible Neurogenin-2 (NGN2) expression cassette. The NGN2 hPSC line was first differentiated to neural crest cell (NCC) progenitors, then incorporated into a cytocompatible gelatin methacryloyl-based bioink for 3D bioprinting. Upregulated NGN2 expression in the bioprinted NCCs resulted in induced SN (iSN) populations that exhibited specific cell markers, with 3D analysis revealing widespread neurite outgrowth through the scaffold volume. Calcium imaging demonstrated functional activity of iSNs, including membrane excitability properties and voltage-gated sodium channel (NaV) activity. This efficient approach to generate 3D bioprinted iSN structures streamlines the development of neural tissue models, useful for the study of neurodevelopment and disease states and offering translational potential.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos , Bioimpressão , Proteínas do Tecido Nervoso , Impressão Tridimensional , Células Receptoras Sensoriais , Alicerces Teciduais , Humanos , Bioimpressão/métodos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Células Receptoras Sensoriais/metabolismo , Células Receptoras Sensoriais/citologia , Proteínas do Tecido Nervoso/metabolismo , Alicerces Teciduais/química , Linhagem Celular , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Diferenciação Celular , Engenharia Tecidual/métodos , Gelatina/química , Crista Neural/citologia , Crista Neural/metabolismoRESUMO
Friedreich's ataxia (FRDA) is a rare neurodegenerative disease caused by an expansion of a GAA repeat sequence within the Frataxin (FXN) gene. Prominent regions of neurodegeneration include sensory neurons within the dorsal root ganglia. Here we present a set of genetically modified FRDA induced pluripotent stem cell (iPSC) lines that carry an inducible neurogenin-2 (NGN2) expression cassette. Exogenous expression of NGN2 in iPSC derived neural crest progenitors efficiently generates functionally mature sensory neurons. These cell lines will provide a streamlined source of FRDA iPSC sensory neurons for studying both disease mechanism and screening potential therapeutics.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos , Ataxia de Friedreich , Células-Tronco Pluripotentes Induzidas , Proteínas do Tecido Nervoso , Ataxia de Friedreich/genética , Ataxia de Friedreich/patologia , Ataxia de Friedreich/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Humanos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , Linhagem Celular , Diferenciação Celular , FrataxinaRESUMO
The pro-neural transcription factor neurogenin-2 (NGN2) possesses the ability to rapidly and effectively transform stem cells into fully operational neurons. Here we report the successful generation of a modified H9 human embryonic H9 stem cell line containing a doxycycline (DOX) inducible NGN2 expression construct featuring a floxed Blasticidin/mApple selection module in the safe-harbor locus CLYBL. This cell line retains its pluripotent state in the absence of DOX, yet readily transitions into a neuronal state upon DOX introduction.