RESUMO
Cockayne Syndrome B (CSB) is a hereditary multiorgan syndrome which-through largely unknown mechanisms-can affect the brain where it clinically presents with microcephaly, intellectual disability and demyelination. Using human induced pluripotent stem cell (hiPSC)-derived neural 3D models generated from CSB patient-derived and isogenic control lines, we here provide explanations for these three major neuropathological phenotypes. In our models, CSB deficiency is associated with (i) impaired cellular migration due to defective autophagy as an explanation for clinical microcephaly; (ii) altered neuronal network functionality and neurotransmitter GABA levels, which is suggestive of a disturbed GABA switch that likely impairs brain circuit formation and ultimately causes intellectual disability; and (iii) impaired oligodendrocyte maturation as a possible cause of the demyelination observed in children with CSB. Of note, the impaired migration and oligodendrocyte maturation could both be partially rescued by pharmacological HDAC inhibition.
Assuntos
Síndrome de Cockayne , Células-Tronco Pluripotentes Induzidas , Oligodendroglia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/patologia , Síndrome de Cockayne/genética , Síndrome de Cockayne/metabolismo , Síndrome de Cockayne/patologia , Oligodendroglia/metabolismo , Oligodendroglia/citologia , Movimento Celular , Enzimas Reparadoras do DNA/metabolismo , Enzimas Reparadoras do DNA/genética , Neurônios/metabolismo , Neurônios/patologia , Autofagia , Encéfalo/metabolismo , Encéfalo/patologia , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , Ácido gama-Aminobutírico/metabolismo , DNA Helicases/metabolismo , DNA Helicases/genética , Microcefalia/patologia , Microcefalia/metabolismo , Microcefalia/genética , Doenças Desmielinizantes/patologia , Doenças Desmielinizantes/metabolismo , Diferenciação CelularRESUMO
The call for a paradigm change in toxicology from the United States National Research Council in 2007 initiates awareness for the invention and use of human-relevant alternative methods for toxicological hazard assessment. Simple 2D in vitro systems may serve as first screening tools, however, recent developments infer the need for more complex, multicellular organotypic models, which are superior in mimicking the complexity of human organs. In this review article most critical organs for toxicity assessment, i.e., skin, brain, thyroid system, lung, heart, liver, kidney, and intestine are discussed with regards to their functions in health and disease. Embracing the manifold modes-of-action how xenobiotic compounds can interfere with physiological organ functions and cause toxicity, the need for translation of such multifaceted organ features into the dish seems obvious. Currently used in vitro methods for toxicological applications and ongoing developments not yet arrived in toxicity testing are discussed, especially highlighting the potential of models based on embryonic stem cells and induced pluripotent stem cells of human origin. Finally, the application of innovative technologies like organs-on-a-chip and genome editing point toward a toxicological paradigm change moves into action.
Assuntos
Células-Tronco Pluripotentes Induzidas , Testes de Toxicidade , Humanos , Técnicas In Vitro , Estados UnidosRESUMO
Animal models have been greatly contributing to our understanding of physiology, mechanisms of diseases, and toxicity. Yet, their limitations due to, e.g., interspecies variation are reflected in the high number of drug attrition rates, especially in central nervous system (CNS) diseases. Therefore, human-based neural in vitro models for studying safety and efficacy of substances acting on the CNS are needed. Human iPSC-derived cells offer such a platform with the unique advantage of reproducing the "human context" in vitro by preserving the genetic and molecular phenotype of their donors. Guiding the differentiation of hiPSC into cells of the nervous system and combining them in a 2D or 3D format allows to obtain complex models suitable for investigating neurotoxicity or brain-related diseases with patient-derived cells. This chapter will give an overview over stem cell-based human 2D neuronal and mixed neuronal/astrocyte models, in vitro cultures of microglia, as well as CNS disease models and considers new developments in the field, more specifically the use of brain organoids and 3D bioprinted in vitro models for safety and efficacy evaluation.
Assuntos
Células-Tronco Pluripotentes Induzidas , Síndromes Neurotóxicas , Animais , Diferenciação Celular , Sistema Nervoso Central , Humanos , NeurôniosRESUMO
Cockayne syndrome (CS) is a rare hereditary autosomal recessive disorder primarily caused by mutations in Cockayne syndrome protein A (CSA) or B (CSB). While many of the functions of CSB have been at least partially elucidated, little is known about the actual developmental dysregulation in this devasting disorder. Of particular interest is the regulation of cerebral development as the most debilitating symptoms are of neurological nature. We generated neurospheres and cerebral organoids utilizing Cockayne syndrome B protein (CSB)-deficient induced pluripotent stem cells derived from two patients with distinct severity levels of CS and healthy controls. The transcriptome of both developmental timepoints was explored using RNA-Seq and bioinformatic analysis to identify dysregulated biological processes common to both patients with CS in comparison to the control. CSB-deficient neurospheres displayed upregulation of the VEGFA-VEGFR2 signalling pathway, vesicle-mediated transport and head development. CSB-deficient cerebral organoids exhibited downregulation of brain development, neuron projection development and synaptic signalling. We further identified the upregulation of steroid biosynthesis as common to both timepoints, in particular the upregulation of the cholesterol biosynthesis branch. Our results provide insights into the neurodevelopmental dysregulation in patients with CS and strengthen the theory that CS is not only a neurodegenerative but also a neurodevelopmental disorder.
Assuntos
Síndrome de Cockayne , Células-Tronco Pluripotentes Induzidas , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , DNA Helicases/genética , Enzimas Reparadoras do DNA/metabolismo , Síndrome de Cockayne/genética , Síndrome de Cockayne/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Encéfalo/metabolismo , Organoides/metabolismoRESUMO
There is a call for a paradigm shift in developmental neurotoxicity (DNT) evaluation, which demands the implementation of faster, more cost-efficient, and human-relevant test systems than current in vivo guideline studies. Under the umbrella of the Organisation for Economic Co-operation and Development (OECD), a guidance document is currently being prepared that instructs on the regulatory use of a DNT in vitro battery (DNT IVB) for fit-for-purpose applications. One crucial issue for OECD application of methods is validation, which for new approach methods (NAMs) requires novel approaches. Here, mechanistic information previously identified in vivo, as well as reported neurodevelopmental adversities in response to disturbances on the cellular and tissue level, are of central importance. In this study, we scientifically validate the Neurosphere Assay, which is based on human primary neural progenitor cells (hNPCs) and an integral part of the DNT IVB. It assesses neurodevelopmental key events (KEs) like NPC proliferation (NPC1ab), radial glia cell migration (NPC2a), neuronal differentiation (NPC3), neurite outgrowth (NPC4), oligodendrocyte differentiation (NPC5), and thyroid hormone-dependent oligodendrocyte maturation (NPC6). In addition, we extend our work from the hNPCs to human induced pluripotent stem cell-derived NPCs (hiNPCs) for the NPC proliferation (iNPC1ab) and radial glia assays (iNPC2a). The validation process we report for the endpoints studied with the Neurosphere Assays is based on 1) describing the relevance of the respective endpoints for brain development, 2) the confirmation of the cell type-specific morphologies observed in vitro, 3) expressions of cell type-specific markers consistent with those morphologies, 4) appropriate anticipated responses to physiological pertinent signaling stimuli and 5) alterations in specific in vitro endpoints upon challenges with confirmed DNT compounds. With these strong mechanistic underpinnings, we posit that the Neurosphere Assay as an integral part of the DNT in vitro screening battery is well poised for DNT evaluation for regulatory purposes.
RESUMO
Stable and predictive neural cell culture models are a necessary premise for many research fields. However, conventional 2D models lack 3D cell-material/-cell interactions and hence do not reflect the complexity of the in vivo situation properly. Here two alginate/gellan gum/laminin (ALG/GG/LAM) hydrogel blends are presented for the fabrication of human induced pluripotent stem cell (hiPSC)-based 3D neural models. For hydrogel embedding, hiPSC-derived neural progenitor cells (hiNPCs) are used either directly or after 3D neural pre-differentiation. It is shown that stiffness and stress relaxation of the gel blends, as well as the cell differentiation strategy influence 3D model development. The embedded hiNPCs differentiate into neurons and astrocytes within the gel blends and display spontaneous intracellular calcium signals. Two fit-for-purpose models valuable for i) applications requiring a high degree of complexity, but less throughput, such as disease modeling and long-term exposure studies and ii) higher throughput applications, such as acute exposures or substance screenings are proposed. Due to their wide range of applications, adjustability, and printing capabilities, the ALG/GG/LAM based 3D neural models are of great potential for 3D neural modeling in the future.
Assuntos
Células-Tronco Pluripotentes Induzidas , Alginatos , Diferenciação Celular , Humanos , Hidrogéis , Laminina , Polissacarídeos Bacterianos , Impressão TridimensionalRESUMO
Human induced pluripotent stem cells (hiPSC) are a promising tool for replacing animal-based experiments. To warrant data reproducibility, quality-controlled research material is recommended. While the need for global harmonization of quality standards for stem cell banking centers, commercial providers, pre-clinical and clinical use of cells is well documented, there are no recommendations available for quality control of hiPSC in an academic research environment to date. To fill this gap, we here give an example of a quality-controlled, two-tiered banking process producing a fully characterized master cell bank (MCB) and partially characterized respective working cell banks (WCB). Characterization includes the study of morphology, mycoplasma contamination, cell line identity, karyotype stability, cell antigen expression and viability, gene expression, pluripotency, and post-thaw recovery. Costs of these procedures are calculated. We present the results of the proposed testing panel of two hiPSC MCBs and show that both fulfil the defined specifications regarding the above-mentioned characterization assays during and upon banking. In conclusion, we propose a panel of eight assays, which are practical and useful for an academic research laboratory working with hiPSCs. Meeting these proposed specifications ensures the quality of pluripotent stem cells throughout diverse experiments at moderate costs.
Assuntos
Células-Tronco Pluripotentes Induzidas , Células-Tronco Pluripotentes , Animais , Técnicas de Cultura de Células , Diferenciação Celular , Linhagem Celular , Humanos , Reprodutibilidade dos TestesRESUMO
Biodegradable hydrogels that promote stem cell differentiation into neurons in three dimensions (3D) are highly desired in biomedical research to study drug neurotoxicity or to yield cell-containing biomaterials for neuronal tissue repair. Here, we demonstrate that oxidized alginate-gelatin-laminin (ADA-GEL-LAM) hydrogels facilitate neuronal differentiation and growth of embedded human induced pluripotent stem cell (hiPSC) derived neurospheres. ADA-GEL and ADA-GEL-LAM hydrogels exhibiting a stiffness close to ~5 kPa at initial cell culture conditions of 37 °C were prepared. Laminin supplemented ADA-GEL promoted an increase in neuronal differentiation in comparison to pristine ADA-GEL, with enhanced neuron migration from the neurospheres to the bulk 3D hydrogel matrix. The presence of laminin in ADA-GEL led to a more than two-fold increase in the number of neurospheres with migrated neurons. Our findings suggest that laminin addition to oxidized alginate-gelatin hydrogel matrices plays a crucial role to tailor oxidized alginate-gelatin hydrogels suitable for 3D neuronal cell culture applications.