Protocol for the derivation, culturing, and differentiation of human iPS-cell-derived neuroepithelial stem cells to study neural differentiation in vitro.
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ABSTRACT:
SUBMITTER: Calvo-Garrido J
PROVIDER: S-EPMC8121988 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Research on human brain development and neurological diseases is limited by the lack of advanced experimental in vitro models that truly recapitulate the complexity of the human brain. Here, we describe a robust human brain organoid system that is highly specific to the midbrain derived from regionally patterned neuroepithelial stem cells. These human midbrain organoids contain spatially organized groups of dopaminergic neurons, which make them an attractive model for the study of Parkinson's disease. Midbrain organoids are characterized in detail for neuronal, astroglial, and oligodendrocyte differentiation. Furthermore, we show the presence of synaptic connections and electrophysiological activity. The complexity of this model is further highlighted by the myelination of neurites. The present midbrain organoid system has the potential to be used for advanced in vitro disease modeling and therapy development.
| S-EPMC5425618 | biostudies-literature
Project description:Research on human brain development and neurological diseases is limited by the lack of advanced experimental in vitro models that truly recapitulate the complexity of the human brain. Here, we describe a robust human brain organoid system that is highly specific to the midbrain derived from regionally patterned neuroepithelial stem cells. These human midbrain organoids contain spatially organized groups of dopaminergic neurons, which make them an attractive model for the study of Parkinson's disease. Midbrain organoids are characterized in detail for neuronal, astroglial, and oligodendrocyte differentiation. Furthermore, we show the presence of synaptic connections and electrophysiological activity. The complexity of this model is further highlighted by the myelination of neurites. The present midbrain organoid system has the potential to be used for advanced in vitro disease modeling and therapy development.
| S-EPMC5425618 | biostudies-literature
Project description:Research on human brain development and neurological diseases is limited by the lack of advanced experimental in vitro models that truly recapitulate the complexity of the human brain. Here, we describe a robust human brain organoid system that is highly specific to the midbrain derived from regionally patterned neuroepithelial stem cells. These human midbrain organoids contain spatially organized groups of dopaminergic neurons, which make them an attractive model for the study of Parkinson's disease. Midbrain organoids are characterized in detail for neuronal, astroglial, and oligodendrocyte differentiation. Furthermore, we show the presence of synaptic connections and electrophysiological activity. The complexity of this model is further highlighted by the myelination of neurites. The present midbrain organoid system has the potential to be used for advanced in vitro disease modeling and therapy development.
| S-EPMC5425618 | biostudies-literature
Project description:Medulloblastoma is the most common malignant brain tumor in children. Here we describe a medulloblastoma model using Induced pluripotent stem (iPS) cell-derived human neuroepithelial stem (NES) cells generated from a Gorlin syndrome patient carrying a germline mutation in the sonic hedgehog (SHH) receptor PTCH1. We found that Gorlin NES cells formed tumors in mouse cerebellum mimicking human medulloblastoma. Retransplantation of tumor-isolated NES (tNES) cells resulted in accelerated tumor formation, cells with reduced growth factor dependency, enhanced neurosphere formation in vitro, and increased sensitivity to Vismodegib. Using our model, we identified LGALS1 to be a GLI target gene that is up-regulated in both Gorlin tNES cells and SHH-subgroup of medulloblastoma patients. Taken together, we demonstrate that NES cells derived from Gorlin patients can be used as a resource to model medulloblastoma initiation and progression and to identify putative targets.
| S-EPMC7443968 | biostudies-literature
Project description:BackgroundA major challenge for the clinical use of human pluripotent stem cells is the development of safe, robust and controlled differentiation protocols. Adaptation of research protocols using reagents designated as research-only to those which are suitable for clinical use, often referred to as good manufacturing practice (GMP) reagents, is a crucial and laborious step in the translational pipeline. However, published protocols to assist this process remain very limited.MethodsWe adapted research-grade protocols for the derivation and differentiation of long-term neuroepithelial stem cell progenitors (lt-NES) to GMP-grade reagents and factors suitable for clinical applications. We screened the robustness of the protocol with six clinical-grade hESC lines deposited in the UK Stem Cell Bank.ResultsHere, we present a new GMP-compliant protocol to derive lt-NES, which are multipotent, bankable and karyotypically stable. This protocol resulted in robust and reproducible differentiation of several clinical-grade embryonic stem cells from which we derived lt-NES. Furthermore, GMP-derived lt-NES demonstrated a high neurogenic potential while retaining the ability to be redirected to several neuronal sub-types.ConclusionsOverall, we report the feasibility of derivation and differentiation of clinical-grade embryonic stem cell lines into lt-NES under GMP-compliant conditions. Our protocols could be used as a flexible tool to speed up translation-to-clinic of pluripotent stem cells for a variety of neurological therapies or regenerative medicine studies.
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Project description:Establishing specific cell lineages from human induced pluripotent stem cells (hiPSCs) is vital for cell therapy approaches in regenerative medicine, particularly for neurodegenerative disorders. While neural precursors have been induced from hiPSCs, the establishment of hiPSC-derived human neural stem cells (hiNSCs), with characteristics that match foetal hNSCs and abide by cGMP standards, thus allowing clinical applications, has not been described. We generated hiNSCs by a virus-free technique, whose properties recapitulate those of the clinical-grade hNSCs successfully used in an Amyotrophic Lateral Sclerosis (ALS) phase I clinical trial. Ex vivo, hiNSCs critically depend on exogenous mitogens for stable self-renewal and amplification and spontaneously differentiate into astrocytes, oligodendrocytes and neurons upon their removal. In the brain of immunodeficient mice, hiNSCs engraft and differentiate into neurons and glia, without tumour formation. These findings now warrant the establishment of clinical-grade, autologous and continuous hiNSC lines for clinical trials in neurological diseases such as Huntington's, Parkinson's and Alzheimer's, among others.
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Project description:Exosomes, a key element of the central nervous system microenvironment, mediate intercellular communication via horizontally transferring bioactive molecules. Emerging evidence has implicated exosomes in the regulation of neurogenesis. Recently, we compared the neurogenic potential of exosomes released from primary mouse embryonic neural stem cells (NSCs) and astrocyte-reprogrammed NSCs, and observed diverse neurogenic potential of those two exosome populations in vitro. However, the roles of NSC-derived exosomes on NSC differentiation and the underlying mechanisms remain largely unknown. In this study, we firstly demonstrated that NSC-derived exosomes facilitate the differentiation of NSCs and the maturation of both neuronal and glial cells in defined conditions. We then identified miR-9, a pro-neural miRNA, as the most abundantly expressed miRNA in NSC-derived exosomes. The silencing of miR-9 in exosomes abrogates the positive effects of NSC-derived exosomes on the differentiation of NSCs. We further identified Hes1 as miR-9 downstream target, as the transfection of Hes1 siRNA restored the differentiation promoting potential of NSC-derived exosomes after knocking down exosomal miR-9. Thus, our data indicate that NSC-derived exosomes facilitate the differentiation of NSCs via transferring miR-9, which sheds light on the development of cell-free therapeutic strategies for treating neurodegeneration.
| S-EPMC8155619 | biostudies-literature
Project description:Recent advances in human induced pluripotent stem cell-derived cardiomyocytes (iPSCM) field offer a novel platform for modeling cardiac metabolism, heart diseases drug candidates screening and cardiac toxicity assessments. These workflows require a fully functional characterization of iPSCMs. Here we report a step by step protocol for iPSCM metabolic characterization. The described assays cover analysis of small metabolites involved in a vital metabolic pathways.
| S-EPMC7046813 | biostudies-literature
Project description:Reports of functional recovery from spinal cord injury after the transplantation of rat fetus-derived neural stem cells and embryonic stem cells has raised great expectations for the successful clinical use of stem cell transplantation therapy. However, the ethical issues involved in destroying human embryos or fertilized oocytes to obtain stem cells have been a major obstacle to developing clinically useful stem cell sources, and the transplantation of stem cells isolated from other human embryonic tissues has not yet been developed for use in clinical applications. Recently, induced pluripotent stem cells, which can serve as a source of cells for autologous transplantation, have been attracting a great deal of attention as a clinically viable alternative to stem cells obtained directly from tissues. In this review, we outline the neural induction of mouse embryonic stem cells and induced pluripotent stem cells, their therapeutic efficacy in spinal cord injury, and their safety in vivo.
| S-EPMC3250290 | biostudies-literature
Project description:Heavy alcohol consumption during pregnancy can cause significant mental retardation and brain damage. We recently showed that ethanol depletes reserve cerebral cortical stem cell capacity. Moreover, proliferating neuroepithelial cells exposed to ethanol were resistant to subsequent retinoic acid-induced differentiation. Emerging evidence suggests that cytokines play a crucial growth-promoting role in the developing neural tube.We cultured murine cortical neurosphere cultures in control or ethanol-supplemented mitogenic medium, to mimic alcohol exposure during the period of neuroepithelial proliferation. Cultures were then treated with a step-wise mitogen-withdrawal, integrin-activation model to mimic subsequent phases of neuronal migration and early differentiation. We examined the impact of alcohol exposure during neurogenesis on the secretion of inflammatory and growth-promoting cytokines.Cortical neurosphere cultures exhibit increasingly complex differentiation phenotypes in response to step-wise mitogen-withdrawal and laminin exposure. Some inflammation-modulating cytokines were secreted independent of differentiation state. However, chemotactic cytokines were specifically secreted at high levels, as a function of differentiation stage. monocyte chemotactic protein-1, vascular endothelial growth factor-A, and interleukin (IL)-10 were coordinately decreased during differentiation compared with neuroepithelial proliferation, while granulocyte macrophage-colony stimulating factor (GM-CSF) was induced during differentiation, compared with the neuroepithelial proliferation period. Ethanol exposure during the period of neuroepithelial proliferation prevented the early differentiation-induced increase in GM-CSF while inducing differentiation-associated increase in IL-12 secretion.Embryonic cerebral cortical neuroepithelial-derived precursors secrete high levels of several angiogenic and neural-growth-promoting cytokines as they differentiate into neurons. Our data collectively suggest that ethanol exposure during the period of neuroepithelial proliferation significantly disrupts cytokine signals that are required for the support of emerging neurovascular networks, and the maintenance of neural stem cell beds.
| S-EPMC2909106 | biostudies-literature