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Human tau mutations in cerebral organoids induce a progressive dyshomeostasis of cholesterol.
Mutations in the MAPT gene that encodes tau lead to frontotemporal dementia (FTD) with pathology evident in both cerebral neurons and glia. Human cerebral organoids (hCOs) from individuals harboring pathogenic tau mutations can reveal the earliest downstream effects on molecular pathways within a developmental context, generating interacting neurons and glia. We found that in hCOs carrying the V337M and R406W tau mutations, the cholesterol biosynthesis pathway in astrocytes was the top upregulated gene set compared with isogenic controls by single-cell RNA sequencing (scRNA-seq). The 15 upregulated genes included HMGCR, ACAT2, STARD4, LDLR, and SREBF2. This result was confirmed in a homozygous R406W mutant cell line by immunostaining and sterol measurements. Cholesterol abundance in the brain is tightly regulated by efflux and cholesterol biosynthetic enzyme levels in astrocytes, and dysregulation can cause aberrant phosphorylation of tau. Our findings suggest that cholesterol dyshomeostasis is an early event in the etiology of neurodegeneration caused by tau mutations.
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Autophagy is required for the development and functionality of lacrimal gland-like organoids.
Lacrimal glands (LGs) serve as pivotal exocrine glands crucial for protecting the ocular surface. Dysfunction in LG cell composition or secretion is implicated in dry eye disease (DED). While autophagy plays a vital role in tissue homeostasis in many organs, how it affects LG development and secretory function is not known. Here, we have undertaken a genetic study by utilizing autophagy-deficient human embryonic stem cells (hESCs) and differentiating them into LG-like organoids. Autophagy-deficient LG-like organoids exhibited improper development and secretion, along with increased protein aggregation, proliferation, and cell death. These phenotypes were associated with an accumulation of PAX6, a transcription factor crucial for brain and eye development, which we identified as an autophagy substrate. Pharmacological interventions with nicotinamide mononucleotide (NMN) and melatonin were able to rescue the cellular dysfunction in autophagy-deficient LG-like organoids. Together, our study highlights the role of autophagy in LG along with potential therapeutic interventions for DED.
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From engineered heart tissue to cardiac organoid.
The advent of human pluripotent stem cells (hPSCs) presented a new paradigm to employ hPSC-derived cardiomyocytes (hPSC-CMs) in drug screening and disease modeling. However, hPSC-CMs differentiated in conventional two-dimensional systems are structurally and functionally immature. Moreover, these differentiation systems generate predominantly one type of cell. Since the heart includes not only CMs but other cell types, such monolayer cultures have limitations in simulating the native heart. Accordingly, three-dimensional (3D) cardiac tissues have been developed as a better platform by including various cardiac cell types and extracellular matrices. Two advances were made for 3D cardiac tissue generation. One type is engineered heart tissues (EHTs), which are constructed by 3D cell culture of cardiac cells using an engineering technology. This system provides a convenient real-time analysis of cardiac function, as well as a precise control of the input/output flow and mechanical/electrical stimulation. The other type is cardiac organoids, which are formed through self-organization of differentiating cardiac lineage cells from hPSCs. While mature cardiac organoids are more desirable, at present only primitive forms of organoids are available. In this review, we discuss various models of hEHTs and cardiac organoids emulating the human heart, focusing on their unique features, utility, and limitations.
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Valproic acid-induced teratogenicity is driven by senescence and prevented by Rapamycin in human spinal cord and animal models.
Valproic acid (VPA) is an effective and widely used anti-seizure medication but is teratogenic when used during pregnancy, affecting brain and spinal cord development for reasons that remain largely unclear. Here we designed a genetic recombinase-based SOX10 reporter system in human pluripotent stem cells that enables tracking and lineage tracing of Neural Crest cells (NCCs) in a human organoid model of the developing neural tube. We found that VPA induces extensive cellular senescence and promotes mesenchymal differentiation of human NCCs. We next show that the clinically approved drug Rapamycin inhibits senescence and restores aberrant NCC differentiation trajectory after VPA exposure in human organoids and in developing zebrafish, highlighting the therapeutic promise of this approach. Finally, we identify the pioneer factor AP1 as a key element of this process. Collectively our data reveal cellular senescence as a central driver of VPA-associated neurodevelopmental teratogenicity and identifies a new pharmacological strategy for prevention. These results exemplify the power of genetically modified human stem cell-derived organoid models for drug discovery.
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3D-Printed Scaffolds Promote Enhanced Spinal Organoid Formation for Use in Spinal Cord Injury.
The transplantation of regionally specific spinal neural progenitor cells (sNPCs) has shown promise for functional restoration after spinal cord injury (SCI) by forming connections with host neural circuits. Here, 3D-printed organoid scaffolds for transplantation using clinically relevant human induced pluripotent stem cell-derived regionally specific sNPCs is developed. Scaffolds with microscale channels are printed, and sNPCs are subsequently printed within these channels. The scaffolds direct axonal projections along the channels and guide the cells to simulate in vivo-like conditions, leading to more effective cell maturation and the development of neuronal networks crucial for restoring function after SCI. The scaffolds, with organoids assembled along their lengths, are transplanted into the transected spinal cords of rats. This significantly promotes the functional recovery of the rats. At 12 weeks post-transplantation, the majority of the cells in the scaffolds differentiate into neurons and integrate into the host spinal cord tissue. These results demonstrate their potential to create a relay system along the spinal cord and form synapses in both the rostral and caudal directions relative to the scaffold. It is envisioned that combining sNPCs, organoid assembly, and 3D printing strategies can ultimately lead to a transformative treatment approach for SCI.
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Generation of innervated cochlear organoid recapitulates early development of auditory unit.
Functional cochlear hair cells (HCs) innervated by spiral ganglion neurons (SGNs) are essential for hearing, whereas robust models that recapitulate the peripheral auditory circuity are still lacking. Here, we developed cochlear organoids with functional peripheral auditory circuity in a staging three-dimensional (3D) co-culture system by initially reprogramming cochlear progenitor cells (CPCs) with increased proliferative potency that could be long-term expanded, then stepwise inducing the differentiation of cochlear HCs, as well as the outgrowth of neurites from SGNs. The function of HCs and synapses within organoids was confirmed by a series of morphological and electrophysiological evaluations. Single-cell mRNA sequencing revealed the differentiation trajectories of CPCs toward the major cochlear cell types and the dynamic gene expression during organoid HC development, which resembled the pattern of native HCs. We established the cochlear organoids with functional synapses for the first time, which provides a platform for deciphering the mechanisms of sensorineural hearing loss.
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Protocol to generate species-specific astrocyte-conditioned medium for human organoid neuron maturation.
Brain organoids have emerged as promising models for neuroscience research; however, their utility is often limited by inadequate maturation. Here, we present a protocol for enhancing the maturation of human 2D and 3D neural cultures using astrocyte-conditioned medium (ACM). We outline the steps for isolating primary mouse astrocytes, culturing both mouse and human astrocytes, and preparing ACM. Additionally, we provide detailed procedures for quality control and downstream applications of ACM derived from different species. For complete details on the use and execution of this protocol, please refer to Zheng et al.1.
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Generating Patient-Derived Gliomas within Cerebral Organoids.
Glioblastoma (GBM) remains a devastating disease with a median survival of less than two years. Current preclinical models are unable to accurately reflect the complexity of human GBM. We recently established a cerebral organoid glioma (GLICO) model to study the invasion and biology of patient-derived glioma stem cells in miniature replicas of the human brain. Through the dissemination of our detailed methodology, we aim to encourage other scientists to further build upon our existing model for studying these destructive tumors. For complete details on the use and execution of this protocol, please refer to Linkous et al. (2019).
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Pax6 isoforms shape eye development: Insights from developmental stages and organoid models.
Pax6 is a critical transcription factor involved in the development of the central nervous system. However, in humans, mutations in Pax6 predominantly result in iris deficiency rather than neurological phenotypes. This may be attributed to the distinct functions of Pax6 isoforms, Pax6a and Pax6b. In this study, we investigated the spatial and temporal expression patterns of Pax6 isoforms during different stages of mouse eye development. We observed a strong correlation between Pax6a expression and the neuroretina gene Sox2, while Pax6b showed a high correlation with iris-component genes, including the mesenchymal gene Foxc1. During early patterning from E10.5, Pax6b was expressed in the hinge of the optic cup and neighboring mesenchymal cells, whereas Pax6a was absent in these regions. At E14.5, both Pax6a and Pax6b were expressed in the future iris and ciliary body, coinciding with the integration of mesenchymal cells and Mitf-positive cells in the outer region. From E18.5, Pax6 isoforms exhibited distinct expression patterns as lineage genes became more restricted. To further validate these findings, we utilized ESC-derived eye organoids, which recapitulated the temporal and spatial expression patterns of lineage genes and Pax6 isoforms. Additionally, we found that the spatial expression patterns of Foxc1 and Mitf were impaired in Pax6b-mutant ESC-derived eye organoids. This in vitro eye organoids model suggested the involvement of Pax6b-positive local mesodermal cells in iris development. These results provide valuable insights into the regulatory roles of Pax6 isoforms during iris and neuroretina development and highlight the potential of ESC-derived eye organoids as a tool for studying normal and pathological eye development.
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Novel Human Meningioma Organoids Recapitulate the Aggressiveness of the Initiating Cell Subpopulations Identified by ScRNA-Seq.
High-grade meningioma has an unsatisfactory outcome despite surgery and postoperative radiotherapy; however, the factors driving its malignancy and recurrence remain largely unknown, which limits the development of systemic treatments. Single-cell RNA sequencing (scRNA-Seq) technology is a powerful tool for studying intratumoral cellular heterogeneity and revealing the roles of various cell types in oncogenesis. In this study, scRNA-Seq is used to identify a unique initiating cell subpopulation (SULT1E1+ ) in high-grade meningiomas. This subpopulation modulates the polarization of M2-type macrophages and promotes meningioma progression and recurrence. A novel patient-derived meningioma organoid (MO) model is established to characterize this unique subpopulation. The resulting MOs fully retain the aggressiveness of SULT1E1+ and exhibit invasiveness in the brain after orthotopic transplantation. By targeting SULT1E1+ in MOs, the synthetic compound SRT1720 is identified as a potential agent for systemic treatment and radiation sensitization. These findings shed light on the mechanism underlying the malignancy of high-grade meningiomas and provide a novel therapeutic target for refractory high-grade meningioma.