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Abnormal mitochondria in Down syndrome iPSC-derived GABAergic interneurons and organoids.
Down syndrome (DS) is caused by trisomy 21, and it is characterized by developmental brain disorders and neurological dysfunction. Clinical studies and basic research have revealed that defects in mitochondrial function contribute to the pathogenesis of DS. However, the underlying mechanisms of mitochondrial dysfunction in DS remain unclear. In this study, we first generated GABAergic interneurons and medial ganglionic eminence (MGE) organoids from DS patients and control induced pluripotent stem cells. The mitochondria were abnormally clustered in the perinuclear region of GABA neurons and cell in MGE organoids from DS patients, which exhibited impaired mitochondrial function as assessed by seahorse oxidative phosphorylation assay. Inhibition of the DSCAM-PAK1 pathway by gene editing or treatment with a small molecule corrected mitochondrial perinuclear aggregation in cells from DS patients. Therefore, our study provides insight into the potential mechanism of mitochondrial dysfunction in DS.
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Proteomic phenotype of cerebral organoids derived from autism spectrum disorder patients reveal disrupted energy metabolism, cellular components, and biological processes.
The way in which brain morphology and proteome are remodeled during embryonal development, and how they are linked to the cellular metabolism, could be a key for elucidating the pathological mechanisms of certain neurodevelopmental disorders. Cerebral organoids derived from autism spectrum disorder (ASD) patients were generated to capture critical time-points in the neuronal development, and metabolism and protein expression were investigated. The early stages of development, when neurogenesis commences (day in vitro 39), appeared to be a critical timepoint in pathogenesis. In the first month of development, increased size in ASD-derived organoids were detected in comparison to the controls. The size of the organoids correlates with the number of proliferating cells (Ki-67 positive cells). A significant difference in energy metabolism and proteome phenotype was also observed in ASD organoids at this time point, specifically, prevalence of glycolysis over oxidative phosphorylation, decreased ATP production and mitochondrial respiratory chain activity, differently expressed cell adhesion proteins, cell cycle (spindle formation), cytoskeleton, and several transcription factors. Finally, ASD patients and controls derived organoids were clustered based on a differential expression of ten proteins-heat shock protein 27 (hsp27) phospho Ser 15, Pyk (FAK2), Elk-1, Rac1/cdc42, S6 ribosomal protein phospho Ser 240/Ser 244, Ha-ras, mTOR (FRAP) phospho Ser 2448, PKCα, FoxO3a, Src family phospho Tyr 416-at day 39 which could be defined as potential biomarkers and further investigated for potential drug development.
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2-Cl-C.OXT-A stimulates contraction through the suppression of phosphodiesterase activity in human induced pluripotent stem cell-derived cardiac organoids.
BACKGROUND: 2-Cl-C.OXT-A (COA-Cl) is a novel synthesized adenosine analog that activates Sphingosine-1-phosphate 1 receptor (S1P1R) and combines with the adenosine A1 receptor (A1R) in G proteins and was shown to enhance angiogenesis and improve the brain function in rat stroke models. However, the role of COA-Cl in hearts remains unclear. COA-Cl, which has a similar structure to xanthine derivatives, has the potential to suppress phosphodiesterase (PDE), which is an important factor involved in the beating of heart muscle. METHODS AND RESULTS: Cardiac organoids with fibroblasts, human induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CMs), and hiPSC-derived endothelial cells (hiPSC-ECs) were cultured until they started beating. The beating and contraction of organoids were observed before and after the application of COA-Cl. COA-Cl significantly increased the beating rate and fractional area change in organoids. To elucidate the mechanism underlying these effects of COA-Cl on cardiac myocytes, pure hiPSC-CM spheroids were evaluated in the presence/absence of Suramin (antagonist of A1R). The effects of COA-Cl, SEW2871 (direct stimulator of S1P1R), two positive inotropes (Isoproterenol [ISO] and Forskolin [FSK]), and negative inotrope (Propranolol [PRP]) on spheroids were assessed based on the beating rates and cAMP levels. COA-Cl stimulated the beating rates about 1.5-fold compared with ISO and FSK, while PRP suppressed the beating rate. However, no marked changes were observed with SEW2871. COA-Cl, ISO, and FSK increased the cAMP level. In contrast, the level of cAMP did not change with PRP or SEW2871 treatment. The results were the same in the presence of Suramin as absence. Furthermore, an enzyme analysis showed that COA-Cl suppressed the PDE activity by half. CONCLUSIONS: COA-Cl, which has neovascularization effects, suppressed PDE and increased the contraction of cardiac organoids, independent of S1P1R and A1R. These findings suggest that COA-Cl may be useful as an inotropic agent for promoting angiogenesis in the future.
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The search for better animal models of Alzheimer's disease.
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Generation of human induced pluripotent stem cell-derived cerebral organoids for cellular and molecular characterization.
Human induced pluripotent stem cell (hiPSC)-derived cerebral organoids (COs) can serve as an in vitro model for studying normal and pathologic human brain development. Here, we optimized existing protocols to streamline the generation of forebrain COs from hiPSCs. We employ these COs to define the impact of disease-causing mutations on cell fate, differentiation, maturation, and morphology relevant to neurodevelopmental disorders. Although limited to forebrain CO identity, this schema requires minimal external interference and is amenable to low-throughput biochemical assays. For complete details on the use and execution of this profile, please refer to Anastasaki et al. (2020) and Wegscheid et al. (2021).
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HOPX regulates the development and lineage progression of human stem cell-derived outer radial glia-like cells.
Outer radial glia (oRG) are abundant in the human fetal cortex but rare in rodents, yet their molecular regulation remains poorly understood. Although the homeodomain-only protein X (HOPX) is a well-established oRG marker, its functional role in human cortical development remains undefined. Using CRISPR-Cas9 gene editing in human pluripotent stem cells, we show that HOPX loss impairs the expansion and lineage progression of oRG-like cells in two-dimensional neural cultures and three-dimensional cortical organoids. HOPX knockout precursors exhibit reduced proliferation, diminished neurogenesis, and premature differentiation toward astrocytic and oligodendrocytic lineages. HOPX-deficient organoids display reduced outer subventricular zone size, decreased neuronal output, and early gliogenesis. Mechanistically, HOPX loss impairs PI3K-mTOR signaling. Transcriptomic analysis identifies HOPX-regulated programs governing gliogenic competence, mTOR, and Wnt signaling. Together, these findings identify HOPX as an important regulator of oRG-like cell maintenance and the neurogenic-gliogenic balance, providing insight into the molecular programs governing human neural progenitor expansion.
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Prenatal organoids for personalised medicine.
Organoids are three-dimensional structures derived from a single stem cell. They self-organize in vitro and mature to recreate microarchitecture and characteristics of their tissues of origin. Since the first culture of mouse intestinal epithelial organoids and the translation to human, this technology has shown vast potential. Applications include developmental biology, disease modelling, drug testing and regenerative medicine making it increasingly relevant to clinical practice. Organoids can now be generated from tissues such as lung, intestine, kidney and brain using adult and fetal stem cells, pluripotent stem cells and cancer samples. Whether to study fetal development, disease physiopathology, regenerative medicine or to develop new therapies in a faster, more ethical and cost-effective way than animal models, organoids represent a powerful new tool. First described in 1999, personalised medicine aims to fine-tune diagnostic, prognosis and treatment to each patient. Nowadays, personalised medicine is spreading to most areas of clinical management, becoming the gold standard for the adoption of new interventions. More recently, these concepts have been applied to fetal medicine for prevention and therapy with initial reports appearing in literature in 2020. By recapitulating patient-specific disease mechanisms, prenatal organoids represent a unique bridge between in vitro-based approaches and personalised fetal medicine. This review presents fetal diseases for which organoids were explored, focusing on the implications of their derivation from prenatal sources such as the amniotic fluid, and discusses their advantages, challenges, and perspectives for prenatal counselling, prognosis or diagnosis, regenerative therapy, in utero surgery, disease modelling and prenatal drug testing.
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3D Neuromodulation in Neural Organoids with Shell MEAs.
Neural organoids (NOs) have emerged as important tissue engineering models for microphysiological systems, brain sciences, and biocomputing. Establishing reliable relationships between stimulation and recording traces of electrical activity is essential for monitoring the functionality of NOs, especially in paradigms such as neural plasticity, learning, or stimulus discrimination. While researchers have demonstrated neuromodulation in NOs, they have primarily used 2D microelectrode arrays (MEAs) with limited access to the entire 3D contour of the NOs. Here, we report neuromodulation using tiny mimics of macroscale EEG caps, or shell MEAs. Specifically, we observe that stimulating current within a specific range (20 to 30 µA) induced a statistically significant increase in neuron firing rate when comparing the activity 5 s before and after stimulation. We detect neuromodulatory behavior using both three- and 16-electrode shells and generated 3D spatiotemporal maps of neuromodulatory activity around the entire surface of the NO. Our studies demonstrate a methodology for investigating 3D spatiotemporal neuromodulation in organoids of broad relevance to biomedical engineering models of neural functionality, plasticity, and learning.
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Redesigning hydrogel geometry for enhanced organoids.
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Dysregulation of Human ClpP Using Small Molecules with Piperazine-Based Scaffold for Diffuse Intrinsic Pontine Glioma Therapy Validated by Patient-Derived Tumor Organoids.
Diffuse intrinsic pontine glioma (DIPG) is a dismal pediatric brainstem tumor with a median survival of approximately 12 months and no curative treatments. ONC201, the most clinically characterized small-molecule, selectively activates human caseinolytic protease P (hClpP). Guided by X-ray structure of the hClpP:ONC201 complex, piperazine-based derivatives were designed and identified compound 26 (DA29) as a lead. Structural studies confirmed its binding to the enzyme hydrophobic allosteric pocket, a finding consistent with its strong proteolytic activation. Permeability under dynamic flow conditions suggested the ability of 26 (DA29) to cross cellular barriers, indicating potential brain tumor penetration. Mechanistically, 26 (DA29) activates hClpP, displaces hClpX from the hClpXP complex, induces mitochondrial dysfunction and ROS accumulation, and exerts cytotoxic effects in patient-derived DIPG cells and organoids. Notably, unlike ONC201, it does not interact with D2/D3 dopamine receptors. Overall, these findings establish a new structural framework for hClpP-targeted therapies and support further development of 26 (DA29) for DIPG treatment.