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Visualization of individual cell division history in complex tissues using iCOUNT.
The division potential of individual stem cells and the molecular consequences of successive rounds of proliferation remain largely unknown. Here, we developed an inducible cell division counter (iCOUNT) that reports cell division events in human and mouse tissues in vitro and in vivo. Analyzing cell division histories of neural stem/progenitor cells (NSPCs) in the developing and adult brain, we show that iCOUNT can provide novel insights into stem cell behavior. Further, we use single-cell RNA sequencing (scRNA-seq) of iCOUNT-labeled NSPCs and their progenies from the developing mouse cortex and forebrain-regionalized human organoids to identify functionally relevant molecular pathways that are commonly regulated between mouse and human cells, depending on individual cell division histories. Thus, we developed a tool to characterize the molecular consequences of repeated cell divisions of stem cells that allows an analysis of the cellular principles underlying tissue formation, homeostasis, and repair.
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Transparency and ongoing communication with participants in brain organoid research: Consensus of an interdisciplinary working group.
Stem cell-based models of the human brain benefit from biospecimens that can be used for a broad range of future research. But current regulations do not address the desire of research participants to remain engaged beyond initial biospecimen donation. We present practicable strategies for engaging participants while preserving scientific potential.
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Axially multifocal metalens for 3D volumetric photoacoustic imaging of neuromelanin in live brain organoid.
Optical resolution photoacoustic imaging of uneven samples without z-scanning is transformative for the fast analysis and diagnosis of diseases. However, current approaches to elongate the depth of field (DOF) typically imply cumbersome postprocessing procedures, bulky optical element ensembles, or substantial excitation beam side lobes. Metasurface technology allows for the phase modulation of light and the miniaturization of imaging systems to wavelength-size thickness. Here, we propose a metalens composed of submicrometer-thick titanium oxide nanopillars, which generates an elongated beam of diffraction-limited diameter with an aspect ratio of 286 and a uniform intensity throughout the DOF. The metalens enhances visualization of phantom samples with tilted surfaces compared to conventional lenses. Moreover, the volumetric imaging of neuromelanin is facilitated for depths of up to 500 micrometers within the human midbrain and forebrain organoids that are 3D biological models of human brain regions. This approach provides a miniaturized platform for neurodegenerative disease diagnosis and drug discovery.
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Broadly Active Antiviral Compounds Disturb Zika Virus Progeny Release Rescuing Virus-Induced Toxicity in Brain Organoids.
RNA viruses have gained plenty of attention during recent outbreaks of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Zika virus (ZIKV), and Ebola virus. ZIKV is a vector borne Flavivirus that is spread by mosquitoes and it mainly infects neuronal progenitor cells. One hallmark of congenital ZIKV disease is a reduced brain size in fetuses, leading to severe neurological defects. The World Health Organization (WHO) is urging the development of new antiviral treatments against ZIKV, as there are no efficient countermeasures against ZIKV disease. Previously, we presented a new class of host-targeting antivirals active against a number of pathogenic RNA viruses, such as SARS-CoV-2. Here, we show the transfer of the image-based phenotypic antiviral assay to ZIKV-infected brain cells, followed by mechanism-of-action studies and a proof-of-concept study in a three-dimensional (3D) organoid model. The novel antiviral compounds showed a therapeutic window against ZIKV in several cell models and rescued ZIKV-induced neurotoxicity in brain organoids. The compound's mechanism-of-action was pinpointed to late steps in the virus life cycle, impairing the formation of new virus particles. Collectively, in this study, we expand the antiviral activity of new small molecule inhibitors to a new virus class of Flaviviruses, but also uncover compounds' mechanism of action, which are important for the further development of antivirals.
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A simple metastatic brain cancer model using human embryonic stem cell-derived cerebral organoids.
Every year, hundreds of thousands of people die because of metastatic brain cancer. Most metastatic cancer research uses 2D cell culture or animal models, but they have a few limitations, such as difficulty reproducing human tissue structures. This study developed a simple 3D in vitro model to better replicate brain metastasis using human cancer cells and human embryonic stem cell-derived cerebral organoids (metastatic brain cancer cerebral organoid [MBCCO]). The MBCCO model successfully reproduced metastatic cancer processes, including cell adhesion, proliferation, and migration, in addition to cell-cell interactions. Using the MBCCO model, we demonstrated that lung-specific X protein (LUNX) plays an important role in cell proliferation and migration or invasion. We also observed astrocyte accumulation around and their interaction with cancer cells through connexin 43 in the MBCCO model. We analyzed whether the MBCCO model can be used to screen drugs by measuring the effects of gefitinib, a well-known anticancer agent. We also examined the toxicity of gefitinib using normal cerebral organoids (COs). Therefore, the MBCCO model is a powerful tool for modeling human metastatic brain cancer in vitro and can also be used to screen drugs.
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Developmental defects and impaired network excitability in a cerebral organoid model of KCNJ11 p.V59M-related neonatal diabetes.
The gene KCNJ11 encodes Kir6.2 a major subunit of the ATP-sensitive potassium channel (KATP) expressed in both the pancreas and brain. Heterozygous gain of function mutations in KCNJ11 can cause neonatal diabetes mellitus (NDM). In addition, many patients exhibit neurological defects ranging from modest learning disorders to severe cognitive dysfunction and seizures. However, it remains unclear to what extent these neurological deficits are due to direct brain-specific activity of mutant KATP. We have generated cerebral organoids derived from human induced pluripotent stem cells (hiPSCs) possessing the KCNJ11 mutation p.Val59Met (V59M) and from non-pathogenic/normal hiPSCs (i.e., control/WT). Control cerebral organoids developed neural networks that could generate stable synchronized bursting neuronal activity whereas those derived from V59M cerebral organoids showed reduced synchronization. Histocytochemical studies revealed a marked reduction in neurons localized to upper cortical layer-like structures in V59M cerebral organoids suggesting dysfunction in the development of cortical neuronal network. Examination of temporal transcriptional profiles of neural stem cell markers revealed an extended window of SOX2 expression in V59M cerebral organoids. Continuous treatment of V59M cerebral organoids with the KATP blocker tolbutamide partially rescued the neurodevelopmental differences. Our study demonstrates the utility of human cerebral organoids as an investigative platform for studying the effects of KCNJ11 mutations on neurophysiological outcome.
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Intelligent acoustofluidics enabled mini-bioreactors for human brain organoids.
Acoustofluidics, by combining acoustics and microfluidics, provides a unique means to manipulate cells and liquids for broad applications in biomedical sciences and translational medicine. However, it is challenging to standardize and maintain excellent performance of current acoustofluidic devices and systems due to a multiplicity of factors including device-to-device variation, manual operation, environmental factors, sample variability, etc. Herein, to address these challenges, we propose "intelligent acoustofluidics" - an automated system that involves acoustofluidic device design, sensor fusion, and intelligent controller integration. As a proof-of-concept, we developed intelligent acoustofluidics based mini-bioreactors for human brain organoid culture. Our mini-bioreactors consist of three components: (1) rotors for contact-free rotation via an acoustic spiral phase vortex approach, (2) a camera for real-time tracking of rotational actions, and (3) a reinforcement learning-based controller for closed-loop regulation of rotational manipulation. After training the reinforcement learning-based controller in simulation and experimental environments, our mini-bioreactors can achieve the automated rotation of rotors in well-plates. Importantly, our mini-bioreactors can enable excellent control over rotational mode, direction, and speed of rotors, regardless of fluctuations of rotor weight, liquid volume, and operating temperature. Moreover, we demonstrated our mini-bioreactors can stably maintain the rotational speed of organoids during long-term culture, and enhance neural differentiation and uniformity of organoids. Comparing with current acoustofluidics, our intelligent system has a superior performance in terms of automation, robustness, and accuracy, highlighting the potential of novel intelligent systems in microfluidic experimentation.
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Single-cell epigenomic reconstruction of developmental trajectories from pluripotency in human neural organoid systems.
Cell fate progression of pluripotent progenitors is strictly regulated, resulting in high human cell diversity. Epigenetic modifications also orchestrate cell fate restriction. Unveiling the epigenetic mechanisms underlying human cell diversity has been difficult. In this study, we use human brain and retina organoid models and present single-cell profiling of H3K27ac, H3K27me3 and H3K4me3 histone modifications from progenitor to differentiated neural fates to reconstruct the epigenomic trajectories regulating cell identity acquisition. We capture transitions from pluripotency through neuroepithelium to retinal and brain region and cell type specification. Switching of repressive and activating epigenetic modifications can precede and predict cell fate decisions at each stage, providing a temporal census of gene regulatory elements and transcription factors. Removing H3K27me3 at the neuroectoderm stage disrupts fate restriction, resulting in aberrant cell identity acquisition. Our single-cell epigenome-wide map of human neural organoid development serves as a blueprint to explore human cell fate determination.
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CX3CL1 (fractalkine)-CX3CR1 interface drives CD8⁺ T cell-induced neurodegeneration in human brain organoids.
Immune-neuron interactions play a critical role in neuroinflammatory and neurodegenerative diseases, such as Alzheimer's disease (AD), but how CD8⁺ T cells contribute to brain pathology remains poorly understood. Amyloid-β (Aβ), a hallmark of AD, is known to trigger neuronal stress and inflammation. Here, we established a human cortical organoid (hCO) model to investigate CD8⁺ T cell responses to Aβ-induced neurodegenerative stress. Aβ exposure increased neuronal expression of CX3CL1 (fractalkine), which engages its receptor CX3CR1 on CD8⁺ T cells. Transwell migration assays showed enhanced recruitment of CX3CR1⁺ CD8⁺ T cells toward Aβ-treated hCOs. In parallel, Aβ upregulated IL-15 expression, a cytokine that supports CD8⁺ T cell activation. The infiltration of CD8⁺ T cells led to neuronal injury, marked by MAP2 loss and cleaved Caspase-3 activation, accompanied by an increase in gene signatures for cell death and inflammation. Exposure to the conditioned medium of activated CX3CR1high CD8⁺ T cell recapitulated this damage, and the neutralization of CD8+ T cell cytokines and cytotoxic molecules rescued the phenotype. These findings identify the CX3CL1-CX3CR1 axis and CD8⁺ T cell effector programs as drivers of neurotoxicity and establish brain organoids as a platform to study immune-driven neurodegeneration.
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LINE-1 retrotransposons mediate cis-acting transcriptional control in human pluripotent stem cells and regulate early brain development.
Long interspersed nuclear element 1 (L1) retrotransposons represent a vast source of genetic variability. However, mechanistic analysis of whether and how L1s contribute to human developmental programs is lacking, in part due to the challenges associated with specific profiling and manipulation of human L1 expression. Here, we show that thousands of hominoid-specific L1 integrants are expressed in human induced pluripotent stem cells and cerebral organoids. The activity levels of individual L1 promoters vary widely and correlate with an active epigenetic state. Efficient on-target CRISPR interference (CRISPRi) silencing of L1s revealed nearly a hundred co-opted L1-derived chimeric transcripts, and L1 silencing resulted in changes in neural differentiation programs and reduced cerebral organoid size. Together, these data implicate L1s and L1-derived transcripts in hominoid-specific CNS developmental processes.