Biology subjects

Iyer, N.

Publications and source records attributed to Iyer, N..

18 recordsLinked to original sources

bioRxiv2026

Non-cell-autonomous removal of protein aggregates via trogocytosis

Protein aggregate formation can cause cell damage and contribute to tissue pathologies including neurodegeneration. Protein aggregates are normally eliminated by cell-intrinsic mechanisms of lysosomal (autophagic) degradation. However, degradative capacity varies between cell types, raising the possibility that high-capacity cell types, such as macrophages, may contribute to aggregate disposal from the low-capacity cells. Here, we show that cellular protein aggregates are removed by macrophages via trogocytosis -- a partial engulfment of the target cells. Lysosomal stress upon protein aggregate accumulation promotes cell surface expression of the transmembrane isoform of the macrophage growth factor CSF1 (tmCSF1), which is detected by macrophages through the CSF1 receptor to drive trogocytosis. Insufficient or impaired degradation of protein aggregates in lysosomes results in their transport along microtubules to the plasma membrane where they physically associate with tmCSF1. This enables macrophages to specifically trogocytose protein aggregates. These findings reveal a protein homeostasis system that eliminates protein aggregates in a non-cell-autonomous manner.

cell biology↗

bioRxiv2026

Non-invasive genomic sampling uncovers novel connectivities and origins of confiscated gorillas

BackgroundGorillas are a group of African great apes with two species and four subspecies that are currently critically endangered or endangered. Previous studies that analysed the genetics of wild gorillas from non-invasive samples, such as faeces or hair, analysed short mitochondrial or nuclear markers, which may not reflect the wider nuclear genome. Recent technical advances in target capture hybridisation, enrich the endogenous DNA content of non-invasive samples, allowing contiguous genomic regions to be sequenced. ResultsHere, we generated georeferenced genetic data from faecal and hair samples of 280 wild gorillas, sampled from three of the four gorilla subspecies, across large parts of their present-day distributions. With this expanded representation of gorilla genetic diversity in the wild, we detected three population clusters in western lowland gorillas, with the Sangha River and its affluents acting as significant barriers to gene flow. We reconstructed patterns of past population connectivity between western lowland gorillas in the north-eastern distribution range and Cross River gorillas, which may have been facilitated by a migration corridor also used by the Central and Nigeria-Cameroon chimpanzee subspecies. Finally, we predicted the geographic origins of wild-born gorillas, achieving a mean prediction error of 65 km, with a population-level resolution for mountain gorillas and some populations of western lowland gorillas. ConclusionOur work characterises fine-scale population structure in western lowland gorillas, which will be informative for future conservation strategies. This proof of concept in predicting geographic locations of wild gorillas, will be useful for future applications to geolocalise trafficked or rescued gorillas.

evolutionary biology↗

bioRxiv2025

Low-Cost, Rapid Fabrication of Customizable Polyethylene Glycol-Based Cell Culture Devices

Biological research groups may face a high barrier to entry when constructing custom 3D cell culture devices to investigate multi-tissue interactions in vitro. Standard fabrication methods such as lithography, etching, or molding are expensive and require specialized equipment and expertise. To address this, we developed an accessible approach for producing polyethylene glycol (PEG)-based cell culture devices using stereolithography (SLA) 3D printing with a polydimethylsiloxane (PDMS) intermediate mold. Both the intermediate molding steps and the sterilized final device show low cytotoxicity, the final device swells to predictable dimensions and retains its shape for at least 10 days. We used this approach to develop a human pluripotent stem cell (hPSC)-derived neural spheroid outgrowth model that supports directed neurite extension over 14 days. Together, this method provides a highly customizable, affordable platform for rapid fabrication of PEG-based microphysiological systems (MPS) for diverse tissue engineering applications. ImpactAs biomedical labs work to complement animal models with tissue-engineered MPSs, there is a growing need for low-cost, rapid, and iterative fabrication workflows. We developed a pipeline combining 3D printing, a PDMS intermediate mold, and PEG casting, avoiding the need for specialized photolithography. The resulting devices support stable, nutrient-permissive cell culture while allowing control over device dimensions and customizable channel or compartment configurations. We demonstrate its utility with reprogrammed hPSC-derived neurons, which remain challenging to support sustained neurite outgrowth in engineered models. This workflow expands access to cell culture device fabrication for MPSs across a broader range of biological laboratories.

bioengineering↗

bioRxiv2025

A connectomic resource for neural cataloguing and circuit dissection of the larval zebrafish brain

We present a correlated light and electron microscopy (CLEM) dataset from a 7-day-old larval zebrafish, integrating confocal imaging of genetically labeled excitatory (vglut2a) and inhibitory (gad1b) neurons with nanometer-resolution serial section EM. The dataset spans the brain and anterior spinal cord, capturing >180,000 segmented soma, >40,000 molecularly annotated neurons, and 30 million synapses, most of which were classified as excitatory, inhibitory, or modulatory. To characterize the directional flow of activity across the brain, we leverage the synaptic and cell body annotations to compute region-wise input and output drive indices at single cell resolution. We illustrate the datasets utility by dissecting and validating circuits in three distinct systems: water flow direction encoding in the lateral line, recurrent excitation and contralateral inhibition in a hindbrain motion integrator, and functionally relevant targeted long-range projections from a tegmental excitatory nucleus, demonstrating that this resource enables rigorous hypothesis testing as well as exploratory-driven circuit analysis. The dataset is integrated into an open-access platform optimized to facilitate community reconstruction and discovery efforts throughout the larval zebrafish brain.

neuroscience↗

bioRxiv2024

Synaptic Specializations at Dopamine Release Sites Orchestrate Efficient and Precise Neuromodulatory Signaling

Neuromodulators are generally understood to act through expansive projections that broadcast signals relatively indiscriminately, in stark contrast to the precise, synapse-bound organization of fast neurotransmitters like glutamate and GABA. This dichotomy has left the local architecture of neuromodulatory release and receptor engagement largely obscure. Here, using dopamine as a model system, we reveal that neuromodulatory signaling can be guided by precise molecular specificity. We show that dopamine axons exhibit a fundamental tropism for neurons expressing D1 or D2 receptors in the striatum and amygdala, a targeting principle that is conserved in dissociated co-cultures. Importantly, single-bouton-resolved optical imaging of release shows that dopamine is not broadcast broadly but is highly localized to varicosities that form direct, synapse-like appositions with receptor-expressing somata and dendrites. In contrast, varicosities lacking such appositions, or contacting receptor-negative neurons, are largely quiescent, indicating that local interactions with receptor-expressing targets help specify dopamine release competence. To directly visualize receptor organization relative to release sites at endogenous expression levels, we generated ALFA-tag knock-in mice (ALFADoR mice) in which the N-termini of D1 and D2 dopamine receptors are epitope-tagged. Combining these mice with immunolabeling and expansion microscopy in dissociated culture and intact tissue, we show that both D1 and D2 receptors are organized into discrete puncta rather than diffuse membrane distributions. Across major terminal fields of midbrain dopamine neurons, including striatum, amygdala, and prefrontal cortex, these receptor nanoclusters are preferentially positioned near dopamine varicosities at distances far smaller than expected from spatial shuffling controls. Moreover, receptor clusters apposed to dopamine varicosities were observed to be larger than non-apposed clusters, consistent with specialized receptor microdomains that may enhance signaling efficacy. Together, our data demonstrate that dopaminergic signaling is spatially organized by receptor-specific targeting and subcellular microdomain specialization. This structured framework challenges the canonical dichotomy between precise fast transmission and diffuse neuromodulation. It reveals that principles of synaptic organization extend to and define a previously hidden layer of specificity in neuromodulatory systems, opening new frontiers in understanding cell-type specific neuromodulation, axon guidance, and subcellular GPCR organization.

neuroscience↗

bioRxiv2024

iTome Volumetric Serial Sectioning Apparatus for TEM

An automated ultra-microtome capable of sectioning thousands of ultrathin sections onto standard TEM slot grids was developed and used to section: a complete Drosophila melanogaster first-instar larva, three sections per grid, into 4,866 34-nm-thick sections with a cutting and pickup success rate of 99.74%; 30 microns of mouse cortex measuring roughly 400 um x 2000 um at 40 nm per slice; and a full adult Drosophila brain and ventral nerve column into 9,300 sections with a pickup success rate of 99.95%. The apparatus uses optical interferometers to monitor a reference distance between the cutting knife and multiple sample blocks. Cut sections are picked up from the knife-boat water surface while they are still anchored to the cutting knife. Blocks without embedded tissue are used to displace tissue-containing sections away from the knife edge so that the tissue regions end up in the grid slot instead of on the grid rim.

neuroscience↗

bioRxiv2024

Metabolic immaturity of newborns and breast milk bile acids are the central determinants of heightened neonatal vulnerability to norovirus diarrhea

Noroviruses are the leading global cause of acute gastroenteritis, responsible for 685 million annual cases. While all age groups are susceptible to noroviruses, children are vulnerable to more severe infections than adults, underscored by 200 million pediatric cases and up to 200,000 deaths in children annually. Understanding the basis for the increased vulnerability of young hosts is critical to developing effective treatments. The pathogenic outcome of any enteric virus infection is governed by a complex interplay between the virus, intestinal microbiota, and host immune factors. A central mediator in these complex relationships are host- and microbiota-derived metabolites. Noroviruses bind a specific class of metabolites, bile acids, which are produced by the host and then modified by commensal bacterial enzymes. Paradoxically, bile acids can have both proviral and antiviral roles during norovirus infections. Considering these opposing effects, the microbiota-regulated balance of the bile acid pool may be a key determinant of the pathogenic outcome of a norovirus infection. The bile acid pool in newborns is unique due to immaturity of host metabolic pathways and developing gut microbiota, which could underlie the vulnerability of these hosts to severe norovirus infections. Supporting this concept, we demonstrate herein that microbiota and their bile acid metabolites protect from severe norovirus diarrhea whereas host-derived bile acids promote disease. Remarkably, we also report that maternal bile acid metabolism determines neonatal susceptibility to norovirus diarrhea during breastfeeding by delivering proviral bile acids to the newborn. Finally, directed targeting of maternal and neonatal bile acid metabolism can protect the neonatal host from norovirus disease. Altogether, these data support the conclusion that metabolic immaturity in newborns and ingestion of proviral maternal metabolites in breast milk are the central determinants of heightened neonatal vulnerability to norovirus disease.

microbiology↗

bioRxiv2024

RosetteArray Platform for Quantitative High-Throughput Screening of Human Neurodevelopmental Risk

Neural organoids have revolutionized how human neurodevelopmental disorders (NDDs) are studied. Yet, their utility for screening chemical hazards and prospective therapeutics for NDDs is limited by a lack of morphological reproducibility and cost-effective scalability. Here, we describe the RosetteArray platform, which can be used as an off-the-shelf, 96-well plate assay that standardizes incipient forebrain and spinal cord organoid morphogenesis as adherent, micropatterned, 3-D, singularly polarized neural rosette tissues ([~]200 and [~]800 per well, respectively). Seeded directly from cryopreserved human pluripotent stem cells, RosetteArrays are cultured over 6-8 days and fixed, immunostained and imaged in situ to enable artificial intelligence-based quantitative analysis. By screening the inception of [~]75,000 neural organoids throughout this manuscript, we provide proof-of-concept demonstrations of the platforms utility for detecting developmental neurotoxicity hazard and screening genetic and environmental factors known to cause clinical Neural Tube Defect risk. Given the documented perturbation of rosette morphogenesis in neural organoid models of several NDDs, the RosetteArray platform could enable quantitative high-throughput screening (qHTS) of human neurodevelopmental risk across regulatory and precision medicine applications.

neuroscience↗

bioRxiv2024

A split-GAL4 driver line resource for Drosophila CNS cell types

Techniques that enable precise manipulations of subsets of neurons in the fly central nervous system have greatly facilitated our understanding of the neural basis of behavior. Split-GAL4 driver lines allow specific targeting of cell types in Drosophila melanogaster and other species. We describe here a collection of 3060 lines targeting a range of cell types in the adult Drosophila central nervous system and 1373 lines characterized in third-instar larvae. These tools enable functional, transcriptomic, and proteomic studies based on precise anatomical targeting. NeuronBridge and other search tools relate light microscopy images of these split-GAL4 lines to connectomes reconstructed from electron microscopy images. The collections are the result of screening over 77,000 split hemidriver combinations. Previously published and new lines are included, all validated for driver expression and curated for optimal cell type specificity across diverse cell types. In addition to images and fly stocks for these well-characterized lines, we make available 300,000 new 3D images of other split-GAL4 lines.

neuroscience↗

bioRxiv2023

cMYC-mediated immune repression is reversed by inhibition of H3K9/H3K27 methylation maintenance

Aberrant cMYC activity is a key driver of cancer, involved in several hallmark processes. Alongside the canonical hallmark of proliferation, cMYC represses immune signalling in a cell-intrinsic manner. The histone methyltransferases EZH2 and G9a interact with cMYC to modulate gene expression, including repression of immune genes via H3K27 and H3K9 histone methylation. Analyses of 565 cell lines derived from solid cancers demonstrated that greater cMYC-G9a/EZH2-mediated repression correlates with lower immune gene scores in a cell-intrinsic manner (innate, Type I and Type II IFN response), an effect most evident in MYC-amplified cell lines. In ovarian high-grade serous carcinoma (HGSC) cell lines and an in vivo murine model of HGSC, HKMTi-1-005, an inhibitor of H3K27/H3K9 methylation maintenance, relieved cMYC-G9a/EZH2 repression whilst inducing an immune response. A 7-gene immune signature (7ISG), related to viral mimicry signalling, is at the core of the HGSC immune response to HKMTi-1-005. In MYC-amplified HGSC patients, a low 7ISG score was associated with poor survival. Additionally, MYC-amplified cell lines were significantly more sensitive to HKMTi-1-005, whilst a low 7ISG score was associated with greater HKMTi-1-005 sensitivity, effects that were independent of canonical cMYC transcriptional activation. Examining the effects of HKMTi-1-005 treatment in a MYC-deregulated lung adenocarcinoma (LuAd) revealed induction of an immune response in vitro and prolonged survival in vivo. This suggests that inhibition of H3K27/H3K9 methylation maintenance will have efficacy in cMYC-deregulated tumours with low 7ISG scores, via disruption of cMYC-mediated repression of cell autonomous immune signalling and induction of an anti-tumour immune response. Statement of significanceOver 70% of cancers are cMYC-deregulated. We show that inhibition of H3K27/H3K9 methylation maintenance relieves cMYC-dependent immune repression and prolongs survival of animal tumour models, suggesting a novel approach to treating cMYC-deregulated tumours.

cancer biology↗

bioRxiv2023

ARF6 as a novel activator of HIF-2alpha in pulmonary arterial hypertension

ADP-ribosylation factor 6 (ARF6), a GTPase associated with cancer metastasis, is activated in the lung endothelium in pulmonary arterial hypertension (PAH). To identify ARF6-regulated pathways relevant to PAH, we performed a state-of-the-art proteomic analysis of human pulmonary artery endothelial cells (HPAECs) overexpressing the wildtype, constitutively active, fast-cycling and dominant negative mutants of ARF6. The analysis revealed a novel link of ARF6 with hypoxia-inducible factor (HIF), in addition to endocytotic vesicle trafficking, cell proliferation, angiogenesis, oxidative stress and lipid metabolism. Active ARF6 markedly increased expression and activity of HIF-2, critical in PAH, with HIF-1 relatively unaffected. Hypoxic ARF6 activation was a prerequisite for HIF-2 activation and HIF-dependent gene expression in HPAECs, PAH blood-derived late outgrowth endothelial colony forming cells (ECFCs) and hypoxic mouse lungs in vivo. A novel ARF6 inhibitor, chlortetracycline (CTC), reduced hypoxia-induced HIF-2 activation, proliferation and angiogenesis in HPAECs and reduced HIF-2 expression in lung and heart tissues of hypoxic mice. PAH ECFCs showed elevated expression and activity of ARF6 and HIF2, which was attenuated by CTC. Oral CTC attenuated development of PH in chronically hypoxic mice. In conclusion, we are first to demonstrate a key role of ARF6 in the regulation of HIF-2 activation in vitro and in vivo and show that HIF-2, a master-regulator of vascular remodelling in PAH, can be targeted by a clinically approved antibiotic chlortetracycline.

cell biology↗

bioRxiv2023

Heme oxygenase-1 expressing omental macrophages as a therapeutic target in ovarian high grade serous carcinoma

Ovarian high grade serous carcinoma (HGSC) remains a disease of poor prognosis that is unresponsive to current immune checkpoint inhibitors. Although PI3K pathway alterations are common in HGSC, attempts to target this pathway have been unsuccessful. We hypothesised aberrant PI3K pathway activation may alter the HGSC immune microenvironment and present a novel targeting strategy. We used both murine models and HGSC patient samples to study the impact of loss of Pten, a negative regulator of PI3K pathway signalling. We identified populations of resident macrophages specifically in Pten null omental tumours. These macrophages derive from peritoneal fluid macrophages and have a unique gene expression programme, marked by high levels of HMOX1 expression, the gene for the enzyme heme oxygenase-1. Targeting resident peritoneal macrophages prevents appearance of HMOX1hi macrophages and in doing so reduces tumour growth. Furthermore, direct inhibition of HMOX1 extends survival in vivo. HMOX1hi macrophages with corresponding gene expression programmes are also identified in human HGSC tumours and their presence correlates with activated tumoural PI3K pathway/mTOR signalling and poor overall survival in HGSC patients. In contrast, tumours with low number of HMOX1hi macrophages are marked by increased adaptive immune response gene expression. Our data suggest that HMOX1hi macrophages represent a potential therapeutic target and biomarker for poor prognosis HGSC.

cancer biology↗

bioRxiv2022

Isolated Grauer's gorilla populations differ in diet and gut microbiome

The animal gut microbiome has been implicated in a number of key biological processes, ranging from digestion to behavior, and has also been suggested to facilitate local adaptation. However, studies in wild animals rarely compare multiple populations that differ ecologically, which is the level at which local adaptation may occur. Further, few studies simultaneously characterize diet and the gut microbiome from the same sample, despite the likely presence of co-dependencies. Here, we investigate the interplay between diet and gut microbiome in three geographically isolated populations of the critically endangered Grauers gorilla, which we show to be genetically differentiated. We find population- and social group-specific dietary and gut microbial profiles and co-variation between diet and gut microbiome, despite the presence of core microbial taxa. There was no detectable effect of age, sex, or genetic relatedness on the microbiome. Diet differed considerably across populations, with the high-altitude population consuming a lower diversity of plants compared to low-altitude populations, consistent with food plant availability constraining diet. The observed pattern of covariation between diet and gut microbiome is likely a result of long-term social and ecological factors. Our study suggests that the gut microbiome is sufficiently plastic to support flexible food selection and hence contribute to local adaptation.

ecology↗

bioRxiv2020

Isotropic 3D electron microscopy reference library of whole cells and tissues

Understanding cellular architecture is essential for understanding biology. Electron microscopy (EM) uniquely visualizes cellular structures with nanometer resolution. However, traditional methods, such as thin-section EM or EM tomography, have limitations inasmuch as they only visualize a single slice or a relatively small volume of the cell, respectively. Focused Ion Beam-Scanning Electron Microscopy (FIB-SEM) demonstrated the ability to image cellular samples at 4-nm isotropic voxels with rather limited imageable volume. Here, we present 3D EM images of whole cells and tissues with two orders of magnitude increases in imageable volume at 4-nm voxels. Such data with a combined fine resolution scale and large sample size do not currently exist, and are enabled by the advances in higher precision and stability of FIB milling, together with enhanced signal detection and faster SEM scanning. More importantly, we have generated a volume EM atlas encompassing ten diverse datasets of whole cells and tissues, from cancer cells to immune cells, and from mouse pancreatic islets to Drosophila neural tissues. These open-access data (via OpenOrganelle) represent a foundation to nucleate a new field of high-resolution whole-cell volume EM and subsequent analyses, and invite biologists to explore this new paradigm and pose fundamentally new questions.

cell biology↗

bioRxiv2019

Correlative three-dimensional super-resolution and block face electron microscopy of whole vitreously frozen cells

Living cells function through the spatial compartmentalization of thousands of distinct proteins serving a multitude of diverse biochemical needs. Correlative super-resolution (SR) fluorescence and electron microscopy (EM) has emerged as a pathway to directly view nanoscale protein relationships to the underlying global ultrastructure, but has traditionally suffered from tradeoffs of structure preservation, fluorescence retention, resolution, and field of view. We developed a platform for three-dimensional correlative cryogenic SR and focused ion beam milled block-face EM across entire vitreously frozen cells that addresses these issues by preserving native ultrastructure and enabling independent SR and EM workflow optimization. Application to a variety of biological systems revealed a number of unexpected protein-ultrastructure relationships and underscored the value of a comprehensive multimodal view of ultrastructural variability across whole cells.

cell biology↗

bioRxiv2019

Epithelium intrinsic vitamin A signaling co-ordinates pathogen clearance in the gut via IL-18

Intestinal epithelial cells (IECs) are at the forefront of host-pathogen interactions, coordinating a cascade of immune responses to confer protection against pathogens. Here we show that IEC-intrinsic vitamin A signaling restricts pathogen invasion early in the infection and subsequently activates immune cells to promote pathogen clearance. Mice blocked for retinoic acid receptor (RAR) signaling selectively in IECs (stop{Delta}IEC) showed significantly higher Salmonella burden in colonic tissues early in the infection associated with higher luminal and systemic loads of the pathogen at later stages. Higher susceptibility of stop{Delta}IEC mice correlated with attenuated mucosal interferon gamma (IFN{gamma}) production by underlying immune cells. We found that, at homeostasis, the intestinal epithelium of stop{Delta}IEC mice produced significantly lower amounts of interleukin 18 (IL-18), a potent inducer of IFN{gamma}. Regulation of IL-18 by vitamin A was also observed in a dietary model of vitamin A supplementation. IL-18 reconstitution in stop{Delta}IEC mice restored resistance to Salmonella by promoting epithelial cell shedding to eliminate infected cells and limit pathogen invasion early in infection. Further, IL-18 augmented IFN{gamma} production by underlying immune cells to restrict pathogen burden and systemic spread. Our work uncovers a critical role for vitamin A in coordinating a biphasic immune response to Salmonella infection by regulating IL-18 production by IECs.\n\nAuthor SummaryEpithelial cells line the intestinal lumen, forming a barrier between the body and dietary and microbial contents in the lumen. Apart from absorbing nutrients from diet, these epithelial cells help mediate a stable, symbiotic relationship between microbes in the gut and the immune cells. During infection, they help co-ordinate the immune response to counter the infection. How dietary micronutrients, such as vitamin A, inform epithelial cell function during infection is poorly understood. Using a model where epithelial cells in the gut cannot respond to vitamin A signals, we find that epithelial vitamin A signaling promotes resistance to Salmonella infection. We show that, vitamin A increases the production of a key cytokine, interleukin 18, by epithelial cells. IL-18 promotes shedding of infected epithelial cells to reduce the pathogen invasion while also inducing the production of interferon gamma by immune cells to mediate pathogen clearance. Thus, epithelial cells dynamically respond to dietary vitamin A to regulate interleukin 18 production and potentiate resistance to infection.

immunology↗

bioRxiv2018

In Vitro Assay for the Detection of Network Connectivity in Embryonic Stem Cell-Derived Cultures

Stem cell transplantation holds great promise as a repair strategy following spinal cord injury. Embryonic stem cell (ESC) transplantation therapies have elicited encouraging though limited improvement in motor and sensory function with the use of heterogeneous mixtures of spinal cord neural progenitors and ESCs. Recently, transgenic lines of ESCs have been developed to allow for purification of specific candidate populations prior to transplantation, but the functional network connectivity of these populations and its relationship to recovery is difficult to examine with current technological limitations. In this study, we combine an ESC differentiation protocol, multi-electrode arrays (MEAs), and previously developed neuronal connectivity detection algorithms to develop an in vitro high-throughput assay of network connectivity in ESC-derived populations of neurons. Neuronal aggregation results in more consistent detection of individual neuronal activity than dissociated cultures. Both aggregated and dissociated culture types exhibited synchronized bursting behaviors at days 17 and 18 on MEAs, and thousands of statistically significance functional connections were detected in both culture types. Aggregate cultures, however, demonstrate a tight linear relationship between the inter-neuron distance of neuronal pairs and the time delay of the neuronal pair functional connections, whereas dissociated cultures do not. These results suggest that ESC-derived aggregated cultures may reflect some of the spatiotemporal connectivity characteristics of in vivo tissue and prove to be useful models of investigating potentially therapeutic populations of ESC-derived neurons in vitro.\n\nNOVELTY AND SIGNIFICANCEPrevious investigations of stem cell-derived network connectivity on multi-electrode arrays (MEAs) have been limited to characterizations of bursting activity or broad averages of overall temporal network correlations, both of which overlook neuronal level interactions. The use of spike-sorting and short-time cross-correlation histograms along with statistical techniques developed specifically for MEAs allows for the characterization of functional connections between individual stem cell-derived neurons. This high-throughput connectivity assay will open doors for future examinations of the differences in functional network formation between various candidate stem cell-derived populations for spinal cord injury transplantation therapies--a critical inquiry into their therapeutic viability.

bioengineering↗

bioRxiv2017

Engineering Human CNS Morphogenesis: Controlled Induction of Singular Neural Rosette Emergence

Human pluripotent stem cell (hPSC)-derived neural organoids have revolutionized in vitro modelling of human neurological disorders. Cell-intrinsic morphogenesis processes displayed within these tissues could serve as the basis for ex vivo manufacture of brain and spinal cord tissues with biomimetic anatomy and physiology. However, we must first understand how to control their emergent properties starting at the genesis of neural organoid formation, i.e. emergence of polarized neuroepithelium. In vivo, all CNS tissues develop from a singular neuroepithelial tube. Yet, current protocols yield organoids with multiple neuroepithelial rings, a.k.a. neural rosettes, each acting as independent centers of morphogenesis and thereby impeding coordinate tissue development. We discovered that the morphology of hPSC-derived neural tissues is a critical biophysical parameter for inducing singular neural rosette emergence. Tissue morphology screens conducted using micropatterned array substrates and an automated image analysis determined that circular morphologies of 200-250 and 150m diameter are optimal for inducing singular neural rosette emergence within 80-85% forebrain and 73.5% spinal tissues, respectively. The discrepancy in optimal circular morphology for Pax6+/N-cadherin+ neuroepithelial forebrain versus spinal tissues was due to previously unknown differences in ROCK-mediated cell contractility. The singular neuroepithelium induced within geometrically confined tissues persisted as the tissues morphed from a 2-D monolayer to multilayered 3-D hemispherical aggregate. Upon confinement release using clickable micropatterned substrates, the tissue displayed radial outgrowth with maintenance of a singular neuroepithelium and peripheral neuronal differentiation. Thus, we have quantitatively defined a pertinent biophysical parameter for effectively inducing a singular neuroepithelium emergence within morphing hPSC-derived neural tissues.\n\nSignificance StatementHuman pluripotent stem cell (hPSC)-derived neural organoids display emergent properties that, if harnessed, could serve as the basis for ex vivo manufacture of brain and spinal cord tissues with biomimetic macroscale anatomy and physiology. Their chaotic terminal structure arises from uncontrolled morphogenesis at their genesis, resulting in spontaneous induction of multiple neuroepithelial morphogenesis centers,a.k.a. neural rosettes. Here, we determined that neural tissue morphology is a pertinent biophysical parameter for controlling subsequent morphogenesis, and identified discrete circular tissue morphologies as optimal and effective at inducing singular neural rosette emergence within forebrain and spinal neural tissues. Thus, we developed an approach to reproducibly control the initial stage of hPSC-derived neural tissue morphogenesis enabling their manufacture with a biomimetic nascent CNS anatomy.

bioengineering↗