RNA velocity of single cells La Manno, Gioele; Soldatov, Ruslan; Zeisel, Amit ...
Nature (London),
08/2018, Letnik:
560, Številka:
7719
Journal Article
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RNA abundance is a powerful indicator of the state of individual cells. Single-cell RNA sequencing can reveal RNA abundance with high quantitative accuracy, sensitivity and throughput
. However, this ...approach captures only a static snapshot at a point in time, posing a challenge for the analysis of time-resolved phenomena such as embryogenesis or tissue regeneration. Here we show that RNA velocity-the time derivative of the gene expression state-can be directly estimated by distinguishing between unspliced and spliced mRNAs in common single-cell RNA sequencing protocols. RNA velocity is a high-dimensional vector that predicts the future state of individual cells on a timescale of hours. We validate its accuracy in the neural crest lineage, demonstrate its use on multiple published datasets and technical platforms, reveal the branching lineage tree of the developing mouse hippocampus, and examine the kinetics of transcription in human embryonic brain. We expect RNA velocity to greatly aid the analysis of developmental lineages and cellular dynamics, particularly in humans.
The dentate gyrus exhibits life-long neurogenesis of granule-cell neurons, supporting hippocampal dependent learning and memory. Both temporal lobe epilepsy patients and animal models frequently have ...hippocampal-dependent learning and memory difficulties and show evidence of reduced neurogenesis. Animal and human temporal lobe epilepsy studies have also shown strong innate immune system activation, which in animal models reduces hippocampal neurogenesis. We sought to determine if and how neuroinflammation signals reduced neurogenesis in the epileptic human hippocampus and its potential reversibility.
We isolated endogenous neural stem cells from surgically resected hippocampal tissue in 15 patients with unilateral hippocampal sclerosis. We examined resultant neurogenesis after growing them either as neurospheres in an ideal environment, in 3D cultures which preserved the inflammatory microenvironment and/or in 2D cultures which mimicked it.
3D human hippocampal cultures largely replicated the cellular composition and inflammatory environment of the epileptic hippocampus. The microenvironment of sclerotic human epileptic hippocampal tissue is strongly anti-neurogenic, with sustained release of the proinflammatory proteins HMGB1 and IL-1β. IL-1β and HMGB1 significantly reduce human hippocampal neurogenesis and blockade of their IL-1R and TLR 2/4 receptors by IL1Ra and Box-A respectively, significantly restores neurogenesis in 2D and 3D culture.
Our results demonstrate a HMGB1 and IL-1β-mediated environmental anti-neurogenic effect in human TLE, identifying both the IL-1R and TLR 2/4 receptors as potential drug targets for restoring human hippocampal neurogenesis in temporal lobe epilepsy.
The adult human brain comprises more than a thousand distinct neuronal and glial cell types, a diversity that emerges during early brain development. To reveal the precise sequence of events during ...early brain development, we used single-cell RNA sequencing and spatial transcriptomics and uncovered cell states and trajectories in human brains at 5 to 14 postconceptional weeks (pcw). We identified 12 major classes that are organized as ~600 distinct cell states, which map to precise spatial anatomical domains at 5 pcw. We described detailed differentiation trajectories of the human forebrain and midbrain and found a large number of region-specific glioblasts that mature into distinct pre-astrocytes and pre–oligodendrocyte precursor cells. Our findings reveal the establishment of cell types during the first trimester of human brain development.
INTRODUCTION
The adult human brain is divided into hundreds of spatial domains, each comprising tens or hundreds of distinct neuronal, glial, and other cell types. This complex arrangement of cells is initially established during the first trimester of development, yet the difficulty of accessing such early embryos has hindered detailed molecular analysis. Dissecting the spatial, temporal, and transcriptional changes that occur in the whole brain during the first trimester promises to reveal the fundamental blueprint of the human brain.
RATIONALE
To comprehensively map brain cell types and gene expression trajectories during the first trimester, we collected 26 brain specimens spanning 5 to 14 postconceptional weeks (pcw) that were dissected into 111 distinct biological samples. Each of these samples was subjected to single-cell RNA sequencing, resulting in a collection of 1,665,937 high-quality single-cell transcriptomes. These data were complemented by a spatial transcriptomic analysis at 5 pcw using highly multiplexed RNA fluorescence in situ hybridization (FISH) and spatial transcriptomics. We identified 616 clusters, which we annotated with metadata, including class and subclass, spatial location, embryonic age distribution, and specific gene expression markers.
RESULTS
The detailed resolution of the dataset allowed us to characterize general principles of brain development as well as delineate the differentiation trajectories of several brain regions. The developing excitatory neuron lineages in the neocortex revealed three different ongoing molecular programs: differentiation from radial glia to neurons, cell cycle, and maturation. We found a delicate balance between progenitor and differentiation factors in intermediate progenitor cells (IPCs), with the induction of neurogenic transcription factors visible after the G
1
cell cycle phase. Our findings support a conserved progressive transcriptional maturation in older specimens. Many genes were induced in late radial glia and glioblasts, making up a program that drives progenitors toward neurogenesis as well as gliogenesis. In the forebrain γ-aminobutyric acid–mediated (GABAergic) neuronal lineage, we found evidence of migration of
CRABP
-expressing cells from the medial ganglionic eminence into the thalamus, which are predicted to give rise to thalamic
PVALB
+
neurons in the adult. Examining ventral midbrain development, we found a diverse set of progenitors already arising at 8 pcw, defining broad
TH
class identity, although adult
TH
subtype identities must arise after 14 pcw. Focusing on developing glia, we found a large set of region-specific glioblasts, of which most showed evidence of maturation into astrocytes. This provides a plausible mechanism for the specification of adult region-specific astrocyte types. We further identified oligodendrocyte precursor cells (OPCs) specific to the forebrain, midbrain, and hindbrain that expressed large numbers of functionally conserved genes.
CONCLUSION
Although previous studies have explored specific regions of the brain during development, this is the first known comprehensive study of the whole human brain during the crucial first trimester. We found that although neurons were the most diverse, both pre-astrocytes and OPCs were regionally distinct, and their gene expression suggests region- and cell type–specific supportive functions. These findings highlight the importance of early patterning events and provide a rich resource for the interpretation of the many brain disorders that show region-specific patterns of occurrence or severity and for identifying therapeutic targets for human disorders that affect specific brain cell populations.
Atlas of the developing human brain.
We studied the human brain using single-cell RNA sequencing from 5 to 14 pcw and multiplexed in situ
RNA detection at 5 pcw. Our findings reveal how early patterning events establish the organization of the future brain and how maturation and differentiation trajectories are superimposed on this basic plan to generate the extraordinary complexity of the adult nervous system.
Abstract Neurofibrillary tangles composed of abnormally hyperphosphorylated tau protein are a hallmark of Alzheimer’s disease (AD) and related tauopathies. Tau hyperphosphorylation is thought to ...promote aggregation with subsequent tangle formation. Reducing tau phosphorylation by boosting the activity of the key phosphatase/s that mediate dephosphorylation of tau could be a viable clinical strategy in AD. One of the key phosphatases implicated in regulating tau protein phosphorylation is the serine–threonine phosphatase PP2A. We have determined that sodium selenate can act as a specific agonist for PP2A, significantly boosting phosphatase activity. Acute treatment of either neuroblastoma cells or normal aged mice with sodium selenate rapidly reduced tau protein phosphorylation. Sodium selenate-treated transgenic TAU441 mice had significantly lower levels of phospho- and total tau levels in the hippocampus and amygdala compared with controls and exhibited significantly improved spatial learning and memory on the Morris Water Maze task. Sodium selenate is a specific activator of PP2A with excellent oral bioavailability, and favourable central nervous system penetrating properties. Clinical studies in patients with AD are envisaged in the near future.
Traumatic brain injury (TBI) is caused by rapid deformation of the brain, resulting in a cascade of pathological events and ultimately neurodegeneration. Understanding how the biomechanics of brain ...deformation leads to tissue damage remains a considerable challenge. We have developed an in vitro model of TBI utilising organotypic hippocampal slice cultures on deformable silicone membranes, and an injury device, which generates tissue deformation through stretching the silicone substrate. Our injury device controls the biomechanical parameters of the stretch via feedback control, resulting in a reproducible and equi-biaxial deformation stimulus. Organotypic cultures remain well adhered to the membrane during deformation, so that tissue strain is 93 and 86% of the membrane strain in the
x- and
y-axis, respectively. Cell damage following injury is positively correlated with strain. In conclusion, we have developed a unique in vitro model to study the effects of mechanical stimuli within a complex cellular environment that mimics the in vivo environment. We believe this model could be a powerful tool to study the acute phases of TBI and the induced cell degeneration could provide a good platform for the development of potential therapeutic approaches and may be a useful in vitro alternative to animal models of TBI.
Deformation of brain tissue in response to mechanical loading of the head is the root-cause of traumatic brain injury (TBI). Even below ultimate failure limits, deformation activates ...pathophysiological cascades resulting in delayed cell death. Injury response of soft tissues, such as the chest and spinal cord, is dependent on the product of deformation and velocity, a parameter termed the viscous criterion. We set out to test if hippocampal cell death could be predicted by a similar combination of strain and strain rate and if the viscous criterion was valid for hippocampus. Quantitative prediction of the brain's biological response to mechanical stimuli is difficult to achieve in animal models of TBI, so we utilized an in vitro model of TBI based on hippocampal slice cultures. We quantified the temporal development of cell death after precisely controlled deformations for 30 combinations of strain (0.05–0.50) and strain rate (0.1–50
s
−1) relevant to TBI. Loading conditions for a subset of cultures were verified by analysis of high-speed video. Cell death was found to be significantly dependent on time-post injury, on strain magnitude, and to a lesser extent, on anatomical region by a repeated-measures, three-way ANOVA. The responses of the CA1 and CA3 regions of the hippocampus were not statistically different in contrast to some in vivo TBI studies. Surprisingly, cell death was not dependent on strain rate leading us to conclude that the viscous criterion is not a valid predictor for hippocampal tissue injury. Given the large data set and extensive combinations of biomechanical parameters, predictive mathematical functions relating independent variables (strain, region, and time post-injury) to the resultant cell death were defined. These functions can be used as tolerance criteria to equip finite element models of TBI with the added capability to predict biological consequences.
New neurones are produced in the adult hippocampus throughout life and are necessary for certain types of hippocampal learning. Little, however, is known about the control of hippocampal ...neurogenesis. We used primary hippocampal cultures from early post‐natal rats and neuropeptide Y Y1 receptor knockout mice as well as selective neuropeptide Y receptor antagonists and agonists to demonstrate that neuropeptide Y is proliferative for nestin‐positive, sphere‐forming hippocampal precursor cells and β‐tubulin‐positive neuroblasts and that the neuroproliferative effect of neuropeptide Y is mediated via its Y1 receptor. Immunohistochemistry confirmed Y1 receptor staining on both nestin‐positive cells and β‐tubulin‐positive cells in culture and short pulse 5‐bromo‐2‐deoxyuridine studies demonstrated that neuropeptide Y has a proliferative effect on both cell types. These studies suggest that the proliferation of hippocampal neuroblasts and precursor cells is increased by neuropeptide Y and, therefore, that hippocampal learning and memory may be modulated by neuropeptide Y‐releasing interneurones.
In the next few decades, brain medicine will present a particular socioeconomic challenge for aging citizens worldwide. Many disorders and ailments that affect the brain--including Alzheimer's ...disease, Parkinson's disease, dementia and stroke--are chronic conditions that persist for years or even decades. To cope with an increasing disease burden, drug discovery needs biologically relevant and predictive testing systems.
The effects of raised brain lactate levels on neuronal survival following hypoxia or ischemia is still a source of controversy among basic and clinical scientists. We have sought to address this ...controversy by studying the effects of glucose and lactate on neuronal survival in acute and cultured hippocampal slices. Following a 1‐h hypoxic episode, neuronal survival in cultured hippocampal slices was significantly higher if glucose was present in the medium compared with lactate. However, when the energy substrate during the hypoxic period was glucose and then switched to lactate during the normoxic recovery period, the level of cell damage in the CA1 region of organotypic cultures was significantly improved from 64.3 ± 2.1 to 74.6 ± 2.1% compared with cultures receiving glucose during and after hypoxia. Extracellular field potentials recorded from the CA1 region of acute slices were abolished during oxygen deprivation for 20 min, but recovered almost fully to baseline levels with either glucose (82.6 ± 10.0%) or lactate present in the reperfusion medium (108.1 ± 8.3%). These results suggest that lactate alone cannot support neuronal survival during oxygen deprivation, but a combination of glucose followed by lactate provides for better neuroprotection than either substrate alone.
Abstract Embryonic stem cells (ESCs) offer attractive prospective as potential source of neurons for cell replacement therapy in human neurodegenerative diseases. Besides, ESCs neural differentiation ...enables in vitro tissue engineering for fundamental research and drug discovery aimed at the nervous system. We have established stable and long-term three-dimensional (3D) culture conditions which can be used to model long latency and complex neurodegenerative diseases. Mouse ESCs-derived neural progenitor cells generated by MS5 stromal cells induction, result in strictly neural 3D cultures of about 120-μm thick, whose cells expressed mature neuronal, astrocytes and myelin markers. Neurons were from the glutamatergic and gabaergic lineages. This nervous tissue was spatially organized in specific layers resembling brain sub-ependymal (SE) nervous tissue, and was maintained in vitro for at least 3.5 months with great stability. Electron microscopy showed the presence of mature synapses and myelinated axons, suggesting functional maturation. Electrophysiological activity revealed biological signals involving action potential propagation along neuronal fibres and synaptic-like release of neurotransmitters. The rapid development and stabilization of this 3D cultures model result in an abundant and long-lasting production that is compatible with multiple and productive investigations for neurodegenerative diseases modeling, drug and toxicology screening, stress and aging research.
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