Simultaneous CRISPR screening and spatial transcriptomics reveals intracellular, intercellular, and functional transcriptional circuits.

Pooled optical screens have enabled the study of cellular interactions, morphology, or dynamics at massive scale, but have not yet leveraged the power of highly-plexed single-cell resolved transcriptomic readouts to inform molecular pathways. Here, we present Perturb-FISH, which bridges these approaches by combining imaging spatial transcriptomics with parallel optical detection of in situ amplified guide RNAs. We show that Perturb-FISH recovers intracellular effects that are consistent with Perturb-seq results in a screen of lipopolysaccharide response in cultured monocytes, and uncover new intercellular and density-dependent regulation of the innate immune response. We further pair Perturb-FISH with a functional readout in a screen of autism spectrum disorder risk genes, showing common calcium activity phenotypes in induced pluripotent stem cell derived astrocytes and their associated genetic interactions and dysregulated molecular pathways. Perturb-FISH is thus a generally applicable method for studying the genetic and molecular associations of spatial and functional biology at single-cell resolution.

Scalable genetic screening for regulatory circuits using compressed Perturb-seq.

Pooled CRISPR screens with single-cell RNA sequencing readout (Perturb-seq) have emerged as a key technique in functional genomics, but they are limited in scale by cost and combinatorial complexity. In this study, we modified the design of Perturb-seq by incorporating algorithms applied to random, low-dimensional observations. Compressed Perturb-seq measures multiple random perturbations per cell or multiple cells per droplet and computationally decompresses these measurements by leveraging the sparse structure of regulatory circuits. Applied to 598 genes in the immune response to bacterial lipopolysaccharide, compressed Perturb-seq achieves the same accuracy as conventional Perturb-seq with an order of magnitude cost reduction and greater power to learn genetic interactions. We identified known and novel regulators of immune responses and uncovered evolutionarily constrained genes with downstream targets enriched for immune disease heritability, including many missed by existing genome-wide association studies. Our framework enables new scales of interrogation for a foundational method in functional genomics.

Robust induction of functional astrocytes using NGN2 expression in human pluripotent stem cells.

Emerging evidence of species divergent features of astrocytes coupled with the relative inaccessibility of human brain tissue underscore the utility of human pluripotent stem cell (hPSC) technologies for the generation and study of human astrocytes. However, existing approaches for hPSC-astrocyte generation are typically lengthy or require intermediate purification steps. Here, we establish a rapid and highly scalable method for generating functional human induced astrocytes (hiAs). These hiAs express canonical astrocyte markers, respond to pro-inflammatory stimuli, exhibit ATP-induced calcium transients and support neuronal network development. Moreover, single-cell transcriptomic analyses reveal the generation of highly reproducible cell populations across individual donors, mostly resembling human fetal astrocytes. Finally, hiAs generated from a trisomy 21 disease model identify expected alterations in cell-cell adhesion and synaptic signaling, supporting their utility for disease modeling applications. Thus, hiAs provide a valuable and practical resource for the study of basic human astrocyte function and dysfunction in disease.

Migration speed of captured breast cancer subpopulations correlates with metastatic fitness.

The genetic alterations contributing to migration proficiency, a phenotypic hallmark of metastatic cells required for colonizing distant organs, remain poorly defined. Here, we used single-cell magneto-optical capture (scMOCa) to isolate fast cells from heterogeneous human breast cancer cell populations, based on their migratory ability alone. We show that captured fast cell subpopulations retain higher migration speed and focal adhesion dynamics over many generations as a result of a motility-related transcriptomic profile. Upregulated genes in isolated fast cells encoded integrin subunits, proto-cadherins and numerous other genes associated with cell migration. Dysregulation of several of these genes correlates with poor survival outcomes in people with breast cancer, and primary tumors established from fast cells generated a higher number of circulating tumor cells and soft tissue metastases in pre-clinical mouse models. Subpopulations of cells selected for a highly migratory phenotype demonstrated an increased fitness for metastasis.

Optimization-based decoding of Imaging Spatial Transcriptomics data.

MOTIVATION: Imaging Spatial Transcriptomics techniques characterize gene expression in cells in their native context by imaging barcoded probes for mRNA with single molecule resolution. However, the need to acquire many rounds of high-magnification imaging data limits the throughput and impact of existing methods. RESULTS: We describe the Joint Sparse method for Imaging Transcriptomics, an algorithm for decoding lower magnification Imaging Spatial Transcriptomics data than that used in standard experimental workflows. Joint Sparse method for Imaging Transcriptomics incorporates codebook knowledge and sparsity assumptions into an optimization problem, which is less reliant on well separated optical signals than current pipelines. Using experimental data obtained by performing Multiplexed Error-Robust Fluorescence in situ Hybridization on tissue from mouse brain, we demonstrate that Joint Sparse method for Imaging Transcriptomics enables improved throughput and recovery performance over standard decoding methods. AVAILABILITY AND IMPLEMENTATION: Software implementation of JSIT, together with example files, is available at https://github.com/jpbryan13/JSIT.

Compressed Perturb-seq: highly efficient screens for regulatory circuits using random composite perturbations.

Pooled CRISPR screens with single-cell RNA-seq readout (Perturb-seq) have emerged as a key technique in functional genomics, but are limited in scale by cost and combinatorial complexity. Here, we reimagine Perturb-seq's design through the lens of algorithms applied to random, low-dimensional observations. We present compressed Perturb-seq, which measures multiple random perturbations per cell or multiple cells per droplet and computationally decompresses these measurements by leveraging the sparse structure of regulatory circuits. Applied to 598 genes in the immune response to bacterial lipopolysaccharide, compressed Perturb-seq achieves the same accuracy as conventional Perturb-seq at 4 to 20-fold reduced cost, with greater power to learn genetic interactions. We identify known and novel regulators of immune responses and uncover evolutionarily constrained genes with downstream targets enriched for immune disease heritability, including many missed by existing GWAS or trans-eQTL studies. Our framework enables new scales of interrogation for a foundational method in functional genomics.

Pyramidal neuron subtype diversity governs microglia states in the neocortex.

Microglia are specialized macrophages in the brain parenchyma that exist in multiple transcriptional states and reside within a wide range of neuronal environments1-4. However, how and where these states are generated remains poorly understood. Here, using the mouse somatosensory cortex, we demonstrate that microglia density and molecular state acquisition are determined by the local composition of pyramidal neuron classes. Using single-cell and spatial transcriptomic profiling, we unveil the molecular signatures and spatial distributions of diverse microglia populations and show that certain states are enriched in specific cortical layers, whereas others are broadly distributed throughout the cortex. Notably, conversion of deep-layer pyramidal neurons to an alternate class identity reconfigures the distribution of local, layer-enriched homeostatic microglia to match the new neuronal niche. Leveraging the transcriptional diversity of pyramidal neurons in the neocortex, we construct a ligand-receptor atlas describing interactions between individual pyramidal neuron subtypes and microglia states, revealing rules of neuron-microglia communication. Our findings uncover a fundamental role for neuronal diversity in instructing the acquisition of microglia states as a potential mechanism for fine-tuning neuroimmune interactions within the cortical local circuitry.

GABA-receptive microglia selectively sculpt developing inhibitory circuits.

Microglia, the resident immune cells of the brain, have emerged as crucial regulators of synaptic refinement and brain wiring. However, whether the remodeling of distinct synapse types during development is mediated by specialized microglia is unknown. Here, we show that GABA-receptive microglia selectively interact with inhibitory cortical synapses during a critical window of mouse postnatal development. GABA initiates a transcriptional synapse remodeling program within these specialized microglia, which in turn sculpt inhibitory connectivity without impacting excitatory synapses. Ablation of GABAB receptors within microglia impairs this process and leads to behavioral abnormalities. These findings demonstrate that brain wiring relies on the selective communication between matched neuronal and glial cell types.

A potent nuclear export mechanism imposes USP16 cytoplasmic localization during interphase.

USP16 (also known as UBP-M) has emerged as a histone H2AK119 deubiquitylase (DUB) implicated in the regulation of chromatin-associated processes and cell cycle progression. Despite this, available evidence suggests that this DUB is also present in the cytoplasm. How the nucleo-cytoplasmic transport of USP16, and hence its function, is regulated has remained elusive. Here, we show that USP16 is predominantly cytoplasmic in all cell cycle phases. We identified the nuclear export signal (NES) responsible for maintaining USP16 in the cytoplasm. We found that USP16 is only transiently retained in the nucleus following mitosis and then rapidly exported from this compartment. We also defined a non-canonical nuclear localization signal (NLS) sequence that plays a minimal role in directing USP16 into the nucleus. We further established that this DUB does not accumulate in the nucleus following DNA damage. Instead, only enforced nuclear localization of USP16 abolishes DNA double-strand break (DSB) repair, possibly due to unrestrained DUB activity. Thus, in contrast to the prevailing view, our data indicate that USP16 is actively excluded from the nucleus and that this DUB might indirectly regulate DSB repair.This article has an associated First Person interview with the first author of the paper.

Opto-magnetic capture of individual cells based on visual phenotypes.

The ability to isolate rare live cells within a heterogeneous population based solely on visual criteria remains technically challenging, due largely to limitations imposed by existing sorting technologies. Here, we present a new method that permits labeling cells of interest by attaching streptavidin-coated magnetic beads to their membranes using the lasers of a confocal microscope. A simple magnet allows highly specific isolation of the labeled cells, which then remain viable and proliferate normally. As proof of principle, we tagged, isolated, and expanded individual cells based on three biologically relevant visual characteristics: i) presence of multiple nuclei, ii) accumulation of lipid vesicles, and iii) ability to resolve ionizing radiation-induced DNA damage foci. Our method constitutes a rapid, efficient, and cost-effective approach for isolation and subsequent characterization of rare cells based on observable traits such as movement, shape, or location, which in turn can generate novel mechanistic insights into important biological processes.

Exploiting Molecular Barcodes in High-Throughput Cellular Assays.

Multiplexing strategies, which greatly increase the number of simultaneously measured parameters in single experiments, are now being widely implemented by both the pharmaceutical industry and academic researchers. Color has long been used to identify biological signals and, when combined with molecular barcodes, has substantially enhanced the depth of multiplexed sample characterization. Moreover, the recent advent of DNA barcodes has led to an explosion of innovative cell sequencing approaches. Novel barcoding strategies also show great promise for encoding spatial information in transcriptomic studies, and for precise assessment of molecular abundance. Both color- and DNA-based barcodes can be conveniently analyzed with either a microscope or a cytometer, or via DNA sequencing. Here we review the basic principles of several technologies used to create barcodes and detail the type of samples that can be identified with such tags.

Opto-magnetic Selection and Isolation of Single Cells.

Capturing single cells from large heterogenous populations based solely on observable traits is necessary for many cell biology applications and remains a major technical challenge. The protocol we present allows the isolation of viable and metabolically active cells selected for their shape, migration speed, contact to other cells, or intracellular protein localization. We previously introduced a method termed Cell Labeling via Photobleaching (CLaP) for the efficient tagging of cells chosen for visual criteria. Here we describe a new protocol for capturing such cells using ferromagnetic beads termed single-cell magneto-optical capture (scMOCa). This technology is especially useful when the number of target cells represents an extremely low fraction of the total population (potentially one single cell), a situation in which conventional sorting techniques like fluorescent or magnetic activated cell sorting (F/MACS) cannot provide satisfactory results in terms of capture efficiency and specificity. scMOCa uses the lasers of a confocal microscope to photobleach and crosslink biotin-4-fluorecein molecules to cell membranes. Streptavidin coated magnetic beads then adhere to biotin moieties and a magnet allows the capture of illuminated cells. By precisely controlling liquid volumes and spacing between the different parts of a simple setup, high cell selectivity and capture efficacy can be achieved. scMOCA allows visual selection and isolation of any number of cells in a microscopy field and captured cells remain viable to generate new colonies of chosen phenotypes for downstream analyses.

Axonal Degeneration in Retinal Ganglion Cells Is Associated with a Membrane Polarity-Sensitive Redox Process.

Axonal degeneration is a pathophysiological mechanism common to several neurodegenerative diseases. The slow Wallerian degeneration (WldS) mutation, which results in reduced axonal degeneration in the central and peripheral nervous systems, has provided insight into a redox-dependent mechanism by which axons undergo self-destruction. We studied early molecular events in axonal degeneration with single-axon laser axotomy and time-lapse imaging, monitoring the initial changes in transected axons of purified retinal ganglion cells (RGCs) from wild-type and WldS rat retinas using a polarity-sensitive annexin-based biosensor (annexin B12-Cys101,Cys260-N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethylenediamine). Transected axons demonstrated a rapid and progressive change in membrane phospholipid polarity, manifested as phosphatidylserine externalization, which was significantly delayed and propagated more slowly in axotomized WldS RGCs compared with wild-type axons. Delivery of bis(3-propionic acid methyl ester)phenylphosphine borane complex, a cell-permeable intracellular disulfide-reducing drug, slowed the onset and velocity of phosphatidylserine externalization in wild-type axons significantly, replicating the WldS phenotype, whereas extracellular redox modulation reversed the WldS phenotype. These findings are consistent with an intra-axonal redox mechanism for axonal degeneration associated with the initiation and propagation of phosphatidylserine externalization after axotomy.SIGNIFICANCE STATEMENT Axonal degeneration is a neuronal process independent of somal apoptosis, the propagation of which is unclear. We combined single-cell laser axotomy with time-lapse imaging to study the dynamics of phosphatidylserine externalization immediately after axonal injury in purified retinal ganglion cells. The extension of phosphatidylserine externalization was slowed and delayed in Wallerian degeneration slow (WldS) axons and this phenotype could be reproduced by intra-axonal disulfide reduction in wild-type axons and reversed by extra-axonal reduction in WldS axons. These results are consistent with a redox mechanism for propagation of membrane polarity asymmetry in axonal degeneration.

Live single-cell laser tag.

The ability to conduct image-based, non-invasive cell tagging, independent of genetic engineering, is key to cell biology applications. Here we introduce cell labelling via photobleaching (CLaP), a method that enables instant, specific tagging of individual cells based on a wide array of criteria such as shape, behaviour or positional information. CLaP uses laser illumination to crosslink biotin onto the plasma membrane, coupled with streptavidin conjugates to label individual cells for genomic, cell-tracking, flow cytometry or ultra-microscopy applications. We show that the incorporated mark is stable, non-toxic, retained for several days, and transferred by cell division but not to adjacent cells in culture. To demonstrate the potential of CLaP for genomic applications, we combine CLaP with microfluidics-based single-cell capture followed by transcriptome-wide next-generation sequencing. Finally, we show that CLaP can also be exploited for inducing transient cell adhesion to substrates for microengineering cultures with spatially patterned cell types.

Approaches for neural tissue regeneration.

There is currently no treatment for neurodegenerative diseases such as Parkinson's or Alzheimer's diseases. While spinal cord injury has no treatment either, nerve injuries are being treated with autologous grafts, a procedure that in turn translates into a loss of function in the donor area. The development of therapies for these pathologies has become urgent as population keeps on ageing. A promising direction of investigation is the use of regenerative techniques to re-grow healthy and functional tissue in the injured area. In this review article, various approaches currently investigated to promote neural regeneration are covered. Those include approaches based on (and many times combining) stem cell therapy, scaffolds made of hydrogel, electrospun fibers and conductive materials as well as the use of soluble or non-diffusible growth factors.

Differentiation of neuronal stem cells into motor neurons using electrospun poly-L-lactic acid/gelatin scaffold.

Neural stem cells (NSCs) provide promising therapeutic potential for cell replacement therapy in spinal cord injury (SCI). However, high increases of cell viability and poor control of cell differentiation remain major obstacles. In this study, we have developed a non-woven material made of co-electrospun fibers of poly L-lactic acid and gelatin with a degradation rate and mechanical properties similar to peripheral nerve tissue and investigated their effect on cell survival and differentiation into motor neuronal lineages through the controlled release of retinoic acid (RA) and purmorphamine. Engineered Neural Stem-Like Cells (NSLCs) seeded on these fibers, with and without the instructive cues, differentiated into ß-III-tubulin, HB-9, Islet-1, and choactase-positive motor neurons by immunostaining, in response to the release of the biomolecules. In addition, the bioactive material not only enhanced the differentiation into motor neuronal lineages but also promoted neurite outgrowth. This study elucidated that a combination of electrospun fiber scaffolds, neural stem cells, and controlled delivery of instructive cues could lead to the development of a better strategy for peripheral nerve injury repair.

Vascularized bone tissue engineering: approaches for potential improvement.

Significant advances have been made in bone tissue engineering (TE) in the past decade. However, classical bone TE strategies have been hampered mainly due to the lack of vascularization within the engineered bone constructs, resulting in poor implant survival and integration. In an effort toward clinical success of engineered constructs, new TE concepts have arisen to develop bone substitutes that potentially mimic native bone tissue structure and function. Large tissue replacements have failed in the past due to the slow penetration of the host vasculature, leading to necrosis at the central region of the engineered tissues. For this reason, multiple microscale strategies have been developed to induce and incorporate vascular networks within engineered bone constructs before implantation in order to achieve successful integration with the host tissue. Previous attempts to engineer vascularized bone tissue only focused on the effect of a single component among the three main components of TE (scaffold, cells, or signaling cues) and have only achieved limited success. However, with efforts to improve the engineered bone tissue substitutes, bone TE approaches have become more complex by combining multiple strategies simultaneously. The driving force behind combining various TE strategies is to produce bone replacements that more closely recapitulate human physiology. Here, we review and discuss the limitations of current bone TE approaches and possible strategies to improve vascularization in bone tissue substitutes.