A multiscale fluidic device for the study of dendrite-mediated cell to cell communication.

Many cell types communicate by means of dendritic extensions via a multi-tiered set of geometric and chemical cues. Until recently, mimicking the compartmentalized in vivo cellular environment of dendrite-expressing cells such as osteocytes and motor neurons in a spatially and temporally controllable manner was limited by the challenges of in vitro device fabrication at submicron scales. Utilizing the improved resolution of current fabrication technology, we have designed a multiscale device, the Macro-micro-nano system, or Mµn, composed of two distinct cell-seeding and interrogation compartments separated by a nanochannel array. The array enables dendrite ingrowth, while providing a mechanism for fluidic sequestration and/or temporally-mediated diffusible signaling between cell populations. Modeling of the Mµn system predicted the ability to isolate diffusible signals, namely ATP. Empirical diffusion studies verified computational modeling. In addition, cell viability, dendrite interaction with the nanoarray, and cellular purinergic response to heat shock were experimentally evaluated within the device for both osteocytes and motor neurons. Our results describe a novel in vitro system in which dendrite-expressing cell types can be studied within nano-environments that mimic in vivo conditions. In particular, the Mµn system enables real-time observation of cell to cell communication between cell populations in distinct, but fluidically coupled regions.

Epigenome editing technologies for discovery and medicine.

Epigenome editing has rapidly evolved in recent years, with diverse applications that include elucidating gene regulation mechanisms, annotating coding and noncoding genome functions and programming cell state and lineage specification. Importantly, given the ubiquitous role of epigenetics in complex phenotypes, epigenome editing has unique potential to impact a broad spectrum of diseases. By leveraging powerful DNA-targeting technologies, such as CRISPR, epigenome editing exploits the heritable and reversible mechanisms of epigenetics to alter gene expression without introducing DNA breaks, inducing DNA damage or relying on DNA repair pathways.

Astrocytes sense glymphatic-level shear stress through the interaction of sphingosine-1-phosphate with Piezo1.

Astrocyte endfeet enwrap brain vasculature, forming a boundary for perivascular glymphatic flow of fluid and solutes along and across the astrocyte endfeet into the brain parenchyma. We evaluated astrocyte sensitivity to shear stress generated by such flow, finding a set point for downstream calcium signaling that is below about 0.1 dyn/cm2. This set point is modulated by albumin levels encountered in cerebrospinal fluid (CSF) under normal conditions and following a blood-brain barrier breach or immune response. The astrocyte mechanosome responsible for the detection of shear stress includes sphingosine-1-phosphate (S1P)-mediated sensitization of the mechanosensor Piezo1. Fluid flow through perivascular channels delimited by vessel wall and astrocyte endfeet thus generates sufficient shear stress to activate astrocytes, thereby potentially controlling vasomotion and parenchymal perfusion. Moreover, S1P receptor signaling establishes a set point for Piezo1 activation that is finely tuned to coincide with CSF albumin levels and to the low shear forces resulting from glymphatic flow.

Activation of the imprinted Prader-Willi Syndrome locus by CRISPR-based epigenome editing.

Epigenome editing with DNA-targeting technologies such as CRISPR-dCas9 can be used to dissect gene regulatory mechanisms and potentially treat associated disorders. For example, Prader-Willi Syndrome (PWS) is caused by loss of paternally expressed imprinted genes on chromosome 15q11.2-q13.3, although the maternal allele is intact but epigenetically silenced. Using CRISPR repression and activation screens in human induced pluripotent stem cells (iPSCs), we identified genomic elements that control expression of the PWS gene SNRPN from the paternal and maternal chromosomes. We showed that either targeted transcriptional activation or DNA demethylation can activate the silenced maternal SNRPN and downstream PWS transcripts. However, these two approaches function at unique regions, preferentially activating different transcript variants and involving distinct epigenetic reprogramming mechanisms. Remarkably, transient expression of the targeted demethylase leads to stable, long-term maternal SNRPN expression in PWS iPSCs. This work uncovers targeted epigenetic manipulations to reprogram a disease-associated imprinted locus and suggests possible therapeutic interventions.

Astrocyte sensitivity to glymphatic shear stress is amplified by albumin and mediated by the interaction of sphingosine 1 phosphate with Piezo1.

Astrocyte endfeet enwrap brain vasculature, forming a boundary for perivascular glymphatic flow of fluid and solutes along and across the astrocyte endfeet into the brain parenchyma. To determine whether astrocytes may sense and respond to the shear forces generated by glymphatic flow, we examined intracellular calcium (Ca 2+ ) changes evoked in astrocytes to brief fluid flow applied in calibrated microfluidic chambers. Shear stresses < 20 dyn/cm 2 failed to evoke Ca 2+ responses in the absence of albumin, but cells responded to shear stress below 1 dyn/cm 2 when as little as 5 µM albumin was present in flow medium. A role for extracellular matrix in mechanotransduction was indicated by reduced sensitivity after degradation of heparan sulfate proteoglycan. Sphingosine-1-phosphate (S1P) amplified shear responses in the absence of albumin, whereas mechanosensitivity was attenuated by the S1P receptor blocker fingolimod. Piezo1 participated in the transduction as revealed by blockade by the spider toxin GsMTX and amplification by the chemical modulator Yoda1, even in absence of albumin or S1P. Our findings that astrocytes are exquisitely sensitive to shear stress and that sensitivity is greatly amplified by albumin concentrations encountered in normal and pathological CSF predict that perivascular astrocytes are responsive to glymphatic shear stress and that responsiveness is augmented by elevated CSF protein. S1P receptor signaling thus establishes a setpoint for Piezo1 activation that is finely tuned to coincide with albumin level in CSF and to the low shear forces resulting from glymphatic flow. Graphical abstract: Astrocyte endfoot responds to glymphatic shear stress when albumin is present. Mechanism involves sphingosine-1-phosphate (S1P) binding to its receptor (S1PR), activating phospholipase C (PLC) and thereby sensitizing the response of Piezo1 to flow. Ca 2+ influx triggers Ca 2+ release from intracellular stores and further downstream signaling, thereby modulating parenchymal perfusion. Illustration created using BioRender.com.

Orthogonal CRISPR screens to identify transcriptional and epigenetic regulators of human CD8 T cell function.

The clinical response to adoptive T cell therapies is strongly associated with transcriptional and epigenetic state. Thus, technologies to discover regulators of T cell gene networks and their corresponding phenotypes have great potential to improve the efficacy of T cell therapies. We developed pooled CRISPR screening approaches with compact epigenome editors to systematically profile the effects of activation and repression of 120 transcription factors and epigenetic modifiers on human CD8+ T cell state. These screens nominated known and novel regulators of T cell phenotypes with BATF3 emerging as a high confidence gene in both screens. We found that BATF3 overexpression promoted specific features of memory T cells such as increased IL7R expression and glycolytic capacity, while attenuating gene programs associated with cytotoxicity, regulatory T cell function, and T cell exhaustion. In the context of chronic antigen stimulation, BATF3 overexpression countered phenotypic and epigenetic signatures of T cell exhaustion. CAR T cells overexpressing BATF3 significantly outperformed control CAR T cells in both in vitro and in vivo tumor models. Moreover, we found that BATF3 programmed a transcriptional profile that correlated with positive clinical response to adoptive T cell therapy. Finally, we performed CRISPR knockout screens with and without BATF3 overexpression to define co-factors and downstream factors of BATF3, as well as other therapeutic targets. These screens pointed to a model where BATF3 interacts with JUNB and IRF4 to regulate gene expression and illuminated several other novel targets for further investigation.

Transcriptional and epigenetic regulators of human CD8+ T cell function identified through orthogonal CRISPR screens.

Clinical response to adoptive T cell therapies is associated with the transcriptional and epigenetic state of the cell product. Thus, discovery of regulators of T cell gene networks and their corresponding phenotypes has potential to improve T cell therapies. Here we developed pooled, epigenetic CRISPR screening approaches to systematically profile the effects of activating or repressing 120 transcriptional and epigenetic regulators on human CD8+ T cell state. We found that BATF3 overexpression promoted specific features of memory T cells and attenuated gene programs associated with cytotoxicity, regulatory T cell function, and exhaustion. Upon chronic antigen stimulation, BATF3 overexpression countered phenotypic and epigenetic signatures of T cell exhaustion. Moreover, BATF3 enhanced the potency of CAR T cells in both in vitro and in vivo tumor models and programmed a transcriptional profile that correlates with positive clinical response to adoptive T cell therapy. Finally, we performed CRISPR knockout screens that defined cofactors and downstream mediators of the BATF3 gene network.

Glioblastoma-Astrocyte Connexin 43 Gap Junctions Promote Tumor Invasion.

Glioblastoma multiforme (GBM), classified as World Health Organization grade IV astrocytoma, is the deadliest adult cancer of the central nervous system. An important contributing factor to poor survival rates in GBM is extensive invasion, which decreases the efficacy of resection and subsequent adjuvant therapies. These treatments could be markedly improved with increased resolution of the genetic and molecular initiators and effectors of invasion. Connexin 43 (Cx43) is the principal astrocytic gap junction (GJ) protein. Despite the heterogeneity of GBM, a subpopulation of cells in almost all GBM tumors express Cx43. Functional GJs between GBM cells and astrocytes at the tumor edge are of critical interest for understanding invasion. In this study, we find that both in vitro and in ex vivo slice cultures, GBM is substantially less invasive when placed in a Cx43-deficient astrocyte environment. Furthermore, when Cx43 is deleted in GBM, the invasive phenotype is recovered. These data strongly suggest that there are opposing roles for Cx43 in GBM migration. We find that Cx43 is localized to the tumor edge in our ex vivo model, suggesting that GBM-astrocyte GJ communication at the tumor border is a driving force for invasion. Finally, we find that by a Cx43-dependent mechanism, but likely not direct channel-mediated diffusion, miRNAs associated with cell-matrix adhesion are transferred from GBM to astrocytes and miR-19b promotes invasion, revealing a role for post-transcriptional manipulation of astrocytes in fostering an invasion-permissive peritumoral niche. IMPLICATIONS: Cx43-mediated communication, specifically miRNA transfer, profoundly impacts glioblastoma invasion and may enable further therapeutic insight.

Generation and Characterization of Immortalized Mouse Cortical Astrocytes From Wildtype and Connexin43 Knockout Mice.

We transduced mouse cortical astrocytes cultured from four litters of embryonic wildtype (WT) and connexin43 (Cx43) null mouse pups with lentiviral vector encoding hTERT and measured expression of astrocyte-specific markers up to passage 10 (p10). The immortalized cell lines thus generated (designated IWCA and IKOCA, respectively) expressed biomarkers consistent with those of neonatal astrocytes, including Cx43 from wildtype but not from Cx43-null mice, lack of Cx30, and presence of Cx26. AQP4, the water channel that is found in high abundance in astrocyte end-feet, was expressed at moderately high levels in early passages, and its mRNA and protein declined to low but still detectable levels by p10. The mRNA levels of the astrocyte biomarkers aldehyde dehydrogenase 1L1 (ALDH1L1), glutamine synthetase (GS) and glial fibrillary acidic protein (GFAP) remained relatively constant during successive passages. GS protein expression was maintained while GFAP declined with cell passaging but was still detectable at p10. Both mRNA and protein levels of glutamate transporter 1 (GLT-1) declined with passage number. Immunostaining at corresponding times was consistent with the data from Western blots and provided evidence that these proteins were expressed at appropriate intracellular locations. Consistent with our goal of generating immortalized cell lines in which Cx43 was either functionally expressed or absent, IWCA cells were found to be well coupled with respect to intercellular dye transfer and similar to primary astrocyte cultures in terms of time course of junction formation, electrical coupling strength and voltage sensitivity. Moreover, barrier function was enhanced in co-culture of the IWCA cell line with bEnd.3 microvascular endothelial cells. In addition, immunostaining revealed oblate endogenous Cx43 gap junction plaques in IWCA that were similar in appearance to those plaques obtained following transfection of IKOCA cells with fluorescent protein tagged Cx43. Re-expression of Cx43 in IKOCA cells allows experimental manipulation of connexins and live imaging of interactions between connexins and other proteins. We conclude that properties of these cell lines resemble those of primary cultured astrocytes, and they may provide useful tools in functional studies by facilitating genetic and pharmacological manipulations in the context of an astrocyte-appropriate cellular environment.

The dynamic Nexus: gap junctions control protein localization and mobility in distinct and surprising ways.

Gap junction (GJ) channels permit molecules, such as ions, metabolites and second messengers, to transfer between cells. Their function is critical for numerous cellular interactions, providing exchange of metabolites, signaling molecules, and ionic currents. GJ channels are composed of Connexin (Cx) hexamers paired across extracellular space and typically form large rafts of clustered channels, called plaques, at cell appositions. Cxs together with molecules that interact with GJ channels make up a supramolecular structure known as the GJ Nexus. While the stability of connexin localization in GJ plaques has been studied, mobility of other Nexus components has yet to be addressed. Colocalization analysis of several nexus components and other membrane proteins reveal that certain molecules are excluded from the GJ plaque (Aquaporin 4, EAAT2b), while others are quite penetrant (lipophilic molecules, Cx30, ZO-1, Occludin). Fluorescence recovery after photobleaching of tagged Nexus-associated proteins showed that mobility in plaque domains is affected by mobility of the Cx proteins. These novel findings indicate that the GJ Nexus is a dynamic membrane organelle, with cytoplasmic and membrane-embedded proteins binding and diffusing according to distinct parameters.

Apoptotic Osteocytes Induce RANKL Production in Bystanders via Purinergic Signaling and Activation of Pannexin Channels.

Localized apoptosis of osteocytes, the tissue-resident cells within bone, occurs with fatigue microdamage and activates bone resorption. Osteoclasts appear to target and remove dying osteocytes, resorbing damaged bone matrix as well. Osteocyte apoptosis similarly activates bone resorption with estrogen loss and in disuse. Apoptotic osteocytes trigger viable neighbor (ie, bystander) osteocytes to produce RANKL, the cytokine required for osteoclast activation. Signals from apoptotic osteocytes that trigger this bystander RANKL expression remain obscure. Studying signaling among osteocytes has been hampered by lack of in vitro systems that model the limited communication among osteocytes in vivo (ie, via gap junctions on cell processes and/or paracrine signals through thin pericellular fluid spaces around osteocytes). Here, we used a novel multiscale fluidic device (the Macro-micro-nano, or Mµn) that reproduces these key anatomical features. Osteocytes in discrete compartments of the device communicate only via these limited pathways, which allows assessment of their roles in triggering osteocytes RANKL expression. Apoptosis of MLOY-4 osteocytes in the Mµn device caused increased osteocyte RANKL expression in the neighboring compartment, consistent with in vivo findings. This RANKL upregulation in bystander osteocytes was prevented by blocking Pannexin 1 channels as well as its ATP receptor. ATP alone caused comparable RANKL upregulation in bystander osteocytes. Finally, blocking Connexin 43 gap junctions did not abolish osteocyte RANKL upregulation, but did alter the distribution of RANKL expressing bystander osteocytes. These findings point to extracellular ATP, released from apoptotic osteocytes via Panx1 channels, as a major signal for triggering bystander osteocyte RANKL expression and activating bone remodeling. © 2020 American Society for Bone and Mineral Research.

Master Regulators and Cofactors of Human Neuronal Cell Fate Specification Identified by CRISPR Gene Activation Screens.

Technologies to reprogram cell-type specification have revolutionized the fields of regenerative medicine and disease modeling. Currently, the selection of fate-determining factors for cell reprogramming applications is typically a laborious and low-throughput process. Therefore, we use high-throughput pooled CRISPR activation (CRISPRa) screens to systematically map human neuronal cell fate regulators. We utilize deactivated Cas9 (dCas9)-based gene activation to target 1,496 putative transcription factors (TFs) in the human genome. Using a reporter of neuronal commitment, we profile the neurogenic activity of these factors in human pluripotent stem cells (PSCs), leading to a curated set of pro-neuronal factors. Activation of pairs of TFs reveals neuronal cofactors, including E2F7, RUNX3, and LHX8, that improve conversion efficiency, subtype specificity, and maturation of neuronal cell types. Finally, using multiplexed gene regulation with orthogonal CRISPR systems, we demonstrate improved neuronal differentiation with concurrent activation and repression of target genes, underscoring the power of CRISPR-based gene regulation for programming complex cellular phenotypes.

CRISPR-Cas Expands Dynamic Range of Gene Expression From T7RNAP Promoters.

Reducing leaky gene expression is critical for improving protein yield of recombinant bacteria and stability of engineered cellular circuits in synthetic biology. Leaky gene expression occurs when a genetic promoter is not fully repressed, leading to unintended protein synthesis in the absence of stimuli. Existing work have devised specific molecular strategies for reducing leaky gene expression of each promoter. In contrast, we describe a repurposed, modular CRISPRi system that attenuates leaky gene expression using a series of single-guide RNAs targeting the PT7/LacO1 promoter. Furthermore, we demonstrate the efficacy of CRISPRi to significantly increase the dynamic range of T7 RNA Polymerase (T7RNAP) promoters. In addition, we demonstrate that the CRISPRi system can be applied to enhance growth of bacteria that suffer from leaky expression of a toxic protein. Our work establishes a new application of CRISPRi in genomic engineering to improve the control of recombinant gene expression. The approach is potentially generalizable to other gene expression system by changing the single-guide RNAs.

In vitro formation of neuroclusters in microfluidic devices and cell migration as a function of stromal-derived growth factor 1 gradients.

Central nervous system (CNS) cells cultured in vitro as neuroclusters are useful models of tissue regeneration and disease progression. However, the role of cluster formation and collective migration of these neuroclusters to external stimuli has been largely unstudied in vitro. Here, 3 distinct CNS cell types, medulloblastoma (MB), medulloblastoma-derived glial progenitor cells (MGPC), and retinal progenitor cells (RPC), were examined with respect to cluster formation and migration in response to Stromal-Derived Growth Factor (SDF-1). A microfluidic platform was used to distinguish collective migration of neuroclusters from that of individual cells in response to controlled concentration profiles of SDF-1. Cell lines were also compared with respect to expression of CXCR4, the receptor for SDF-1, and the gap junction protein Connexin 43 (Cx43). All cell types spontaneously formed clusters and expressed both CXCR4 and Cx43. RPC clusters exhibited collective chemotactic migration (i.e. movement as clusters) along SDF-1 concentration gradients. MGPCs clusters did not exhibit adhesion-based migration, and migration of MB clusters was inconsistent. This study demonstrates how controlled microenvironments can be used to examine the formation and collective migration of CNS-derived neuroclusters in varied cell populations.

EGF as a New Therapeutic Target for Medulloblastoma Metastasis.

Medulloblastoma (MB) is a malignant pediatric brain tumor known for its aggressive metastatic potential. Despite the well-documented migration of MB cells to other parts of the brain and spinal column, MB chemotaxis is poorly understood. Herein, we examined the in vitro migratory and cellular responses of MB-derived cells to external signaling of Epidermal Growth Factor (EGF), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF-BB), and the stromal cell-derived factors 1-alpha (SDF-1). Experiments utilized transwell assays and immunocytochemistry to identify receptor activation in MB migration, and used a microfluidic platform to examine directionality, trajectory, and gradient-dependence of motile cells. Data illustrates that MB-derived cells respond strongly to EGF in a dosage and gradient-dependent manner with increased EGF-R activation, and show that high EGF gradient fields cause an increased number of cells to migrate longer directed distances. Our results provide evidence that EGF and its receptor play an important role than previously documented in MB chemotactic migration than previously documented and should be considered for developing migration-target therapies against MB metastasis.