Rudolf Jaenisch

Heritable immunization of mice against Lyme disease enables ecological disease prevention

A human blood-brain barrier model reveals pericytes as critical regulators of viral neuroinvasion

The blood-brain barrier (BBB) plays a vital role in regulating the passage of biomolecules between the bloodstream and the central nervous system (CNS) while also protecting the CNS from pathogens. Pericytes reside at the interface between the endothelial cells that form the vessel walls and the brain parenchyma. These cells are critical for maintaining BBB integrity and play key roles in regulating vessel permeability, blood flow, and immune cell migration. In this study, we developed a novel serum-free protocol to generate neural crest cell-derived pericytes (NCC-PCs) from human pluripotent stem cells (hPSCs). These NCC-PCs enhance BMEC barrier function and can be co-cultured with hPSC-derived brain microvascular endothelial cells (BMECs) in a contact co-culture BBB model that recapitulates the in vivo cellular interactions at the BBB. We used this model to evaluate the pathological consequences of BBB cell infection by highly neuroinvasive flaviviruses. Our results identify a previously undescribed role for NCC-PCs in maintaining BMEC barrier integrity during infection and reducing the spread of viral infection to the CNS.

Human iPSC‐Derived Microglia Integrate Into Cerebral Organoids and Assume an In Vivo‐Like Phenotype

Microglia, the brain-resident macrophages, are critically involved in numerous physiological and pathological brain processes, including neoplasms, epilepsy, and neurodegeneration. However, investigating microglial function is notoriously difficult because they are extremely sensitive to changes in their environment and drastically alter their transcriptional state and morphology once they are isolated from the brain and cultured in vitro. In vivo experiments in mice are likewise limited because of vast differences between mouse and human microglia, particularly regarding the expression of disease-associated genes. To overcome this issue, we developed a highly controlled in vitro cerebral organoid platform where human microglia adopt an in vivo-like phenotype. This approach allows long-term studies and high-throughput analysis of in vivo-like human microglia suitable for disease modeling and drug testing.

Oxidative Stress and Interferon Signaling Drive Differential Pathogenesis of Ancestral and Contemporary Zika Viruses in Human Cerebral Organoids

Functional genomic dissection of MS risk loci reveals convergence of cis and trans gene regulatory mechanisms in microglia

Summary Neurological diseases (NDs) are a major source of unmet medical need, and translational insights have been hampered by complex underlying pathophysiologies and limitations of experimental models. Noncoding single nucleotide polymorphisms (SNPs) at hundreds of loci have been linked to ND risk by genome-wide association studies (GWAS), but the causal genes and pathways are largely unknown. Despite the multicellular pathology of complex traits like multiple sclerosis (MS), functional studies that aim to characterize the molecular impact of disease-associated SNPs often investigate all SNPs linked to disease in the same cellular context. Here, we combine a computational approach to predict the pathogenic cell type of individual risk loci with functional CRISPR perturbation studies in iPSC-derived microglia cells (iMGLs). ND SNP enrichment in cell type-specific enhancers is similar between primary and iPSC-derived cells, and mechanistically supported by shared enhancer-promoter interactions. We apply a novel Perturb-seq platform to interrogate MS risk SNPs in iMGL, identifying likely cis -acting causal risk genes at 5 of 9 loci, as well as downstream differentially expressed genes (DEGs). Despite being found in trans to MS risk SNPs, downstream DEGs are substantially enriched for MS heritability. Downstream DEGs from all 5 target genes show significant overlap, converging on genes related to cytokinesis, phagocytosis, and mitochondrial metabolism. We then compared downstream DEGs to gene expression patterns observed in MS patient tissue studies and observed marked similarities, demonstrating that genes dysregulated as the result of GWAS loci perturbation mirrored effects observed in microglia found in MS patient lesions. Collectively, these results demonstrate that cell type aware functional studies can be used to translate ND SNP associations into mechanistic insights and reveal novel convergent biological mechanisms underlying complex traits.

A novel human blood-brain barrier model reveals pericytes as critical regulators of viral neuroinvasion

The blood-brain barrier (BBB) plays a vital role in regulating the passage of biomolecules between the bloodstream and the central nervous system (CNS) while also protecting the CNS from pathogens. Pericytes reside at the interface between the endothelial cells that form the vessel walls and the brain parenchyma. These cells are critical for maintaining BBB integrity and play key roles in regulating vessel permeability, blood flow, and immune cell migration. In this study, we developed a novel serum-free protocol to generate neural crest cell-derived pericytes (NCC-PCs) from human pluripotent stem cells (hPSCs). These NCC-PCs enhance BMEC barrier function and can be co-cultured with hPSC-derived brain microvascular endothelial cells (BMECs) in a contact co-culture BBB model that recapitulates the in vivo cellular interactions at the BBB. We used this model to evaluate the pathological consequences of BBB cell infection by highly neuroinvasive flaviviruses. Our results identify a previously undescribed role for NCC-PCs in maintaining BMEC barrier integrity during infection and reducing the spread of viral infection to the CNS.

SARS-CoV-2 infection of human pluripotent stem cell-derived vascular cells reveals smooth muscle cells as key mediators of vascular pathology during infection

Although respiratory symptoms are the most prevalent disease manifestation of infection by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), nearly 20% of hospitalized patients are at risk for thromboembolic events. This prothrombotic state is considered a key factor in the increased risk of stroke, which is observed clinically during both acute infection and long after symptoms clear. Here, we develop a model of SARS-CoV-2 infection using human-induced pluripotent stem cell-derived endothelial cells (ECs), pericytes (PCs), and smooth muscle cells (SMCs) to recapitulate the vascular pathology associated with SARS-CoV-2 exposure. Our results demonstrate that perivascular cells, particularly SMCs, are a susceptible vascular target for SARS-CoV-2 infection. Utilizing RNA sequencing, we characterize the transcriptomic changes accompanying SARS-CoV-2 infection of SMCs, PCs, and ECs. We observe that infected SMCs shift to a pro-inflammatory state and increase the expression of key mediators of the coagulation cascade. Further, we show human ECs exposed to the secretome of infected SMCs produce hemostatic factors that contribute to vascular dysfunction despite not being susceptible to direct infection. The findings here recapitulate observations from patient sera in human COVID-19 patients and provide mechanistic insight into the unique vascular implications of SARS-CoV-2 infection at a cellular level. This study uses a novel vascular model with human induced pluripotent stem cell-derived endothelial cells, pericytes, and smooth muscle cells to explore the vascular complications associated with SARS-CoV-2. It reveals that smooth muscle cells as the primary sites of infection and inflammation.

Heritable Immunization Establishes a New Model for Pathogen Control

Heritable immunization represents a promising approach for controlling infectious diseases by embedding immunity directly into the genomes of wild species that spread human pathogens. Here, we report the genetic engineering of Mus musculus to produce a neutralizing, protective single-chain antibody against Borrelia burgdorferi , the causative agent of Lyme disease. Engineered mice stably produced a LA-2 scFv-albumin fusion protein across multiple generations, demonstrating robust heritability and stability of gene expression. Following sequential challenges with infected and uninfected ticks, heterozygous mice exhibited strong resistance to infection, effectively interrupting the Borrelia burgdorferi disease transmission cycle. Having recently established novel protocols to genetically engineer the white-footed mouse, Peromyscus leucopus , a key reservoir of Lyme disease, these findings demonstrate the feasibility of heritable immunization as a potential strategy for mitigating Lyme disease transmission in the environment. More broadly, engineered reservoir immunity may offer a generalizable approach to controlling vector-borne and zoonotic disease with profound potential to improve human health.

Multi-species genome-wide CRISPR screens identify conserved suppressors of cold-induced cell death

Summary Cells must adapt to environmental changes to maintain homeostasis. One of the most striking environmental adaptations is entry into hibernation during which core body temperature can decrease from 37°C to as low at 4°C. How mammalian cells, which evolved to optimally function within a narrow range of temperatures, adapt to this profound decrease in temperature remains poorly understood. In this study, we conducted the first genome-scale CRISPR-Cas9 screen in cells derived from Syrian hamster, a facultative hibernator, as well as human cells to investigate the genetic basis of cold tolerance in a hibernator and a non-hibernator in an unbiased manner. Both screens independently revealed glutathione peroxidase 4 (GPX4), a selenium-containing enzyme, and associated proteins as critical for cold tolerance. We utilized genetic and pharmacological approaches to demonstrate that GPX4 is active in the cold and its catalytic activity is required for cold tolerance. Furthermore, we show that the role of GPX4 as a suppressor of cold-induced cell death extends across hibernating species, including 13-lined ground squirrels and greater horseshoe bats, highlighting the evolutionary conservation of this mechanism of cold tolerance. This study identifies GPX4 as a central modulator of mammalian cold tolerance and advances our understanding of the evolved mechanisms by which cells mitigate cold-associated damage—one of the most common challenges faced by cells and organisms in nature.

Multi-species genome-wide CRISPR screens identify conserved suppressors of cold-induced cell death

Summary Cells must adapt to environmental changes to maintain homeostasis. One of the most striking environmental adaptations is entry into hibernation during which core body temperature can decrease from 37°C to as low at 4°C. How mammalian cells, which evolved to optimally function within a narrow range of temperatures, adapt to this profound decrease in temperature remains poorly understood. In this study, we conducted the first genome-scale CRISPR-Cas9 screen in cells derived from Syrian hamster, a facultative hibernator, as well as human cells to investigate the genetic basis of cold tolerance in a hibernator and a non-hibernator in an unbiased manner. Both screens independently revealed glutathione peroxidase 4 (GPX4), a selenium-containing enzyme, and associated proteins as critical for cold tolerance. We utilized genetic and pharmacological approaches to demonstrate that GPX4 is active in the cold and its catalytic activity is required for cold tolerance. Furthermore, we show that the role of GPX4 as a suppressor of cold-induced cell death extends across hibernating species, including 13-lined ground squirrels and greater horseshoe bats, highlighting the evolutionary conservation of this mechanism of cold tolerance. This study identifies GPX4 as a central modulator of mammalian cold tolerance and advances our understanding of the evolved mechanisms by which cells mitigate cold-associated damage—one of the most common challenges faced by cells and organisms in nature.

Tuning porosity of macroporous hydrogels enables rapid rates of stress relaxation and promotes cell expansion and migration

Extracellular matrix (ECM) viscoelasticity broadly regulates cell behavior. While hydrogels can approximate the viscoelasticity of native ECM, it remains challenging to recapitulate the rapid stress relaxation observed in many tissues without limiting the mechanical stability of the hydrogel. Here, we develop macroporous alginate hydrogels that have an order of magnitude increase in the rate of stress relaxation as compared to bulk hydrogels. The increased rate of stress relaxation occurs across a wide range of polymer molecular weights (MWs), which enables the use of high MW polymer for improved mechanical stability of the hydrogel. The rate of stress relaxation in macroporous hydrogels depends on the volume fraction of pores and the concentration of bovine serum albumin, which is added to the hydrogels to stabilize the macroporous structure during gelation. Relative to cell spheroids encapsulated in bulk hydrogels, spheroids in macroporous hydrogels have a significantly larger area and smaller circularity because of increased cell migration. A computational model provides a framework for the relationship between the macroporous architecture and morphogenesis of encapsulated spheroids that is consistent with experimental observations. Taken together, these findings elucidate the relationship between macroporous hydrogel architecture and stress relaxation and help to inform the design of macroporous hydrogels for materials-based cell therapies.

Abortive infection of bat fibroblasts with SARS-CoV-2

Bats are tolerant to highly pathogenic viruses such as Marburg, Ebola, and Nipah, suggesting the presence of a unique immune tolerance toward viral infection. Here, we compared severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection of human and bat ( Rhinolophus ferrumequinum ) pluripotent cells and fibroblasts. Since bat cells do not express an angiotensin-converting enzyme 2 (ACE2) receptor that allows virus infection, we transduced the human ACE2 (hA) receptor into the cells and found that transduced cells can be infected with SARS-CoV-2. Compared to human embryonic stem cells-hA, infected bat induced Pluripotent Stem Cells (iPSCs)-hA produced about a 100-fold lower level of infectious virus and displayed lower toxicity. In contrast, bat embryonic fibroblast-hA produced no infectious virus while being infectable and synthesizing viral RNA and proteins, suggesting abortive infection. Indeed, electron microscopy failed to detect virus-like particles in infected bat fibroblasts in contrast to bat iPSCs or human cells, consistent with the latter producing infectious viruses. This suggests that bat somatic but not pluripotent cells have an effective mechanism to control virus replication. Consistent with previous results by others, we find that bat cells have a constitutively activated innate immune system, which might limit SARS-CoV-2 infection compared to human cells.

Pharmacological inhibition of Fms-like kinase 3 (FLT3) promotes neuronal maturation and suppresses seizure in a mouse model

Fms-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase predominantly expressed in blood and brain cells. While the FLT3 signaling pathway has been extensively studied in blood cell development and leukemia, its role in the brain remains largely unexplored. Through our previous high-throughput drug screening work unexpectedly found that several small molecule FLT3 inhibitors (FLT3i), including KW-2449 and Sunitinib, enhance expression of the gene encoding chloride transporter KCC2 in neurons. KCC2 is crucial for brain development and function, and its dysregulation is linked to many brain diseases. These findings suggest previously unrecognized roles of FLT3 signaling in brain health and disease that have yet to be systematically studied. In this study, we utilized a functional genomics approach to investigate the transcriptomic changes induced by pharmacological inhibition of the FLT3 pathway in brain cells, including cultured primary mouse neurons, a human stem cell-derived neuronal model of Rett syndrome (RTT), and human stem cell-derived microglia cultures. Our results show that treating human or mouse neurons with FLT3i drugs significantly upregulates genes crucial for brain development while downregulating genes linked to neuroinflammation. In contrast, FLT3i treatment of human microglia, which do not express FLT3, has no effect on their gene expression, highlighting the cell type-specific roles of FLT3 signaling in the brain. To further understand how FLT3 signaling regulates the expression of neuronal maturation genes such as KCC2, we conducted a curated CRISPR screen that identified a number of transcription factors involved in FLT3i-mediated KCC2 activation in neurons. The mRNA and protein levels of several neurodevelopmental disorder (NDD) risk genes are significantly upregulated in FLT3i-treated neurons, indicating potential therapeutic applications of FLT3i in rescuing underexpression and/or haploinsufficiency of disease-associated genes. In our in vivo studies, we evaluated the efficacy of the FLT3i drug KW-2449 in mice, demonstrating that it can effectively cross the blood-brain barrier, induce KCC2 protein expression for up to 24 hours after a single injection, and reduces seizure activity in a chemoconvulsant-induced mouse model of temporal lobe epilepsy. Collectively, our findings uncover previously unrecognized roles of neuron-specific FLT3 signaling in promoting neuronal maturation and reducing neuroinflammation. These results suggest that FLT3 kinase signaling regulates a transcriptional program vital for brain development and function, position it as a promising therapeutic target for NDD treatment.

Mitochondrial dysfunction and increased reactive oxygen species production in MECP2 mutant astrocytes and their impact on neurons

Studies on MECP2 function and its implications in Rett Syndrome (RTT) have traditionally centered on neurons. Here, using human embryonic stem cell (hESC) lines, we modeled MECP2 loss-of-function to explore its effects on astrocyte (AST) development and dysfunction in the brain. Ultrastructural analysis of RTT hESC-derived cerebral organoids revealed significantly smaller mitochondria compared to controls (CTRs), particularly pronounced in glia versus neurons. Employing a multiomics approach, we observed increased gene expression and accessibility of a subset of nuclear-encoded mitochondrial genes upon mutation of MECP2 in ASTs compared to neurons. Analysis of hESC-derived ASTs showed reduced mitochondrial respiration and altered key proteins in the tricarboxylic acid cycle and electron transport chain in RTT versus CTRs. Additionally, RTT ASTs exhibited increased cytosolic amino acids under basal conditions, which were depleted upon increased energy demands. Notably, mitochondria isolated from RTT ASTs exhibited increased reactive oxygen species and influenced neuronal activity when transferred to cortical neurons. These findings underscore MECP2 mutation's differential impact on mitochondrial and metabolic pathways in ASTs versus neurons, suggesting that dysfunctional AST mitochondria may contribute to RTT pathophysiology by affecting neuronal health.

Rapid iPSC inclusionopathy models shed light on formation, consequence, and molecular subtype of α-synuclein inclusions

Multi-species genome-wide CRISPR screens identify conserved suppressors of cold-induced cell death

Cells must adapt to environmental changes to maintain homeostasis. One of the most striking environmental adaptations is entry into hibernation during which core body temperature can decrease from 37°C to as low at 4°C. How mammalian cells, which evolved to optimally function within a narrow range of temperatures, adapt to this profound decrease in temperature remains poorly understood. In this study, we conducted the first genome-scale CRISPR-Cas9 screen in cells derived from Syrian hamster, a facultative hibernator, as well as human cells to investigate the genetic basis of cold tolerance in a hibernator and a non-hibernator in an unbiased manner. Both screens independently revealed glutathione peroxidase 4 (GPX4), a selenium-containing enzyme, and associated proteins as critical for cold tolerance. We utilized genetic and pharmacological approaches to demonstrate that GPX4 is active in the cold and its catalytic activity is required for cold tolerance. Furthermore, we show that the role of GPX4 as a suppressor of cold-induced cell death extends across hibernating species, including 13-lined ground squirrels and greater horseshoe bats, highlighting the evolutionary conservation of this mechanism of cold tolerance. This study identifies GPX4 as a central modulator of mammalian cold tolerance and advances our understanding of the evolved mechanisms by which cells mitigate cold-associated damage-one of the most common challenges faced by cells and organisms in nature.

Human-specific paralogs of SRGAP2 induce neotenic features of microglia structural and functional maturation

Microglia play key roles in shaping synaptic connectivity during neural circuits development. Whether microglia display human-specific features of structural and functional maturation is currently unknown. We show that the ancestral gene

Approaches to pandemic prevention – the chromatin vaccine

Developing effective vaccines against viral infections have significant impacts on development, prosperity and well-being of human populations. Thus, successful vaccines such as smallpox and polio vaccines, have promoted global societal well-being. In contrast, ineffective vaccines may fuel arguments that retard scientific progress. We aim to stimulate a multilevel discussion on how to develop effective vaccines against recent and future pandemics by focusing on acquired immunodeficiency syndrome (AIDS), coronavirus disease (COVID) and other viral infections. We appeal to harnessing recent achievements in this field specifically towards a cure for current pandemics and prevention of the next pandemics. Among these, we propose to apply the HIV DNA in chromatin format - an end product of aborted HIV integration in episomal forms, i.e., the chromatin vaccines (cVacc), to elicit the epigenetic silencing and memory that prevent viral replication and infection.

Genomic and biological variation in bat IFNs: An antiviral treatment approach

Bat-borne viruses have attracted considerable research, especially in relation to the Covid-19 pandemic. Although bats can carry multiple zoonotic viruses that are lethal to many mammalian species, they appear to be asymptomatic to viral infection despite the high viral loads contained in their bodies. There are several differences between bats and other mammals. One of the major differences between bats and other mammals is the bats' ability to fly, which is believed to have induced evolutionary changes. It may have also favoured them as suitable hosts for viruses. This is related to their tolerance to viral infection. Innate immunity is the first line of defence against viral infection, but bats have metamorphosed the type of responses induced by innate immunity factors such as interferons. The expression patterns of interferons differ, as do those of interferon-related genes such as interferon regulatory factors and interferon-stimulated genes that contribute to the antiviral response of infected cells. In addition, the signalling pathways related to viral infection and immune responses have been subject to evolutionary changes, including mutations compared to their homologues in other mammals and gene selection. This article discusses the differences in the interferon-mediated antiviral response in bats compared to that of other mammals and how these differences are correlated to viral tolerance in bats. The effect of bat interferons related genes on human antiviral response against bat-borne viruses is also discussed.

Low-cost camera-based estrous tracking enables transgenesis in Peromyscus leucopus , the primary reservoir for Lyme disease

CRISPR/Cas9 technology has revolutionized the production of animal models by reducing experimental timelines, slashing costs and streamlining gene editing, leading to a rapid expansion in the number of unique models for the study of human disease and gene function. However, most non-model animals, many of which are important in cancer and aging research, remain recalcitrant to genome engineering due to our limited understanding of their reproductive biology. Many wild rodents that transmit human diseases remain particularly challenging to engineer due to low pregnancy rates, the lack of external copulatory plugs, and susceptibility to premature termination of pregnancy. Here, we present low-cost activity-based estrous tracking for the efficient generation of timed pregnant and pseudopregnant white-footed mice and extend this tracking method to both lab mice and hamsters. Leveraging this technology, we demonstrate the generation of engineered Peromyscus leucopus , the primary reservoir for Lyme disease-causing bacteria and a putative model organism for studies of aging. These tools have broad implications for biomedical research and ecological engineering.