Homogeneous Cell Population Research Articles

Recombinant human FGF18 preserves depth-dependent mechanical inhomogeneity in articular cartilage

Articular cartilage is a specialised tissue that has a relatively homogenous endogenous cell population but a diverse extracellular matrix (ECM), with depth-dependent mechanical properties. Repair of this tissue remains an elusive clinical goal, with biological interventions preferred to arthroplasty in younger patients. Osteochondral transplantation (OCT) has emerged for the treatment of cartilage defects and osteoarthritis. Fresh allografts stored at 4 °C have been utilised, though matrix and cell viability loss remains an issue. To address this, several studies have developed media formulations to maintain cartilage explants in vitro. One promising factor for these applications is sprifermin, a human-recombinant fibroblast growth factor-18, which stimulates chondrocyte proliferation and matrix synthesis and is in clinical trials for the treatment of osteoarthritis. The study hypothesis was that addition of sprifermin during storage would maintain the unique depth-dependent mechanical profile of articular cartilage explants, a feature not often evaluated. Explants were maintained for up to 6 weeks with or without a weekly 24 h exposure to sprifermin (100 ng/mL) and the compressive modulus was assessed. Results showed that sprifermin-treated samples maintained their depth-dependent mechanical profile through 3 weeks, whereas untreated samples lost their mechanical integrity over 1 week of culture. Sprifermin also affected ECM balance by maintaining the levels of extracellular collagen and suppressing matrix metalloproteinase production. These findings support the use of sprifermin as a medium additive for OCT allografts during in vitro storage and present a potential mechanism where sprifermin may impact a functional characteristic of articular cartilage in repair strategies.

Nonclassical actions of estradiol-17beta are not detectable in the alphaT3-1 and LbetaT2 immortalized gonadotrope cell lines†.

The immortalized mouse gonadotrope cell lines alphaT3-1 and LbetaT2 cells have been a substitute model for primary gonadotropes. These cell lines have provided a homogeneous cell population, as compared to the dissociated anterior pituitaries, which contain a heterogeneous population of cells potentially responsive to estradiol-17beta (E2). Nonclassical actions of E2 assumed to occur through the plasma membrane estrogen receptor 1 (ESR1, also known as ERalpha). These actions have included inhibition of gonadotropin-releasing hormone (GnRH)-induced increases in intracellular calcium concentrations and phosphorylation of p44/42 mitogen-activated protein kinase (ERK-1/2) in ovine pituitaries including primary gonadotropes in vitro. The objective of the present experiment was to determine if alphaT3-1 and LbetaT2 are cell models with limitations to examine the nonclassical actions of E2 occurring in gonadotropes. Experiments were conducted to determine if the cells have ESR1 at the plasma membrane using biotinylation cell and isolation of surface protein and staining with a fluorescently labeled E2 conjugate. The alphaT3-1 cells contain ESR1 associated with but not enriched within lipid rafts of the plasma membrane and do not translocate to lipid rafts upon binding of E2. In contrast, LbetaT2 cells lack ESR1 associated with the plasma membrane. Pretreatment with E2 did not cause inhibition of GnRH-stimulated increases in intracellular concentrations of calcium for either cell type. Phosphorylation of ERK-1/2 was not stimulated by E2 in either cell type. Although these cells lines have been used extensively to study GnRH signaling, in vitro or in vivo effects of nonclassical actions of E2 cannot be replicated in either cell line.

Focus on dedifferentiated adipocytes: characteristics, mechanisms, and possible applications.

It is largely believed that after undergoing differentiation, adipocytes can no longer divide. Yet, using ceiling culture, it was demonstrated in vitro that some adipocytes are able to regain proliferative abilities by becoming fibroblast-like cells called dedifferentiated adipocytes. Mature adipocytes are abundant, can be easily isolated, and represent a homogenous cell population. Because of these advantageous characteristics, dedifferentiated adipocytes are clinically attractive in tissue engineering as a potential treatment resource for conditions such as type 2 diabetes, cardiac and kidney diseases, as well as autoimmune diseases. The aim of this review article is to summarize current knowledge on adipocyte dedifferentiation by accurately describing dedifferentiated adipocyte characteristics such as morphological appearance, gene expression, antigen signature, pluripotency, and functionality. Current hypotheses possibly explaining the biological mechanisms and cellular reprogramming of the dedifferentiation process are summarized. Dedifferentiated adipocytes show a stem cell-like antigen profile and genome signature which add to their proliferative capacities and their ability to re-differentiate into diverse cell lineages. The dedifferentiation process likely involves liposecretion, that is, the rapid secretion of the cell's lipid droplet. Dedifferentiated adipocytes may allow development of new uses in tissue engineering.

Single-cell RNA sequencing of a European and an African lymphoblastoid cell line

In biomedical research, lymphoblastoid cell lines (LCLs), often established by in vitro infection of resting B cells with Epstein-Barr virus, are commonly used as surrogates for peripheral blood lymphocytes. Genomic and transcriptomic information on LCLs has been used to study the impact of genetic variation on gene expression in humans. Here we present single-cell RNA sequencing (scRNA-seq) data on GM12878 and GM18502—two LCLs derived from the blood of female donors of European and African ancestry, respectively. Cells from three samples (the two LCLs and a 1:1 mixture of the two) were prepared separately using a 10x Genomics Chromium Controller and deeply sequenced. The final dataset contained 7,045 cells from GM12878, 5,189 from GM18502, and 5,820 from the mixture, offering valuable information on single-cell gene expression in highly homogenous cell populations. This dataset is a suitable reference for population differentiation in gene expression at the single-cell level. Data from the mixture provide additional valuable information facilitating the development of statistical methods for data normalization and batch effect correction.

Abstract 5094: SCOPE: A normalization and copy number estimation method for single-cell DNA sequencing

Abstract Whole genome single-cell DNA sequencing (scDNA-seq) enables characterization of copy number profiles at the cellular level. This circumvents the averaging effects associated with bulk-tissue sequencing and has increased resolution yet decreased ambiguity in deconvolving cancer subclones and elucidating cancer evolutionary history. ScDNA-seq data is, however, sparse, noisy, and highly variable even within a homogeneous cell population, due to the biases and artifacts that are introduced during the library preparation and sequencing procedure. Here, we propose SCOPE, a normalization and copy number estimation method for scDNA-seq data. The distinguishing features of SCOPE include: (i) utilization of cell-specific Gini coefficients for quality controls and for identification of normal/diploid cells, which are further used as negative control samples in a Poisson latent factor model for normalization; (ii) modeling of GC content bias using an expectation-maximization algorithm embedded in the Poisson generalized linear models, which accounts for the different copy number states along the genome; (iii) a cross-sample iterative segmentation procedure to identify breakpoints that are shared across cells from the same genetic background. We evaluate performance of SCOPE on real scDNA-seq data sets from cancer genomic studies. Compared to existing methods, SCOPE more accurately estimates subclonal copy number aberrations and is shown to have higher correlation with array-based copy number profiles of purified bulk samples from the same patient. We further demonstrate SCOPE on three recently released data sets using the 10X Genomics single-cell CNV pipeline and show that it can reliably recover 1% of the cancer cells from a background of normal and recover the cancer subclonal compositions of 10,000 breast cancer cells. Citation Format: Rujin Wang, Danyu Lin, Yuchao Jiang. SCOPE: A normalization and copy number estimation method for single-cell DNA sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5094.

Abstract 1898: TIMP-2 function is regulated by the composition of the cellular microenvironment

Abstract The activity of mammalian matrix metalloproteinases (MMPs) is regulated by a family of four endogenous inhibitors known as the tissue inhibitor(s) of metalloproteinases (TIMPs). Since their original discovery and characterization several additional biological roles have been ascribed to TIMP family members, such as regulation of receptor tyrosine kinase-dependent cell growth and migration. TIMP-2 is abundantly expressed in normal tissues with low or absent MMP expression. In addition to this unique expression pattern TIMP-2 demonstrates broad spectrum inhibition of MMPs in vitro. Recent studies in our lab have shown that TIMP-2 is a promising candidate for biological cancer therapy. However, the role of TIMP-2 in cancer progression has been subject to debate with conflicting reports correlating enhanced TIMP-2 expression levels with both good and poor clinical outcomes. TIMP-2 exhibits multiple interactions with components of the extracellular matrix (ECM). Our working hypothesis is that TIMP-2 acts as a homeostatic mediator via modulation of ECM and cellular activities. Analysis of expression levels in tumor samples shows that TIMP-2 displays a strong positive correlation with a number of genes involved in regulation of the ECM. Using sample datasets from The Cancer Genome Atlas, we have identified a multi-gene expression pattern suggesting that the MMP-independent activities of TIMP-2 are sequestered in the tumor microenvironment. Using fluorescent labeled TIMP-2, we have conducted live cell imaging to visualize TIMP-2 localization in vitro. Our data reveals that TIMP-2 is highly dynamic both within the extracellular microenvironment and intracellular compartments. In addition, TIMP-2 displays a diverse pattern of localization within homogeneous cell populations and between cells of epithelial or mesenchymal lineages. Other in vitro observations highlight an inconsistent cellular response following TIMP-2 treatment. We speculated that these variations are a result of irregularities in ECM deposition by target cells. To obviate these inconsistencies, we have 1) generated TIMP-2 knockout cell lines; 2) cultured these cells on purified ECM in the presence or absence of exogenous TIMP-2 and/or mitogenic factors; 3) analyzed cell signaling response under these well-defined culture conditions. Our results show that TIMP-2 can differentially modulate intracellular signaling in a manner that is dependent on specific ECM components and the additional presence or absence of a mitogenic stimulus. Current work focuses on details of extracellular TIMP-2 molecular interactions and cellular uptake/signaling. Through further understanding of the extracellular molecular interactions of TIMP-2, and the factors that regulate its cellular functions, researchers will be able to more effectively target the aberrant extracellular environment that is associated with many human diseases. Citation Format: David Peeney. TIMP-2 function is regulated by the composition of the cellular microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1898.

Analysis of glycoconjugates and morphological characterization of the descending colon and rectum of the plains viscacha, Lagostomus maximus

Herbivores exhibit specializations at the intestinal level that facilitate the bacterial fermentation. The available information on the digestive physiology of Lagostomus maximus makes this rodent an interesting model to evaluate morpho-functional adaptations to herbivory. The general objective of this work was centered on the study of the morphology and histochemistry of the descending colon and rectum of L. maximus. To do so, a comparative analysis of the morphology, ultrastructure and glycosylation pattern of both anatomical regions was carried out. Histochemical results revealed that in both sectors of the large intestine, there are goblet cells with different glycosylation pattern within a morphologically homogeneous cell population. The main difference between both intestinal segments lay in the fact that the most distal region of the large intestine showed a greater proportion of sialomucins, characterized by being slightly O-acetylated. Further specific differences were revealed by lectin histochemistry. These data allowed to perform a functional interpretation of the cell types and secreted substances, thus contributing to a better understanding of the role of mucins in the intestinal tract functioning.

Engineering Stem Cell Self-organization to Build Better Organoids.

Organoids form through self-organization processes in which initially homogeneous populations of stem cells spontaneously break symmetry and undergo in-vivo-like pattern formation and morphogenesis, though the processes controlling this are poorly characterized. While these invitro self-organized tissues far exceed the microscopic and functional complexity obtained by current tissue engineering technologies, they are non-physiological in shape and size and have limited function and lifespan. Here, we discuss how engineering efforts for guiding stem-cell-based development at multiple stages can form the basis for the assembly of highly complex and rationally designed self-organizing multicellular systems with increased robustness and physiological relevance.

Comparative analysis of FBS containing media and serum free chemically defined media for adipose derived stem cells production

Background & Aim For the successful development of production effective stem cell therapy, mass production of consistent quality cells is required. Excellent culture medium is important in mass production of quality cells. Classically, fetal bovine serum (FBS) has been used as culture supplement for stem cells. However, due to the undefined and heterologous composition of FBS, researchers have transitioned to more chemically defined serum-free media. In this study, we aimed to investigate and compare the stem cell characteristics cultured in both media as well as media performance tests. Methods, Results & Conclusion Methods Media performance was compared with cell growth rate, accumulated cell number, population homogeneity and cell viability. Comparative analysis of adipose derived mesenchymal stem cells (ADSCs) was performed based on stem cell surface marker expression, differentiation potency, genetic stability, senescence analysis, mRNA expression change, and protein expression change. Results Culture using SFCDM provided more stable population doubling time (PDT), more homogenous cell population, and more cells in a shorter time compared to FBS containing media (FBSCM). The cultured ADSCs were shown to be positive for CD73, CD90, and CD105, but negative for CD14, CD34, CD45, and HLA-DR in both FBSCM and SFCDM. Adipogenic differentiation capability was similar in both media groups, but chondrogenic and osteogenc differentiation capabilities of ADSCs cultured in SFCDM were higher than those cultured in FBSCM. ADSCs positive for β-galactosidase were about 3 times higher in FBSCM . These results indicate that culture in SFCDM has an advantage in reducing cellular senescence of ADSCs compared to FBS media. ADSCs cultured in FBSCM showed a significant increase in the formation frequency of nucleoplasmic bridges comparable to those cultured in SFCDM, suggesting that culture using SFCDM is more genetically stable. The mRNA expression levels of ADSCs cultured in FBSCM were significantly increased for KEGG classification of aging, apoptotic process, cell death, immune response and inflammatory response compared to SFCDM. Conclusion SFCDM can provide higher-speed cell production rate than the classical FBSCM. SFCDM enables the maintenance of higher population homogeneity, genetic stability, and excellent differentiation potency. Taken together, these results suggest that culture using SFCDM provides various advantages through which it is possible to obtain safer, stable, and larger amounts of ADSCs.

Single cell transcriptomic analysis of human mesenchymal stem cells reveals limited heterogeneity

Mesenchymal stem cells (MSCs) are a population of multipotent cells with a superior ability to promote tissue repair by regulating regeneration and inflammation. Effective application of MSCs in disease treatment relies on the production of relatively homogeneous cell population. However, the cellular heterogeneity and the differentiation trajectories of in vitro expanded MSCs remain largely unclear. We profiled the transcriptomes of 361 single MSCs derived from two umbilical cords (UC-MSCs). These UC-MSCs were harvested at different passages and stimulated with or without inflammatory cytokines. Weighted gene correlation network analysis revealed that UC-MSCs surprisingly possess only limited heterogeneity, regardless of donors, and passages. We also found that upon pretreatment with inflammatory cytokines (IFNγ and TNFα), a classical strategy that can improve the efficiency of MSC-based therapy, MSCs exhibited uniformed changes in gene expression. Cell cycle-based principal component analysis showed that the limited heterogeneity identified in these UC-MSCs was strongly associated with their entrance into the G2/M phase. This was further proven by the observation that one featured gene, CD168, was expressed in a cell cycle-dependent manner. When CD168high UC-MSCs were sorted and cultured in vitro, they again showed similar CD168 expression patterns. Our results demonstrated that in vitro expanded UC-MSCs are a well-organized population with limited heterogeneity dominated by cell cycle status. Thus, our studies provided information for standardization of MSCs for disease treatment.

Extent, Heritability, and Functional Relevance of Single Cell Expression Variability in Highly Homogeneous Populations of Human Cells

Because of recent technological developments, single-cell assays such as single-cell RNA sequencing (scRNA-seq) have become much more widely available and have achieved unprecedented resolution in revealing cell heterogeneity. The extent of intrinsic cell-to-cell variability in gene expression, or single cell expression variability (scEV), has thus been increasingly appreciated. However, it remains unclear whether this variability is functionally important and, if so, what its implications are for multi-cellular organisms. We therefore analyzed multiple scRNA-seq data sets from lymphoblastoid cell lines (LCLs), lung airway epithelial cells (LAECs), and dermal fibroblasts (DFs). For each of the three cell types, we estimated scEV in homogeneous populations of cells; we identified 465, 466, and 291 highly variable genes (HVGs), respectively. These HVGs were enriched with specific functions precisely relevant to the cell types, from which the scRNA-seq data used to identify HVGs were generated — e.g., HVGs identified in lymphoblastoid cells were enriched in cytokine signaling pathways, LAECs collagen formation, and DFs keratinization. HVGs were deeply embedded in gene regulatory networks specific to corresponding cell types. We also found that scEV is a heritable trait, partially determined by cell donors’ genetic makeups. Furthermore, across genes, especially immune genes, levels of scEV and between-individual variability in gene expression were positively correlated, suggesting a potential link between the two variabilities measured at different organizational levels. Taken together, our results support the “variation is function” hypothesis, which postulates that scEV is required for higher-level system function. Thus, we argue that quantifying and characterizing scEV in relevant cell types may deepen our understating of normal as well as pathological cellular processes.

Adipose-Derived Stromal Vascular Fraction and Cultured Stromal Cells as Trophic Mediators for Tendon Healing.

Adipose-derived stromal vascular fraction (SVF) is a heterogeneous population of cells that yields a homogeneous population of plastic-adherent adipose tissue-derived stromal cells (ASC) when culture-expanded. SVF and ASC have been used clinically to improve tendon healing, yet their mechanism of action is not fully elucidated. The objective of this study was to investigate the potential for ASC to act as trophic mediators for tendon healing. Flexor digitorum superficialis tendons and adipose tissue were harvested from adult horses to obtain SVF, ASC, and tenocytes. Growth factor gene expression was quantified in SVF and ASC in serial passages and growth factors were quantified in ASC-conditioned medium (CM). Microchemotaxis assays were performed using ASC-CM. Tenocytes were grown in co-culture with autologous ASC or allogeneic SVF. Gene expression for insulin-like growth factor 1 (IGF-1), stromal cell-derived factor-1α (SDF-1α), transforming growth factor-β1 (TGF-β1) and TGF-β3 was significantly higher in SVF compared to ASC. Concentrations were significantly increased in ASC-CM compared to controls for IGF-1 (4-fold) and SDF-1α (6-fold). Medium conditioned by ASC induced significant cell migration in a dose-dependent manner. Gene expression for collagen types I and III, decorin, and cartilage oligomeric matrix protein was modestly, but significantly increased following co-culture of tenocytes with autologous ASC. Our findings support the ability of SVF and ASC to act as trophic mediators in tendon healing, particularly through chemotaxis, which stands to critically impact the intrinsic healing response. In vivo studies to further delineate the potential for SVF and/or ASC to improve tendon healing are warranted. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1429-1439, 2019.

Discovery and characterization of variance QTLs in human induced pluripotent stem cells.

Quantification of gene expression levels at the single cell level has revealed that gene expression can vary substantially even across a population of homogeneous cells. However, it is currently unclear what genomic features control variation in gene expression levels, and whether common genetic variants may impact gene expression variation. Here, we take a genome-wide approach to identify expression variance quantitative trait loci (vQTLs). To this end, we generated single cell RNA-seq (scRNA-seq) data from induced pluripotent stem cells (iPSCs) derived from 53 Yoruba individuals. We collected data for a median of 95 cells per individual and a total of 5,447 single cells, and identified 235 mean expression QTLs (eQTLs) at 10% FDR, of which 79% replicate in bulk RNA-seq data from the same individuals. We further identified 5 vQTLs at 10% FDR, but demonstrate that these can also be explained as effects on mean expression. Our study suggests that dispersion QTLs (dQTLs) which could alter the variance of expression independently of the mean can have larger fold changes, but explain less phenotypic variance than eQTLs. We estimate 4,015 individuals as a lower bound to achieve 80% power to detect the strongest dQTLs in iPSCs. These results will guide the design of future studies on understanding the genetic control of gene expression variance.

Detection of human papillomavirus type 18 oncogen E7 in patients with prostate cancer

Introduction . High prostate cancer (PC) incidence rates testify to importance of research into genesis of the given disease and means of its prevention. The item of oncogenic human papillomaviruses (HPVs) participance in PC origination is still being the subject of debates. If association of PC with HPV is proven prophylaxis of the given disease becomes possible by means of inoculating boys with the vaccines made for cervical cancer (CC) prevention.Objective: to test whether prostate tissues surgically removed from PC patients harbor oncogen E7 of HPV18 – the second most common HPV type responsible for CC.Materials and methods . The study was carried out on prostate glands of 17 PC patients surgically treated in N.N. Blokhin National Medical Research Center of Oncology. Detection of HPV18 oncogen E7 was done by polymerase chain reaction. To elevate polymerase chain reaction sensitivity DNA was isolated from homogeneous cell populations collected by means of microdissections from cryopreserved PC specimens.Results . Amplification products corresponding to HPV18 oncogen E7 were registered in tests from 2 PC patients.Conclusion . HPV18 oncogen E7 was detected in surgically removed prostate tissues of 2 PC patients. In the aggregate with our previous result (7 HPV16-positive PC cases in the same group of 17 patients) the given result enables one to presume participance of oncogenic HPVs in PC genesis. The problem deserves further study.

Heterogeneity of the Human Pancreatic Islet.

Pancreatic β-cells play a pivotal role in maintaining normoglycemia. Recent studies have revealed that the β-cell is not a homogeneous cell population but, rather, is heterogeneous in a number of properties such as electrical activity, gene expression, and cell surface markers. Identification of specific β-cell subpopulations altered in diabetic conditions would open a new avenue to develop targeted therapeutic interventions. As intense studies of β-cell heterogeneity are anticipated in the next decade, it is important that heterogeneity of the islet be recognized. Many studies in the past were undertaken with a small sample of islets, which might overlook important individual variance. In this study, by systematic analyses of the human islet in two and three dimensions, we demonstrate islet heterogeneity in size, number, architecture, cellular composition, and capillary density. There is no stereotypic human islet, and thus, a sufficient number of islets should be examined to ensure study reproducibility.

Dopaminergic Circuits in Reward and Aversion

The mesocorticolimbic dopamine (DA) system, which is composed of DA neurons in the ventral tegmental area (VTA) projecting to nucleus accumbens and medial prefrontal cortex, has been intensively studied because of its importance in reward processing, drug addiction and other neuropsychiatric diseases. Importantly, while VTA DA neurons were thought to form a homogeneous cell population, recent research has demonstrated a much greater diversity of VTA DA cell type and function. Accordingly, VTA DA neurons encode much more than reward and also contribute to negative affect such as aversion and social defeat stress. How VTA DA neurons could mediate both reward and aversion is currently unknown and an important goal of my research. In my presentation, I will discuss work from the lab that has elucidated the circuit architecture and function of the reciprocal connectivity in the mesolimbic DA system (Yang et al., 2018; Neuron). Moreover, I will discuss recent data that demonstrate the identification of a subcircuit within the mesolimbic DA system that contributes to the encoding of conditioned and unconditioned aversive stimuli (de Jong et al., Neuron, in press). Collectively, our work suggest that we need to develop a new perspective on the DA circuitry that will guide future treatment strategies for addiction and other neuropsychiatric disorders where dysfunction of the neural systems underlying motivated behaviors have been strongly implicated.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Multiplexed, Sequential Secretion Analysis of the Same Single Cells Reveals Distinct Effector Response Dynamics Dependent on the Initial Basal State.

The effector response of immune cells dictated by an array of secreted proteins is a highly dynamic process, requiring sequential measurement of all relevant proteins from single cells. Herein, a microchip‐based, 10‐plexed, sequential secretion assay on the same single cells and at the scale of ≈5000 single cells measured simultaneously over 4 time points are shown. It is applied to investigating the time course of single human macrophage response to toll‐like receptor 4 (TLR4) ligand lipopolysaccharide (LPS) and reveals four distinct activation modes for different proteins in single cells. Protein secretion dynamics classifies the cells into two major activation states dependent on the basal state of each cell. Single‐cell RNA sequencing performed on the same samples at the matched time points further demonstrates the existence of two major activation states at the transcriptional level, which are enriched for translation versus inflammatory programs, respectively. These results show a cell‐intrinsic heterogeneous response in a phenotypically homogeneous cell population. This work demonstrates the longitudinal tracking of protein secretion signature in thousands of single cells at multiple time points, providing dynamic information to better understand how individual immune cells react to pathogenic challenges over time and how they together constitute a population response.

Modeling Variability in Populations of Cells Using Approximated Multivariate Distributions.

We are interested in studying the evolution of large homogeneous populations of cells, where each cell is assumed to be composed of a group of biological players (species) whose dynamics is governed by a complex biological pathway, identical for all cells. Modeling the inherent variability of the species concentrations in different cells is crucial to understand the dynamics of the population. In this work, we focus on handling this variability by modeling each species by a random variable that evolves over time. This appealing approach runs into the curse of dimensionality since exactly representing a joint probability distribution involving a large set of random variables quickly becomes intractable as the number of variables grows. To make this approach amenable to biopathways, we explore different techniques to (i) approximate the exact joint distribution at a given time point, and (ii) to track its evolution as time elapses. We start with the problem of approximating the probability distribution of biological species in a population of cells at some given time point. Data come from different fine-grained models of biological pathways of increasing complexities, such as (perturbed) Ordinary Differential Equations (ODEs). Classical approximations rely on the strong and unrealistic assumption that variables/species are independent, or that they can be grouped into small independent clusters. We propose instead to use the Chow-Liu tree representation, based on overlapping clusters of two variables, which better captures correlations between variables. Our experiments show that the proposed approximation scheme is more accurate than existing ones to model probability distributions deriving from biopathways. Then we address the problem of tracking the dynamics of a population of cells, that is computing from an initial distribution the evolution of the (approximate) joint distribution of species over time, called the inference problem. We evaluate several approximate inference algorithms (e.g., [14] , [17] ) for coarse-grained abstractions [12], [16] of biological pathways. Using the Chow-Liu tree approximation, we develop a new inference algorithm which is very accurate according to the experiments we report, for a minimal computation overhead. Our implementation is available at https://codeocean.com/capsule/6491669/tree.

West Nile Virus-Inclusive Single-Cell RNA Sequencing Reveals Heterogeneity in the Type I Interferon Response within Single Cells.

West Nile virus (WNV) is a neurotropic mosquito-borne flavivirus of global importance. Neuroinvasive WNV infection results in encephalitis and can lead to prolonged neurological impairment or death. Type I interferon (IFN-I) is crucial for promoting antiviral defenses through the induction of antiviral effectors, which function to restrict viral replication and spread. However, our understanding of the antiviral response to WNV infection is mostly derived from analysis of bulk cell populations. It is becoming increasingly apparent that substantial heterogeneity in cellular processes exists among individual cells, even within a seemingly homogenous cell population. Here, we present WNV-inclusive single-cell RNA sequencing (scRNA-seq), an approach to examine the transcriptional variation and viral RNA burden across single cells. We observed that only a few cells within the bulk population displayed robust transcription of IFN-β mRNA, and this did not appear to depend on viral RNA abundance within the same cell. Furthermore, we observed considerable transcriptional heterogeneity in the IFN-I response, with genes displaying high unimodal and bimodal expression patterns. Broadly, IFN-stimulated genes negatively correlated with viral RNA abundance, corresponding with a precipitous decline in expression in cells with high viral RNA levels. Altogether, we demonstrated the feasibility and utility of WNV-inclusive scRNA-seq as a high-throughput technique for single-cell transcriptomics and WNV RNA detection. This approach can be implemented in other models to provide insights into the cellular features of protective immunity and identify novel therapeutic targets.IMPORTANCE West Nile virus (WNV) is a clinically relevant pathogen responsible for recurrent epidemics of neuroinvasive disease. Type I interferon is essential for promoting an antiviral response against WNV infection; however, it is unclear how heterogeneity in the antiviral response at the single-cell level impacts viral control. Specifically, conventional approaches lack the ability to distinguish differences across cells with varying viral abundance. The significance of our research is to demonstrate a new technique for studying WNV infection at the single-cell level. We discovered extensive variation in antiviral gene expression and viral abundance across cells. This protocol can be applied to primary cells or in vivo models to better understand the underlying cellular heterogeneity following WNV infection for the development of targeted therapeutic strategies.

Separate cortical and hippocampal cell populations target the rat nucleus reuniens and mammillary bodies.

Nucleus reuniens receives dense projections from both the hippocampus and the frontal cortices. Reflecting these connections, this nucleus is thought to enable executive functions, including those involving spatial learning. The mammillary bodies, which also support spatial learning, again receive dense hippocampal inputs, as well as lighter projections from medial frontal areas. The present study, therefore, compared the sources of these inputs to nucleus reuniens and the mammillary bodies. Retrograde tracer injections in rats showed how these two diencephalic sites receive projections from separate cell populations, often from adjacent layers in the same cortical areas. In the subiculum, which projects strongly to both sites, the mammillary body inputs originate from a homogenous pyramidal cell population in more superficial levels, while the cells that target nucleus reuniens most often originate from cells positioned at a deeper level. In these deeper levels, a more morphologically diverse set of subiculum cells contributes to the thalamic projection, especially at septal levels. While both diencephalic sites also receive medial frontal inputs, those to nucleus reuniens are especially dense. The densest inputs to the mammillary bodies appear to arise from the dorsal peduncular cortex, where the cells are mostly separate from deeper neurons that project to nucleus reuniens. Again, in those other cortical regions that innervate both nucleus reuniens and the mammillary bodies, there was no evidence of collateral projections. The findings support the notion that these diencephalic nuclei represent components of distinct, but complementary, systems that support different aspects of cognition.