Publications by authors named "Peter A Sims"

65 Publications

Heterogeneity of human anti-viral immunity shaped by virus, tissue, age, and sex.

Cell Rep 2021 Nov;37(9):110071

Department of Microbiology and Immunology, Columbia University Irving Medical Center, New York, NY 10032, USA; Department of Surgery, Columbia University Irving Medical Center, New York, NY 10032, USA. Electronic address:

The persistence of anti-viral immunity is essential for protection and exhibits profound heterogeneity across individuals. Here, we elucidate the factors that shape maintenance and function of anti-viral T cell immunity in the body by comprehensive profiling of virus-specific T cells across blood, lymphoid organs, and mucosal tissues of organ donors. We use flow cytometry, T cell receptor sequencing, single-cell transcriptomics, and cytokine analysis to profile virus-specific CD8 T cells recognizing the ubiquitous pathogens influenza and cytomegalovirus. Our results reveal that virus specificity determines overall magnitude, tissue distribution, differentiation, and clonal repertoire of virus-specific T cells. Age and sex influence T cell differentiation and dissemination in tissues, while T cell tissue residence and functionality are highly correlated with the site. Together, our results demonstrate how the covariates of virus, tissue, age, and sex impact the anti-viral immune response, which is important for targeting, monitoring, and predicting immune responses to existing and emerging viruses.
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http://dx.doi.org/10.1016/j.celrep.2021.110071DOI Listing
November 2021

Protein kinase C and SRC signaling define reciprocally related subgroups of glioblastoma with distinct therapeutic vulnerabilities.

Cell Rep 2021 Nov;37(8):110054

Department of Cancer Biology, Mayo Clinic, Jacksonville, FL 32224, USA. Electronic address:

We report that atypical protein kinase Cι (PKCι) is an oncogenic driver of glioblastoma (GBM). Deletion or inhibition of PKCι significantly impairs tumor growth and prolongs survival in murine GBM models. GBM cells expressing elevated PKCι signaling are sensitive to PKCι inhibitors, whereas those expressing low PKCι signaling exhibit active SRC signaling and sensitivity to SRC inhibitors. Resistance to the PKCι inhibitor auranofin is associated with activated SRC signaling and response to a SRC inhibitor, whereas resistance to a SRC inhibitor is associated with activated PKCι signaling and sensitivity to auranofin. Interestingly, PKCι- and SRC-dependent cells often co-exist in individual GBM tumors, and treatment of GBM-bearing mice with combined auranofin and SRC inhibitor prolongs survival beyond either drug alone. Thus, we identify PKCι and SRC signaling as distinct therapeutic vulnerabilities that are directly translatable into an improved treatment for GBM.
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http://dx.doi.org/10.1016/j.celrep.2021.110054DOI Listing
November 2021

Cellular origins and lineage relationships of the intestinal epithelium.

Am J Physiol Gastrointest Liver Physiol 2021 10 25;321(4):G413-G425. Epub 2021 Aug 25.

Columbia Stem Cell Initiative, Division of Digestive and Liver Diseases, Department of Medicine, Columbia Center for Human Development, Columbia University Irving Medical Center, New York, New York.

Knowledge of the development and hierarchical organization of tissues is key to understanding how they are perturbed in injury and disease, as well as how they may be therapeutically manipulated to restore homeostasis. The rapidly regenerating intestinal epithelium harbors diverse cell types and their lineage relationships have been studied using numerous approaches, from classical label-retaining and genetic lineage tracing methods to novel transcriptome-based annotations. Here, we describe the developmental trajectories that dictate differentiation and lineage specification in the intestinal epithelium. We focus on the most recent single-cell RNA-sequencing (scRNA-seq)-based strategies for understanding intestinal epithelial cell lineage relationships, underscoring how they have refined our view of the development of this tissue and highlighting their advantages and limitations. We emphasize how these technologies have been applied to understand the dynamics of intestinal epithelial cells in homeostatic and injury-induced regeneration models.
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http://dx.doi.org/10.1152/ajpgi.00188.2021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8560372PMC
October 2021

A MYC and RAS co-activation signature in localized prostate cancer drives bone metastasis and castration resistance.

Nat Cancer 2020 Nov 19;1(11):1082-1096. Epub 2020 Oct 19.

Department of Molecular Pharmacology and Therapeutics, Columbia University Irving Medical Center, New York, NY, USA.

Understanding the intricacies of lethal prostate cancer poses specific challenges due to difficulties in accurate modeling of metastasis in vivo. Here we show that mice (for -) develop prostate cancer with a high penetrance of metastasis to bone, thereby enabling detection and tracking of bone metastasis in vivo and ex vivo. Transcriptomic and whole-exome analyses of bone metastasis from these mice revealed distinct molecular profiles conserved between human and mouse and specific patterns of subclonal branching from the primary tumor. Integrating bulk and single-cell transcriptomic data from mouse and human datasets with functional studies in vivo unravels a unique MYC/RAS co-activation signature associated with prostate cancer metastasis. Finally, we identify a gene signature with prognostic value for time to metastasis and predictive of treatment response in human patients undergoing androgen receptor therapy across clinical cohorts, thus uncovering conserved mechanisms of metastasis with potential translational significance.
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http://dx.doi.org/10.1038/s43018-020-00125-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8171279PMC
November 2020

Single-cell characterization of macrophages in glioblastoma reveals MARCO as a mesenchymal pro-tumor marker.

Genome Med 2021 05 19;13(1):88. Epub 2021 May 19.

Department of Systems Biology, Columbia University Irving Medical Center, New York, NY, USA.

Background: Macrophages are the most common infiltrating immune cells in gliomas and play a wide variety of pro-tumor and anti-tumor roles. However, the different subpopulations of macrophages and their effects on the tumor microenvironment remain poorly understood.

Methods: We combined new and previously published single-cell RNA-seq data from 98,015 single cells from a total of 66 gliomas to profile 19,331 individual macrophages.

Results: Unsupervised clustering revealed a pro-tumor subpopulation of bone marrow-derived macrophages characterized by the scavenger receptor MARCO, which is almost exclusively found in IDH1-wild-type glioblastomas. Previous studies have implicated MARCO as an unfavorable marker in melanoma and non-small cell lung cancer; here, we find that bulk MARCO expression is associated with worse prognosis and mesenchymal subtype. Furthermore, MARCO expression is significantly altered over the course of treatment with anti-PD1 checkpoint inhibitors in a response-dependent manner, which we validate with immunofluorescence imaging.

Conclusions: These findings illustrate a novel macrophage subpopulation that drives tumor progression in glioblastomas and suggest potential therapeutic targets to prevent their recruitment.
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http://dx.doi.org/10.1186/s13073-021-00906-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8136167PMC
May 2021

Deconvolution of cell type-specific drug responses in human tumor tissue with single-cell RNA-seq.

Genome Med 2021 05 11;13(1):82. Epub 2021 May 11.

Department of Systems Biology, Columbia University Irving Medical Center, New York, NY, 10032, USA.

Background: Preclinical studies require models that recapitulate the cellular diversity of human tumors and provide insight into the drug sensitivities of specific cellular populations. The ideal platform would enable rapid screening of cell type-specific drug sensitivities directly in patient tumor tissue and reveal strategies to overcome intratumoral heterogeneity.

Methods: We combine multiplexed drug perturbation in acute slice culture from freshly resected tumors with single-cell RNA sequencing (scRNA-seq) to profile transcriptome-wide drug responses in individual patients. We applied this approach to drug perturbations on slices derived from six glioblastoma (GBM) resections to identify conserved drug responses and to one additional GBM resection to identify patient-specific responses.

Results: We used scRNA-seq to demonstrate that acute slice cultures recapitulate the cellular and molecular features of the originating tumor tissue and the feasibility of drug screening from an individual tumor. Detailed investigation of etoposide, a topoisomerase poison, and the histone deacetylase (HDAC) inhibitor panobinostat in acute slice cultures revealed cell type-specific responses across multiple patients. Etoposide has a conserved impact on proliferating tumor cells, while panobinostat treatment affects both tumor and non-tumor populations, including unexpected effects on the immune microenvironment.

Conclusions: Acute slice cultures recapitulate the major cellular and molecular features of GBM at the single-cell level. In combination with scRNA-seq, this approach enables cell type-specific analysis of sensitivity to multiple drugs in individual tumors. We anticipate that this approach will facilitate pre-clinical studies that identify effective therapies for solid tumors.
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http://dx.doi.org/10.1186/s13073-021-00894-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8114529PMC
May 2021

Tumor restriction by type I collagen opposes tumor-promoting effects of cancer-associated fibroblasts.

J Clin Invest 2021 06;131(11)

Department of Medicine, Columbia University, New York, New York, USA.

Cancer-associated fibroblasts (CAF) may exert tumor-promoting and tumor-suppressive functions, but the mechanisms underlying these opposing effects remain elusive. Here, we sought to understand these potentially opposing functions by interrogating functional relationships among CAF subtypes, their mediators, desmoplasia, and tumor growth in a wide range of tumor types metastasizing to the liver, the most common organ site for metastasis. Depletion of hepatic stellate cells (HSC), which represented the main source of CAF in mice and patients in our study, or depletion of all CAF decreased tumor growth and mortality in desmoplastic colorectal and pancreatic metastasis but not in nondesmoplastic metastatic tumors. Single-cell RNA-Seq in conjunction with CellPhoneDB ligand-receptor analysis, as well as studies in immune cell-depleted and HSC-selective knockout mice, uncovered direct CAF-tumor interactions as a tumor-promoting mechanism, mediated by myofibroblastic CAF-secreted (myCAF-secreted) hyaluronan and inflammatory CAF-secreted (iCAF-secreted) HGF. These effects were opposed by myCAF-expressed type I collagen, which suppressed tumor growth by mechanically restraining tumor spread, overriding its own stiffness-induced mechanosignals. In summary, mechanical restriction by type I collagen opposes the overall tumor-promoting effects of CAF, thus providing a mechanistic explanation for their dual functions in cancer. Therapeutic targeting of tumor-promoting CAF mediators while preserving type I collagen may convert CAF from tumor promoting to tumor restricting.
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http://dx.doi.org/10.1172/JCI146987DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8159701PMC
June 2021

Human plasmacytoid dendritic cells mount a distinct antiviral response to virus-infected cells.

Sci Immunol 2021 Apr;6(58)

Department of Pathology, New York University Grossman School of Medicine, New York, NY 10016, USA.

Plasmacytoid dendritic cells (pDCs) can rapidly produce interferons and other soluble factors in response to extracellular viruses or virus mimics such as CpG-containing DNA. pDCs can also recognize live cells infected with certain RNA viruses, but the relevance and functional consequences of such recognition remain unclear. We studied the response of primary DCs to the prototypical persistent DNA virus, human cytomegalovirus (CMV). Human pDCs produced high amounts of type I interferon (IFN-I) when incubated with live CMV-infected fibroblasts but not with free CMV; the response involved integrin-mediated adhesion, transfer of DNA-containing virions to pDCs, and the recognition of DNA through TLR9. Compared with transient polyfunctional responses to CpG or free influenza virus, pDC response to CMV-infected cells was long-lasting, dominated by the production of IFN-I and IFN-III, and lacked diversification into functionally distinct populations. Similarly, pDC activation by influenza-infected lung epithelial cells was highly efficient, prolonged, and dominated by interferon production. Prolonged pDC activation by CMV-infected cells facilitated the activation of natural killer cells critical for CMV control. Last, patients with CMV viremia harbored phenotypically activated pDCs and increased circulating IFN-I and IFN-III. Thus, recognition of live infected cells is a mechanism of virus detection by pDCs that elicits a unique antiviral immune response.
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http://dx.doi.org/10.1126/sciimmunol.abc7302DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8221820PMC
April 2021

Longitudinal profiling of respiratory and systemic immune responses reveals myeloid cell-driven lung inflammation in severe COVID-19.

Immunity 2021 04 11;54(4):797-814.e6. Epub 2021 Mar 11.

Department of Microbiology and Immunology, Columbia University Irving Medical Center, New York, NY 10032, USA; Department of Surgery, Columbia University Irving Medical Center, New York, NY 10032, USA. Electronic address:

Immune response dynamics in coronavirus disease 2019 (COVID-19) and their severe manifestations have largely been studied in circulation. Here, we examined the relationship between immune processes in the respiratory tract and circulation through longitudinal phenotypic, transcriptomic, and cytokine profiling of paired airway and blood samples from patients with severe COVID-19 relative to heathy controls. In COVID-19 airways, T cells exhibited activated, tissue-resident, and protective profiles; higher T cell frequencies correlated with survival and younger age. Myeloid cells in COVID-19 airways featured hyperinflammatory signatures, and higher frequencies of these cells correlated with mortality and older age. In COVID-19 blood, aberrant CD163 monocytes predominated over conventional monocytes, and were found in corresponding airway samples and in damaged alveoli. High levels of myeloid chemoattractants in airways suggest recruitment of these cells through a CCL2-CCR2 chemokine axis. Our findings provide insights into immune processes driving COVID-19 lung pathology with therapeutic implications for targeting inflammation in the respiratory tract.
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http://dx.doi.org/10.1016/j.immuni.2021.03.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7951561PMC
April 2021

The harsh microenvironment in early breast cancer selects for a Warburg phenotype.

Proc Natl Acad Sci U S A 2021 01;118(3)

Department of Cancer Physiology, Moffitt Cancer Center and Research Institute, Tampa, FL 33612;

The harsh microenvironment of ductal carcinoma in situ (DCIS) exerts strong evolutionary selection pressures on cancer cells. We hypothesize that the poor metabolic conditions near the ductal center foment the emergence of a Warburg Effect (WE) phenotype, wherein cells rapidly ferment glucose to lactic acid, even in normoxia. To test this hypothesis, we subjected low-glycolytic breast cancer cells to different microenvironmental selection pressures using combinations of hypoxia, acidosis, low glucose, and starvation for many months and isolated single clones for metabolic and transcriptomic profiling. The two harshest conditions selected for constitutively expressed WE phenotypes. RNA sequencing analysis of WE clones identified the transcription factor KLF4 as potential inducer of the WE phenotype. In stained DCIS samples, KLF4 expression was enriched in the area with the harshest microenvironmental conditions. We simulated in vivo DCIS phenotypic evolution using a mathematical model calibrated from the in vitro results. The WE phenotype emerged in the poor metabolic conditions near the necrotic core. We propose that harsh microenvironments within DCIS select for a WE phenotype through constitutive transcriptional reprogramming, thus conferring a survival advantage and facilitating further growth and invasion.
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http://dx.doi.org/10.1073/pnas.2011342118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7826394PMC
January 2021

Integrating single-cell RNA-seq and imaging with SCOPE-seq2.

Sci Rep 2020 11 10;10(1):19482. Epub 2020 Nov 10.

Department of Systems Biology, Columbia University Irving Medical Center, New York, NY, 10032, USA.

Live cell imaging allows direct observation and monitoring of phenotypes that are difficult to infer from transcriptomics. However, existing methods for linking microscopy and single-cell RNA-seq (scRNA-seq) have limited scalability. Here, we describe an upgraded version of Single Cell Optical Phenotyping and Expression (SCOPE-seq2) for combining single-cell imaging and expression profiling, with substantial improvements in throughput, molecular capture efficiency, linking accuracy, and compatibility with standard microscopy instrumentation. We introduce improved optically decodable mRNA capture beads and implement a more scalable and simplified optical decoding process. We demonstrate the utility of SCOPE-seq2 for fluorescence, morphological, and expression profiling of individual primary cells from a human glioblastoma (GBM) surgical sample, revealing relationships between simple imaging features and cellular identity, particularly among malignantly transformed tumor cells.
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http://dx.doi.org/10.1038/s41598-020-76599-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7655825PMC
November 2020

A map of tumor-host interactions in glioma at single-cell resolution.

Gigascience 2020 10;9(10)

Department of Electrical Engineering and Information Technology (DIETI), University of Naples "Federico II", Via Claudio 21, 80128 Naples, Italy.

Background: Single-cell RNA sequencing is the reference technique for characterizing the heterogeneity of the tumor microenvironment. The composition of the various cell types making up the microenvironment can significantly affect the way in which the immune system activates cancer rejection mechanisms. Understanding the cross-talk signals between immune cells and cancer cells is of fundamental importance for the identification of immuno-oncology therapeutic targets.

Results: We present a novel method, single-cell Tumor-Host Interaction tool (scTHI), to identify significantly activated ligand-receptor interactions across clusters of cells from single-cell RNA sequencing data. We apply our approach to uncover the ligand-receptor interactions in glioma using 6 publicly available human glioma datasets encompassing 57,060 gene expression profiles from 71 patients. By leveraging this large-scale collection we show that unexpected cross-talk partners are highly conserved across different datasets in the majority of the tumor samples. This suggests that shared cross-talk mechanisms exist in glioma.

Conclusions: Our results provide a complete map of the active tumor-host interaction pairs in glioma that can be therapeutically exploited to reduce the immunosuppressive action of the microenvironment in brain tumor.
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http://dx.doi.org/10.1093/gigascience/giaa109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7645027PMC
October 2020

Analysis of respiratory and systemic immune responses in COVID-19 reveals mechanisms of disease pathogenesis.

medRxiv 2020 Oct 18. Epub 2020 Oct 18.

Immune responses to respiratory viruses like SARS-CoV-2 originate and function in the lung, yet assessments of human immunity are often limited to blood. Here, we conducted longitudinal, high-dimensional profiling of paired airway and blood samples from patients with severe COVID-19, revealing immune processes in the respiratory tract linked to disease pathogenesis. Survival from severe disease was associated with increased CD4 T cells and decreased monocyte/macrophage frequencies in the airway, but not in blood. Airway T cells and macrophages exhibited tissue-resident phenotypes and activation signatures, including high level expression and secretion of monocyte chemoattractants CCL2 and CCL3 by airway macrophages. By contrast, monocytes in blood expressed the CCL2-receptor CCR2 and aberrant CD163 and immature phenotypes. Extensive accumulation of CD163 monocyte/macrophages within alveolar spaces in COVID-19 lung autopsies suggested recruitment from circulation. Our findings provide evidence that COVID-19 pathogenesis is driven by respiratory immunity, and rationale for site-specific treatment and prevention strategies.
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http://dx.doi.org/10.1101/2020.10.15.20208041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7587837PMC
October 2020

Single-Cell Genomics Reveals a Novel Cell State During Smooth Muscle Cell Phenotypic Switching and Potential Therapeutic Targets for Atherosclerosis in Mouse and Human.

Circulation 2020 11 23;142(21):2060-2075. Epub 2020 Sep 23.

Division of Cardiology, Department of Medicine (H.P., C.X., A.C.B., J.C., D.Y.Y., S.B.T., W.L., J.S., C.O.I., H.Z., M.P.R.), Columbia University Irving Medical Center, New York.

Background: Smooth muscle cells (SMCs) play significant roles in atherosclerosis via phenotypic switching, a pathological process in which SMC dedifferentiation, migration, and transdifferentiation into other cell types. Yet how SMCs contribute to the pathophysiology of atherosclerosis remains elusive.

Methods: To reveal the trajectories of SMC transdifferentiation during atherosclerosis and to identify molecular targets for disease therapy, we combined SMC fate mapping and single-cell RNA sequencing of both mouse and human atherosclerotic plaques. We also performed cell biology experiments on isolated SMC-derived cells, conducted integrative human genomics, and used pharmacological studies targeting SMC-derived cells both in vivo and in vitro.

Results: We found that SMCs transitioned to an intermediate cell state during atherosclerosis, which was also found in human atherosclerotic plaques of carotid and coronary arteries. SMC-derived intermediate cells, termed "SEM" cells (stem cell, endothelial cell, monocyte), were multipotent and could differentiate into macrophage-like and fibrochondrocyte-like cells, as well as return toward the SMC phenotype. Retinoic acid (RA) signaling was identified as a regulator of SMC to SEM cell transition, and RA signaling was dysregulated in symptomatic human atherosclerosis. Human genomics revealed enrichment of genome-wide association study signals for coronary artery disease in RA signaling target gene loci and correlation between coronary artery disease risk alleles and repressed expression of these genes. Activation of RA signaling by all-trans RA, an anticancer drug for acute promyelocytic leukemia, blocked SMC transition to SEM cells, reduced atherosclerotic burden, and promoted fibrous cap stability.

Conclusions: Integration of cell-specific fate mapping, single-cell genomics, and human genetics adds novel insights into the complexity of SMC biology and reveals regulatory pathways for therapeutic targeting of SMC transitions in atherosclerotic cardiovascular disease.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.120.048378DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8104264PMC
November 2020

Elongation inhibitors do not prevent the release of puromycylated nascent polypeptide chains from ribosomes.

Elife 2020 08 26;9. Epub 2020 Aug 26.

Department of Systems Biology, Columbia University Irving Medical Center, New York, United States.

Puromycin is an amino-acyl transfer RNA analog widely employed in studies of protein synthesis. Since puromycin is covalently incorporated into nascent polypeptide chains, anti-puromycin immunofluorescence enables visualization of nascent protein synthesis. A common assumption in studies of local messenger RNA translation is that the anti-puromycin staining of puromycylated nascent polypeptides in fixed cells accurately reports on their original site of translation, particularly when ribosomes are stalled with elongation inhibitors prior to puromycin treatment. However, when we attempted to implement a proximity ligation assay to detect ribosome-puromycin complexes, we found no evidence to support this assumption. We further demonstrated, using biochemical assays and live cell imaging of nascent polypeptides in mammalian cells, that puromycylated nascent polypeptides rapidly dissociate from ribosomes even in the presence of elongation inhibitors. Our results suggest that attempts to define precise subcellular translation sites using anti-puromycin immunostaining may be confounded by release of puromycylated nascent polypeptide chains prior to fixation.
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http://dx.doi.org/10.7554/eLife.60048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7490010PMC
August 2020

Single-Cell Transcriptional Profiling of the Intestinal Epithelium.

Methods Mol Biol 2020 ;2171:129-153

Columbia Center for Human Development, Columbia Stem Cell Initiative, Division of Digestive & Liver Diseases, Department of Medicine, Columbia University Irving Medical Center, New York, NY, USA.

Emerging single-cell technologies, like single-cell RNA sequencing (scRNA-seq), enable the study of heterogeneous biological systems at cellular resolution. By profiling the set of expressed transcripts in each cell, single-cell transcriptomics has allowed for the cataloging of the cellular constituents of multiple organs and tissues, both in health and disease. In addition, these technologies have provided mechanistic insights into cellular function, cell state transitions, developmental trajectories and lineage relationships, as well as helped to dissect complex, population-level responses to environmental perturbations. scRNA-seq is particularly useful for characterizing the intestinal epithelium because it is a dynamic, rapidly self-renewing tissue comprised of more than a dozen specialized cell types. Here we discuss the fundamentals of single-cell transcriptomics of the murine small intestinal epithelium. We review the principles of proper experimental design and provide methods for the dissociation of the small intestinal epithelium into single cells followed by fluorescence-activated cell sorting (FACS) and for scRNA-seq using the 10× Genomics Chromium platform.
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http://dx.doi.org/10.1007/978-1-0716-0747-3_8DOI Listing
March 2021

Single-Cell Profiling and SCOPE-Seq Reveal Lineage Dynamics of Adult Ventricular-Subventricular Zone Neurogenesis and NOTUM as a Key Regulator.

Cell Rep 2020 06;31(12):107805

Department of Systems Biology, Columbia University Irving Medical Center, New York, NY 10032, USA; Department of Biochemistry & Molecular Biophysics, Columbia University Irving Medical Center, New York, NY 10032, USA. Electronic address:

In the adult ventricular-subventricular zone (V-SVZ), neural stem cells (NSCs) generate new olfactory bulb (OB) neurons and glia throughout life. To map adult neuronal lineage progression, we profiled >56,000 V-SVZ and OB cells by single-cell RNA sequencing (scRNA-seq). Our analyses reveal the molecular diversity of OB neurons, including fate-mapped neurons, lineage progression dynamics, and an NSC intermediate enriched for Notum, which encodes a secreted WNT antagonist. SCOPE-seq technology, which links live-cell imaging with scRNA-seq, uncovers cell-size transitions during NSC differentiation and preferential NOTUM binding to proliferating neuronal precursors. Consistently, application of NOTUM protein in slice cultures and pharmacological inhibition of NOTUM in slice cultures and in vivo demonstrated that NOTUM negatively regulates V-SVZ proliferation. Timely, context-dependent neurogenesis demands adaptive signaling among neighboring progenitors. Our findings highlight a critical regulatory state during NSC activation marked by NOTUM, which attenuates WNT-stimulated proliferation in NSC progeny.
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http://dx.doi.org/10.1016/j.celrep.2020.107805DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7396151PMC
June 2020

Gene expression analysis of the cerebellar cortex in essential tremor.

Neurosci Lett 2020 03 7;721:134540. Epub 2019 Nov 7.

Department of Pathology and Cell Biology, Columbia University Medical Center and the New York Presbyterian Hospital, 630 W 168th Street, P&S 15-405, New York, NY, USA; College of Physicians and Surgeons, Columbia University Medical Center and the New York Presbyterian Hospital, 630 W 168th Street, New York, NY, USA. Electronic address:

Essential tremor (ET) is one of the most common neurological diseases, with a central feature of an 8-12 Hz kinetic tremor. While previous postmortem studies have identified a cluster of morphological changes in the ET cerebellum centered in/around the Purkinje cell (PC) population, including a loss of PCs in some studies, the underlying molecular mechanisms for these changes are not clear. As genomic studies of ET patients have yet to identify major genetic contributors and animal models that fully recapitulate the human disease do not yet exist, the study of human tissue is currently the most applicable method to gain a mechanistic insight into ET disease pathogenesis. To begin exploration of an underlying molecular source of ET disease pathogenesis, we have performed the first transcriptomic analysis by direct sequencing of RNA from frozen cerebellar cortex tissue in 33 ET patients compared to 21 normal controls. Principal component analysis showed a heterogenous distribution of the expression data in ET patients that only partially overlapped with control patients. Differential expression analysis identified 231 differentially expressed gene transcripts ('top gene hits'), a subset of which has defined expression profiles in the cerebellum across neuronal and glial cell types but a largely unknown relationship to cerebellar function and/or ET pathogenesis. Gene set enrichment analysis (GSEA) identified dysregulated pathways of interest and stratified dysregulation among ET cases. By GSEA and mining curated databases, we compiled major categories of dysregulated processes and clustered string networks of known interacting proteins. Here we demonstrate that these 'top gene hits' contribute to regulation of four main biological processes, which are 1) axon guidance, 2) microtubule motor activity, 3) endoplasmic reticulum (ER) to Golgi transport and 4) calcium signaling/synaptic transmission. The results of our transcriptomic analysis suggest there is a range of different processes involved among ET cases, and draws attention to a particular set of genes and regulatory pathways that provide an initial platform to further explore the underlying biology of ET.
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http://dx.doi.org/10.1016/j.neulet.2019.134540DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7593093PMC
March 2020

Single-cell transcriptomics of human T cells reveals tissue and activation signatures in health and disease.

Nat Commun 2019 10 17;10(1):4706. Epub 2019 Oct 17.

Department of Systems Biology, Columbia University Irving Medical Center, New York, NY, USA.

Human T cells coordinate adaptive immunity in diverse anatomic compartments through production of cytokines and effector molecules, but it is unclear how tissue site influences T cell persistence and function. Here, we use single cell RNA-sequencing (scRNA-seq) to define the heterogeneity of human T cells isolated from lungs, lymph nodes, bone marrow and blood, and their functional responses following stimulation. Through analysis of >50,000 resting and activated T cells, we reveal tissue T cell signatures in mucosal and lymphoid sites, and lineage-specific activation states across all sites including distinct effector states for CD8 T cells and an interferon-response state for CD4 T cells. Comparing scRNA-seq profiles of tumor-associated T cells to our dataset reveals predominant activated CD8 compared to CD4 T cell states within multiple tumor types. Our results therefore establish a high dimensional reference map of human T cell activation in health for analyzing T cells in disease.
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http://dx.doi.org/10.1038/s41467-019-12464-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6797728PMC
October 2019

Spatial metagenomic characterization of microbial biogeography in the gut.

Nat Biotechnol 2019 08 22;37(8):877-883. Epub 2019 Jul 22.

Department of Systems Biology, Columbia University Medical Center, New York, NY, USA.

Spatial structuring is important for the maintenance of natural ecological systems. Many microbial communities, including the gut microbiome, display intricate spatial organization. Mapping the biogeography of bacteria can shed light on interactions that underlie community functions, but existing methods cannot accommodate the hundreds of species that are found in natural microbiomes. Here we describe metagenomic plot sampling by sequencing (MaPS-seq), a culture-independent method to characterize the spatial organization of a microbiome at micrometer-scale resolution. Intact microbiome samples are immobilized in a gel matrix and cryofractured into particles. Neighboring microbial taxa in the particles are then identified by droplet-based encapsulation, barcoded 16S rRNA amplification and deep sequencing. Analysis of three regions of the mouse intestine revealed heterogeneous microbial distributions with positive and negative co-associations between specific taxa. We identified robust associations between Bacteroidales taxa in all gut compartments and showed that phylogenetically clustered local regions of bacteria were associated with a dietary perturbation. Spatial metagenomics could be used to study microbial biogeography in complex habitats.
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http://dx.doi.org/10.1038/s41587-019-0183-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6679743PMC
August 2019

Contextualizing the pathology in the essential tremor cerebellar cortex: a patholog-omics approach.

Acta Neuropathol 2019 11 17;138(5):859-876. Epub 2019 Jul 17.

Department of Pathology and Cell Biology, Columbia University Irving Medical Center and the New York Presbyterian Hospital, 622 West 168th Street, PH Stem 1564, New York, NY, 10032, USA.

Several morphological changes, centered in/around Purkinje cells (PCs), have been identified in the cerebellum of essential tremor (ET) patients. These changes have not been contextualized within a broader degenerative disease spectrum, limiting their interpretability. To address this, we compared the severity and patterning of degenerative changes within the cerebellar cortex in patients with ET, other neurodegenerative disorders of the cerebellum (spinocerebellar ataxias (SCAs), multiple system atrophy (MSA)], and other disorders that may involve the cerebellum [Parkinson's disease (PD), dystonia]. Using a postmortem series of 156 brains [50 ET, 23 SCA (6 SCA3; 17 SCA 1, 2 or 6), 15 MSA, 29 PD, 14 dystonia, 25 controls], we generated data on 37 quantitative morphologic metrics, which were grouped into 8 broad categories: (1) PC loss, (2) heterotopic PCs, (3) PC dendritic changes, (4) PC axonal changes (torpedoes), (5) PC axonal changes (other than torpedoes), (6) PC axonal changes (torpedo-associated), (7) basket cell axonal hypertrophy, (8) climbing fiber-PC synaptic changes. Our analyses used z scored raw data for each metric across all diagnoses (5772 total data items). Principal component analysis revealed that diagnostic groups were not uniform with respect to cerebellar pathology. Dystonia and PD each differed from controls in only 2/37 metrics, whereas ET differed in 21, SCA3 in 8, MSA in 19, and SCA1/2/6 in 26 metrics. Comparing ET with primary disorders of cerebellar degeneration (i.e., SCAs), we observed a spectrum of changes reflecting differences of degree, being generally mild in ET and SCA3 and more severe in SCA1/2/6. Comparative analyses across morphologic categories demonstrated differences in relative expression, defining distinctive patterns of changes in these groups. Thus, the degree of cerebellar degeneration in ET aligns it with a milder end in the spectrum of cerebellar degenerative disorders, and a somewhat distinctive signature of degenerative changes marks each of these disorders.
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http://dx.doi.org/10.1007/s00401-019-02043-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7285399PMC
November 2019

Dissecting lung development and fibrosis at single-cell resolution.

Genome Med 2019 05 24;11(1):33. Epub 2019 May 24.

Department of Systems Biology, Columbia University Irving Medical Center, New York, NY, 10032, USA.

Single-cell transcriptome profiling has enabled high-resolution analysis of cellular populations in tissues during development, health, and disease. Recent studies make innovative use of single-cell RNA sequencing (scRNAseq) to investigate mechanisms that allow immune cells to interact with tissue components in the lung during development and fibrotic lung disease.
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http://dx.doi.org/10.1186/s13073-019-0645-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6534905PMC
May 2019

Critical Analysis of Particle Detection Artifacts in Synaptosome Flow Cytometry.

eNeuro 2019 May/Jun;6(3). Epub 2019 Jun 12.

Department of Systems Biology, Columbia University Irving Medical Center, New York, NY 10032

Flow cytometry and fluorescence-activated sorting are powerful techniques that hold great promise for studying heterogeneous populations of submicron particles such as synaptosomes, but many technical challenges arise in these experiments. To date, most flow cytometry studies of synaptosomes have relied on particle detection using forward scatter (FSC) measurements and size estimation with polystyrene (PS) bead standards. However, these practices have serious limitations, and special care must be taken to overcome the poor sensitivity of conventional flow cytometers in the analysis of submicron particles. Technical artifacts can confound these experiments, especially the detection of multiple particles as a single event. Here, we compared analysis of P2 crude synaptosomal preparations from murine forebrain on multiple flow cytometers using both FSC-triggered and fluorescence-triggered detection. We implemented multicolor fluorescent dye-based assays to quantify coincident particle detection and aggregation, and we assessed the false colocalization of antigens in immunostaining analyses. Our results demonstrate that fluorescence triggering and proper dilution can control for coincident particle detection, but not particle aggregation. We confirmed previous studies showing that FSC-based size estimation with PS beads underestimates biological particle size, and we identified pervasive aggregation in the FSC range analyzed in most synaptosome flow cytometry studies. We found that analyzing P2 samples in sucrose/EDTA/tris (SET) buffer reduces aggregation compared to PBS, but does not completely eliminate the presence of aggregates, especially in immunostaining experiments. Our study highlights challenges and pitfalls in synaptosome flow cytometry and provides a methodological framework for future studies.
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http://dx.doi.org/10.1523/ENEURO.0009-19.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6565374PMC
March 2020

Widespread Alterations in Translation Elongation in the Brain of Juvenile Fmr1 Knockout Mice.

Cell Rep 2019 03;26(12):3313-3322.e5

Department of Systems Biology, Columbia University Medical Center, New York, NY 10032, USA; Department of Biochemistry & Molecular Biophysics, Columbia University Medical Center, New York, NY 10032, USA; Sulzberger Columbia Genome Center, Columbia University Medical Center, New York, NY 10032, USA. Electronic address:

FMRP (fragile X mental retardation protein) is a polysome-associated RNA-binding protein encoded by Fmr1 that is lost in fragile X syndrome. Increasing evidence suggests that FMRP regulates both translation initiation and elongation, but the gene specificity of these effects is unclear. To elucidate the impact of Fmr1 loss on translation, we utilize ribosome profiling for genome-wide measurements of ribosomal occupancy and positioning in the cortex of 24-day-old Fmr1 knockout mice. We find a remarkably coherent reduction in ribosome footprint abundance per mRNA for previously identified, high-affinity mRNA binding partners of FMRP and an increase for terminal oligopyrimidine (TOP) motif-containing genes canonically controlled by mammalian target of rapamycin-eIF4E-binding protein-eIF4E binding protein-eukaryotic initiation factor 4E (mTOR-4E-BP-eIF4E) signaling. Amino acid motif- and gene-level analyses both show a widespread reduction of translational pausing in Fmr1 knockout mice. Our findings are consistent with a model of FMRP-mediated regulation of both translation initiation through eIF4E and elongation that is disrupted in fragile X syndrome.
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http://dx.doi.org/10.1016/j.celrep.2019.02.086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6457272PMC
March 2019

Generation and persistence of human tissue-resident memory T cells in lung transplantation.

Sci Immunol 2019 03;4(33)

Columbia Center for Translational Immunology, Columbia University Medical Center, New York, NY 10032, USA.

Tissue-resident memory T cells (T) maintain immunity in diverse sites as determined in mouse models, whereas their establishment and role in human tissues have been difficult to assess. Here, we investigated human lung T generation, maintenance, and function in airway samples obtained longitudinally from human leukocyte antigen (HLA)-disparate lung transplant recipients, where donor and recipient T cells could be localized and tracked over time. Donor T cells persist specifically in the lungs (and not blood) of transplant recipients and express high levels of T signature markers including CD69, CD103, and CD49a, whereas lung-infiltrating recipient T cells gradually acquire T phenotypes over months in vivo. Single-cell transcriptome profiling of airway T cells reveals that donor T cells comprise two T-like subsets with varying levels of expression of T-associated genes, whereas recipient T cells comprised non-T and similar T-like subpopulations, suggesting de novo T generation. Transplant recipients exhibiting higher frequencies of persisting donor T experienced fewer adverse clinical events such as primary graft dysfunction and acute cellular rejection compared with recipients with low donor T persistence, suggesting that monitoring T dynamics could be clinically informative. Together, our results provide spatial and temporal insights into how human T develop, function, persist, and affect tissue integrity within the complexities of lung transplantation.
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http://dx.doi.org/10.1126/sciimmunol.aav5581DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6435356PMC
March 2019

gene signature identification from single-cell RNA-seq with hierarchical Poisson factorization.

Mol Syst Biol 2019 02 22;15(2):e8557. Epub 2019 Feb 22.

Department of Systems Biology, Columbia University Irving Medical Center, New York, NY, USA

Common approaches to gene signature discovery in single-cell RNA-sequencing (scRNA-seq) depend upon predefined structures like clusters or pseudo-temporal order, require prior normalization, or do not account for the sparsity of single-cell data. We present single-cell hierarchical Poisson factorization (scHPF), a Bayesian factorization method that adapts hierarchical Poisson factorization (Gopalan , 2015, , 326) for discovery of both continuous and discrete expression patterns from scRNA-seq. scHPF does not require prior normalization and captures statistical properties of single-cell data better than other methods in benchmark datasets. Applied to scRNA-seq of the core and margin of a high-grade glioma, scHPF uncovers marked differences in the abundance of glioma subpopulations across tumor regions and regionally associated expression biases within glioma subpopulations. scHFP revealed an expression signature that was spatially biased toward the glioma-infiltrated margins and associated with inferior survival in glioblastoma.
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http://dx.doi.org/10.15252/msb.20188557DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6386217PMC
February 2019

Single-Cell Analysis of Regional Differences in Adult V-SVZ Neural Stem Cell Lineages.

Cell Rep 2019 01;26(2):394-406.e5

Biozentrum, University of Basel, 4056 Basel, Switzerland. Electronic address:

The ventricular-subventricular zone (V-SVZ) harbors adult neural stem cells. V-SVZ neural stem cells exhibit features of astrocytes, have a regional identity, and depending on their location in the lateral or septal wall of the lateral ventricle, generate different types of neuronal and glial progeny. We performed large-scale single-cell RNA sequencing to provide a molecular atlas of cells from the lateral and septal adult V-SVZ of male and female mice. This revealed regional and sex differences among adult V-SVZ cells. We uncovered lineage potency bias at the single-cell level among lateral and septal wall astrocytes toward neurogenesis and oligodendrogenesis, respectively. Finally, we identified transcription factor co-expression modules marking key temporal steps in neurogenic and oligodendrocyte lineage progression. Our data suggest functionally important spatial diversity in neurogenesis and oligodendrogenesis in the adult brain and reveal molecular correlates of adult NSC dormancy and lineage specialization.
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http://dx.doi.org/10.1016/j.celrep.2018.12.044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6368857PMC
January 2019

SCOPE-Seq: a scalable technology for linking live cell imaging and single-cell RNA sequencing.

Genome Biol 2018 12 24;19(1):227. Epub 2018 Dec 24.

Department of Systems Biology, Columbia University Irving Medical Center, New York, NY, 10032, USA.

Optically decodable beads link the identity of a sample to a measurement through an optical barcode, enabling libraries of biomolecules to be captured on beads in solution and decoded by fluorescence. This approach has been foundational to microarray, sequencing, and flow-based expression profiling technologies. We combine microfluidics with optically decodable beads and show that phenotypic analysis of living cells can be linked to single-cell sequencing. As a proof-of-concept, we demonstrate the accuracy and scalability of our tool called Single Cell Optical Phenotyping and Expression sequencing (SCOPE-Seq) to combine live cell imaging with single-cell RNA sequencing.
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http://dx.doi.org/10.1186/s13059-018-1607-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6305572PMC
December 2018

Cardiac recovery via extended cell-free delivery of extracellular vesicles secreted by cardiomyocytes derived from induced pluripotent stem cells.

Nat Biomed Eng 2018 May 23;2(5):293-303. Epub 2018 Apr 23.

Department of Medicine, Columbia University, New York, NY, USA.

The ability of extracellular vesicles (EVs) to regulate a broad range of cellular processes has recently been exploited for the treatment of diseases. For example, EVs secreted by stem cells injected into infarcted hearts can induce recovery through the delivery of stem-cell-specific miRNAs. However, the retention of the EVs and the therapeutic effects are short-lived. Here, we show that an engineered hydrogel patch capable of slowly releasing EVs secreted from cardiomyocytes derived from induced pluripotent stem (iPS) cells reduced arrhythmic burden, promoted ejection-fraction recovery, decreased cardiomyocyte apoptosis 24 hours after infarction, and reduced infarct size and cell hypertrophy 4 weeks post-infarction when implanted onto infarcted rat hearts. We also show that the EVs are enriched with cardiac-specific miRNAs known to modulate cardiomyocyte-specific processes. The extended delivery of EVs secreted from iPS-cell-derived cardiomyocytes into the heart may help understand heart recovery and treat heart injury.
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http://dx.doi.org/10.1038/s41551-018-0229-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6159913PMC
May 2018

Single-cell transcriptome analysis of lineage diversity in high-grade glioma.

Genome Med 2018 07 24;10(1):57. Epub 2018 Jul 24.

Department of Systems Biology, Columbia University Medical Center, New York, NY, 10032, USA.

Background: Despite extensive molecular characterization, we lack a comprehensive understanding of lineage identity, differentiation, and proliferation in high-grade gliomas (HGGs).

Methods: We sampled the cellular milieu of HGGs by profiling dissociated human surgical specimens with a high-density microwell system for massively parallel single-cell RNA-Seq. We analyzed the resulting profiles to identify subpopulations of both HGG and microenvironmental cells and applied graph-based methods to infer structural features of the malignantly transformed populations.

Results: While HGG cells can resemble glia or even immature neurons and form branched lineage structures, mesenchymal transformation results in unstructured populations. Glioma cells in a subset of mesenchymal tumors lose their neural lineage identity, express inflammatory genes, and co-exist with marked myeloid infiltration, reminiscent of molecular interactions between glioma and immune cells established in animal models. Additionally, we discovered a tight coupling between lineage resemblance and proliferation among malignantly transformed cells. Glioma cells that resemble oligodendrocyte progenitors, which proliferate in the brain, are often found in the cell cycle. Conversely, glioma cells that resemble astrocytes, neuroblasts, and oligodendrocytes, which are non-proliferative in the brain, are generally non-cycling in tumors.

Conclusions: These studies reveal a relationship between cellular identity and proliferation in HGG and distinct population structures that reflects the extent of neural and non-neural lineage resemblance among malignantly transformed cells.
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http://dx.doi.org/10.1186/s13073-018-0567-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6058390PMC
July 2018
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