Single‐cell transcriptomics reveals immune response of intestinal cell types to viral infection
2021; Springer Nature; Volume: 17; Issue: 7 Linguagem: Inglês
10.15252/msb.20209833
ISSN1744-4292
AutoresSergio Triana, Megan L. Stanifer, Camila Metz‐Zumaran, Mohammed Shahraz, Markus Mukenhirn, Carmon Kee, Clara Serger, Ronald Koschny, Diana Ordoñez‐Rueda, Malte Paulsen, Vladimı́r Beneš, Steeve Boulant, Theodore Alexandrov,
Tópico(s)IL-33, ST2, and ILC Pathways
ResumoArticle26 July 2021Open Access Source DataTransparent process Single-cell transcriptomics reveals immune response of intestinal cell types to viral infection Sergio Triana Sergio Triana orcid.org/0000-0003-0370-7821 Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany Faculty of Biosciences, Collaboration for Joint PhD degree between EMBL and Heidelberg University, Heidelberg, Germany These authors contributed equally to this work Search for more papers by this author Megan L Stanifer Megan L Stanifer orcid.org/0000-0002-5606-1297 Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany Research Group "Cellular Polarity and Viral Infection", German Cancer Research Center (DKFZ), Heidelberg, Germany These authors contributed equally to this work Search for more papers by this author Camila Metz-Zumaran Camila Metz-Zumaran orcid.org/0000-0002-6981-0728 Department of Infectious Diseases, Virology, Heidelberg University, Heidelberg, Germany Search for more papers by this author Mohammed Shahraz Mohammed Shahraz Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany Search for more papers by this author Markus Mukenhirn Markus Mukenhirn Department of Infectious Diseases, Virology, Heidelberg University, Heidelberg, Germany Search for more papers by this author Carmon Kee Carmon Kee orcid.org/0000-0001-5847-5509 Research Group "Cellular Polarity and Viral Infection", German Cancer Research Center (DKFZ), Heidelberg, Germany Department of Infectious Diseases, Virology, Heidelberg University, Heidelberg, Germany Search for more papers by this author Clara Serger Clara Serger Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany Search for more papers by this author Ronald Koschny Ronald Koschny Department of Internal Medicine IV, Interdisciplinary Endoscopy Center, University Hospital Heidelberg, Heidelberg, Germany Search for more papers by this author Diana Ordoñez-Rueda Diana Ordoñez-Rueda Flow Cytometry Core Facility, European Molecular Biology Laboratory, Heidelberg, Germany Search for more papers by this author Malte Paulsen Malte Paulsen Flow Cytometry Core Facility, European Molecular Biology Laboratory, Heidelberg, Germany Search for more papers by this author Vladimir Benes Vladimir Benes Genomics Core Facility, European Molecular Biology Laboratory, Heidelberg, Germany Search for more papers by this author Steeve Boulant Corresponding Author Steeve Boulant [email protected] orcid.org/0000-0001-8614-4993 Research Group "Cellular Polarity and Viral Infection", German Cancer Research Center (DKFZ), Heidelberg, Germany Department of Infectious Diseases, Virology, Heidelberg University, Heidelberg, Germany Search for more papers by this author Theodore Alexandrov Corresponding Author Theodore Alexandrov [email protected] orcid.org/0000-0001-9464-6125 Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany Molecular Medicine Partnership Unit, European Molecular Biology Laboratory, Heidelberg, Germany Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, CA, USA Search for more papers by this author Sergio Triana Sergio Triana orcid.org/0000-0003-0370-7821 Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany Faculty of Biosciences, Collaboration for Joint PhD degree between EMBL and Heidelberg University, Heidelberg, Germany These authors contributed equally to this work Search for more papers by this author Megan L Stanifer Megan L Stanifer orcid.org/0000-0002-5606-1297 Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany Research Group "Cellular Polarity and Viral Infection", German Cancer Research Center (DKFZ), Heidelberg, Germany These authors contributed equally to this work Search for more papers by this author Camila Metz-Zumaran Camila Metz-Zumaran orcid.org/0000-0002-6981-0728 Department of Infectious Diseases, Virology, Heidelberg University, Heidelberg, Germany Search for more papers by this author Mohammed Shahraz Mohammed Shahraz Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany Search for more papers by this author Markus Mukenhirn Markus Mukenhirn Department of Infectious Diseases, Virology, Heidelberg University, Heidelberg, Germany Search for more papers by this author Carmon Kee Carmon Kee orcid.org/0000-0001-5847-5509 Research Group "Cellular Polarity and Viral Infection", German Cancer Research Center (DKFZ), Heidelberg, Germany Department of Infectious Diseases, Virology, Heidelberg University, Heidelberg, Germany Search for more papers by this author Clara Serger Clara Serger Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany Search for more papers by this author Ronald Koschny Ronald Koschny Department of Internal Medicine IV, Interdisciplinary Endoscopy Center, University Hospital Heidelberg, Heidelberg, Germany Search for more papers by this author Diana Ordoñez-Rueda Diana Ordoñez-Rueda Flow Cytometry Core Facility, European Molecular Biology Laboratory, Heidelberg, Germany Search for more papers by this author Malte Paulsen Malte Paulsen Flow Cytometry Core Facility, European Molecular Biology Laboratory, Heidelberg, Germany Search for more papers by this author Vladimir Benes Vladimir Benes Genomics Core Facility, European Molecular Biology Laboratory, Heidelberg, Germany Search for more papers by this author Steeve Boulant Corresponding Author Steeve Boulant [email protected] orcid.org/0000-0001-8614-4993 Research Group "Cellular Polarity and Viral Infection", German Cancer Research Center (DKFZ), Heidelberg, Germany Department of Infectious Diseases, Virology, Heidelberg University, Heidelberg, Germany Search for more papers by this author Theodore Alexandrov Corresponding Author Theodore Alexandrov [email protected] orcid.org/0000-0001-9464-6125 Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany Molecular Medicine Partnership Unit, European Molecular Biology Laboratory, Heidelberg, Germany Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, CA, USA Search for more papers by this author Author Information Sergio Triana1,2, Megan L Stanifer3,4, Camila Metz-Zumaran5, Mohammed Shahraz1, Markus Mukenhirn5, Carmon Kee4,5, Clara Serger1, Ronald Koschny6, Diana Ordoñez-Rueda7, Malte Paulsen7, Vladimir Benes8, Steeve Boulant *,4,5 and Theodore Alexandrov *,1,9,10 1Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany 2Faculty of Biosciences, Collaboration for Joint PhD degree between EMBL and Heidelberg University, Heidelberg, Germany 3Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany 4Research Group "Cellular Polarity and Viral Infection", German Cancer Research Center (DKFZ), Heidelberg, Germany 5Department of Infectious Diseases, Virology, Heidelberg University, Heidelberg, Germany 6Department of Internal Medicine IV, Interdisciplinary Endoscopy Center, University Hospital Heidelberg, Heidelberg, Germany 7Flow Cytometry Core Facility, European Molecular Biology Laboratory, Heidelberg, Germany 8Genomics Core Facility, European Molecular Biology Laboratory, Heidelberg, Germany 9Molecular Medicine Partnership Unit, European Molecular Biology Laboratory, Heidelberg, Germany 10Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, CA, USA **Corresponding author. Tel: +49 6221 56 7865; E-mail: [email protected] ***Corresponding author. Tel: +49 6221 387 8690; E-mail: [email protected] Molecular Systems Biology (2021)17:e9833https://doi.org/10.15252/msb.20209833 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Human intestinal epithelial cells form a primary barrier protecting us from pathogens, yet only limited knowledge is available about individual contribution of each cell type to mounting an immune response against infection. Here, we developed a framework combining single-cell RNA-Seq and highly multiplex RNA FISH and applied it to human intestinal organoids infected with human astrovirus, a model human enteric virus. We found that interferon controls the infection and that astrovirus infects all major cell types and lineages and induces expression of the cell proliferation marker MKI67. Intriguingly, each intestinal epithelial cell lineage exhibits a unique basal expression of interferon-stimulated genes and, upon astrovirus infection, undergoes an antiviral transcriptional reprogramming by upregulating distinct sets of interferon-stimulated genes. These findings suggest that in the human intestinal epithelium, each cell lineage plays a unique role in resolving virus infection. Our framework is applicable to other organoids and viruses, opening new avenues to unravel roles of individual cell types in viral pathogenesis. SYNOPSIS Single-cell sequencing and multiplex single-molecule RNA FISH analyses of human astrovirus 1 (HAstV1)-infected human intestinal organoids characterize viral tropism and unravel the cell lineage-specific immune response to viral infection. A single-cell RNA-Seq reference dataset of human ileum biopsies is established. An integrative framework to investigate cell-type-specific viral pathogenesis in a tissue-like environment is developed. HAstV1 infects all lineages from the human intestinal epithelium, causing an interferon-mediated immune response. HAstV1 evokes a cell lineage-specific intrinsic immune response. Each intestinal cell lineage has a different steady expression of interferon-stimulated genes (ISGs). Introduction The small intestine is responsible for most nutrient absorption in humans. It is composed of various cell types each performing specific functions contributing to homeostasis (Peterson & Artis, 2014). The main cell types found in the intestinal epithelium are the absorptive enterocytes, the mucus-secreting goblet cells, the hormone-producing enteroendocrine cells, the antimicrobial peptide secreting Paneth cells and the stem cells. Due to the constant challenges present in the lumen of the gut, intestinal epithelial cells are turned over every 5 days. This constant self-renewal is organized along the crypt-villus axis and is supported by the stem cells located in the crypts, themselves supported by interlaying Paneth cells (Kretzschmar & Clevers, 2016). Differentiation of the stem cells to the various intestinal cell lineages requires a Notch/Wnt-dependent bifurcation toward either absorptive or secretory progenitor cells. Absorptive progenitor cells give rise to the enterocyte cells while the secretory progenitors differentiate into enteroendocrine, goblet, tuft, or Paneth cells (Sancho et al, 2015; Gehart & Clevers, 2019). Over the past 10 years, intestinal organoids have been developed and have emerged as the best surrogate model that mimics the differentiation and function of the intestinal epithelium (Sato et al, 2009, 2011). Human intestinal epithelial cells (hIECs) play key roles in protecting us from environmental pathogen- and commensal-related challenges. They act as a first-layer physical barrier of the host defense and mount response upon infection (Martens et al, 2018). Humans are exposed to enteric viruses through contaminated food and water sources as well as through direct fecal–oral transmission from infected patients (Tatte & Gopalkrishna, 2019). People in developing countries are at high risk due to the discharge of untreated waste into the environment and lack of medical care after the onset of infection. Diarrheal diseases kill more than 1.5 million people each year worldwide (Tatte & Gopalkrishna, 2019). Depending on the type of virus, infection can lead to gastroenteritis, vomiting, and/or watery diarrhea due to leakage of the intestinal lining. Human astrovirus 1 (HAstV1) is a small non-enveloped, positive-strand RNA virus which is found worldwide (Appleton & Higgins, 1975). These viruses cause a range of symptoms from asymptomatic infections to diarrhea to encephalitis. HAstV1 infections lead to gastroenteritis and account for 2–9% of non-bacterial diarrhea in children (Cortez et al, 2017). Most children are exposed to the virus, and by the age of 5 years old, 90% of children have serum antibodies against astrovirus (Walter & Mitchell, 2003; Cortez et al, 2017). Human astrovirus infections are often not identified due to lack of diagnostic methods to detect all circulating strains. Work using murine astrovirus in a mouse model of infection has used single-cell approaches to reveal the tropism of murine astrovirus (Cortez et al, 2020). It was found that this virus favors goblet cells in the gastrointestinal tract of mice. Interestingly, work using the neurotropic human astrovirus VA1 strain and human intestinal organoids has shown that VA1 has a broader tropism and can potentially infect all major cell types in the gastrointestinal tract (Kolawole et al, 2019). However, how individual cell types respond to astrovirus infection was not investigated. However, only limited knowledge is available about viral pathogenesis in the intestinal epithelium in the context of the cell types, in particular how different human intestinal epithelial cell (hIEC) types contribute to the immune response and clearance of the viral infection. This gap of knowledge is caused by the challenges associated with reproducing the multi-cellular complexity of the human intestinal epithelium. This leads to the current situation where for a majority of enteric viruses, essential questions of viral pathogenesis have been mostly addressed in immortalized cell lines, a model with a limited capacity to reproduce intestinal epithelium in its multi-cellular complexity and missing key phenomena such as cell differentiation. Interestingly, increasing evidence suggests that stem cells are intrinsically resistant to viral infection (Wolf & Goff, 2009; Belzile et al, 2014). It was recently shown that pluripotent and multipotent stem cells exhibit intrinsic expression of interferon-stimulated genes (ISGs) in an interferon-independent manner. This basal expression of ISGs has been proposed to be responsible for the resistance of stem cells to viral infection (Wu et al, 2018). Upon differentiation, stem cells lose expression of these intrinsic ISGs and become responsive to interferon (IFN) (Wu et al, 2018). These observations suggest that, at least in vitro, stem cells and differentiated cells use different strategies to fight viral infection. Whether such cell-type-specific antiviral strategies exist in-vivo where stem cells differentiate into different tissue-specific lineages remains unknown. Answering these questions requires using physiologically relevant ex vivo models as well as single-cell methods able to dissect the heterogeneity of host–pathogen interactions of different cell types and of individual cells within a population. Here, we established a framework to investigate cell-type-specific viral pathogenesis in a tissue-like environment. The framework integrates single-cell RNA sequencing and multiplex RNA in situ hybridization imaging of enteric virus-infected human ileum-derived organoids. In order to enable cell-type-specific analyzes in organoids, we have created a single-cell RNA-Seq reference dataset of human ileum biopsies. Applying this framework to organoids infected by human enteric virus astrovirus (HAstV1), we have characterized HAstV1 infection of various primary hIEC types and determined that HAstV1 is able to infect all intestinal cell types with a preference for proliferating cells. Single-cell transcriptomic analysis revealed a cell-type-specific transcriptional pattern of immune response in human intestinal organoids. We found that both at steady state and during viral infection, each intestinal cell type has unique expression profiles of interferon-stimulated genes creating a distinct antiviral environment. Results HAstV1 infects human intestinal epithelial cells causing IFN-mediated response To unravel how intestinal epithelium cells respond in a cell-type-specific manner, we investigated human ileum-derived organoids infected with the human pathogen HAstV1. To confirm the ability of HAstV1 to replicate in intestinal cells, we showed that HAstV1 is fully capable of infecting the transformed hIECs, Caco-2 cells, as can be seen by the increase in the number of infected cells and the increase in the amount of viral genome copy number over time (Fig EV1-EV5). Following infection with HAstV1, Caco-2 cells mounted an intrinsic immune response characterized by the production of both type I (IFNβ1) and type III (IFNλ) interferons (IFNs) (Fig EV1D). This IFN-mediated response constitutes an antiviral strategy by Caco-2 cells as a pre-treatment of cells with either IFN controlled HAstV1 infection (Fig EV1E and F). This is consistent with previous work reporting that IFN controls HAstV1 infection (Guix et al, 2015; Marvin et al, 2016). Complementarily, investigating another human intestinal epithelial cell type, T84, which is described to be more immunoresponsive (Stanifer et al, 2020a), we found it to be less infectable by HAstV1 unless the IFN-mediated signaling was suppressed by the loss of both the type I and III IFN receptors (dKO) (Fig EV1G). Together, these data confirm the function of IFNs in controlling HAstV1 infection in human intestinal epithelial cells and illustrate the importance of investigating how different cell types mount an interferon response to counteract viral infection. Click here to expand this figure. Figure EV1. Interferons protect human intestinal organoids from HastV1 infection Caco-2 cells were infected with HastV1. At indicated times, the virus was visualized by indirect immunofluorescence for HastV1 (red). Nuclei were stained with DAPI (blue). Scale bar 10 μm. Quantification of the number of HAstV1-infected cells from A. Caco-2 cells were infected with HAstV1. At indicated times, the replication of HAstV1 was assessed by the genome copy number over time using qRT–PCR. Caco-2 cells were infected with HAstV1. At indicated times, the intrinsic innate immune induction of type I (IFNβ1) and III (IFNλ) IFN was evaluated. Caco-2 cells were pre-treated for 24 h with 2,000 IU/ml of IFNβ1 or 300 ng/ml of IFNλ-3. Interferons were maintained during the course of infection and HastV1-infected cells were visualized with indirect immunofluorescence (left) and the number of HAStV1-infected cells was quantified (right). Scale bar 10 μm. Caco-2 cells were pre-treated for 24 h with 2,000 IU/ml of IFNβ1 or 300 ng/ml of IFNλ-3. Interferons were maintained during the course of infection and replication of HAstV1 was assessed by qRT–PCR for the genome copy number. Caco-2 cells, T84 wild-type, and T84 cells lacking both the type I and type III IFN receptors (dKO) were infected with HAstV1. 24 hpi HAstV1 genome replication was evaluated by qRT–PCR for the genome copy number. Data information: A–G Three biological replicates were performed for each experiment. Representative immunofluorescence images are shown. Error bars indicate the standard deviation. Statistics are from unpaired t-test. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. HAstV infection induces MKI67 expression in intestinal organoids Representative images showing multiplex in situ RNA FISH of the proliferation marker MKI67 (green) and HAstV1 infection (red). DAPI is in blue. Scale bar 200 µm. Fluorescence intensity (arbitrary units, a.u.) of the proliferation marker MKI67 plotted against fluorescence intensity (a.u.) of HAstV1 RNA probe in mock-treated organoids and at 4 hpi and 16 hpi. Each dot represents a single cell, infected cells are in red and bystander cells in blue. Line shows a linear regression fit with a 95% confidence interval. Fluorescence intensity (a.u.) of the proliferation marker MKI67 expression in stem cells (OLFM4 positive), goblet cells (FCGBP positive), enterocyte lineage cells (FABP6 positive), and mature enterocytes (APOA4-positive). Infected cells are in red and bystander cells in blue. Floating bar (min and max), line is the mean. Blue statistics show comparison of bystander cells between cell lineages, and red statistics show comparison of infected cells between cell lineages. Ordinary one-way ANOVA and Tukey's multiple comparisons test were used. Gray statistics show comparison between infected and bystander cells within each cell lineage. Unpaired t-test with Welch's correction was used. n.s nonsignificant, **P < 0.01, ***P < 0.001, ****P < 0.0001. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Lineage-specific expression changes in mock versus 4 hpi and 16 hpi Volcano plots of genes that are differentially expressed in cells in one time point relative to the other, showing the statistical significance (−log10 adjusted P-value) versus log2 fold change and gene enrichment analysis results for significantly changing genes (FDR < 0.05) A–D. Enterocyte lineages. E–H. Transit-amplifying (TA) cells. I–K. Enteroendocrine cells. Selected genes are shown, all genes are reported in Dataset EV3. Download figure Download PowerPoint Click here to expand this figure. Figure EV4. Lineage-specific expression changes in mock versus 4 hpi and 16 hpi Volcano plots of genes that are differentially expressed in cells in one time point relative to the other, showing the statistical significance (−log10 adjusted P-value) versus log2 fold change and gene enrichment analysis results for significantly changing genes (FDR < 0.05). A–D. Stem cells. E–H. Goblet cells I–K. Best4+ enterocytes. Selected genes are shown, all genes are reported in Dataset EV3. Download figure Download PowerPoint Click here to expand this figure. Figure EV5. Lineage-specific interferon patterns are conserved across conditions A–C. UMAP embedding of scRNA-Seq data from human ileum-derived organoids based on the significantly changing ISGs for cells at A. Mock-infected, B. 4 hpi and C. 16 hpi. D–F. Unsupervised clustering of the UMAP data from A–C. G–I. The distribution of cell lineages and types in the clusters from D–F. J–L. A heatmap of differentially expressed ISGs across the clusters from D–F. Download figure Download PowerPoint HAstV1 infects human ileum-derived organoids To investigate whether different cell types in the human intestinal epithelium respond to pathogen challenges by mounting a distinct IFN-mediated response, we exploited human intestinal organoids. Human intestinal organoids are an advanced primary cellular system recapitulating the cellular complexity, organization, and function of the human gut and enabling controlled investigations of enteric infection not feasible in the human tissue (Stanifer et al, 2020b). Intestinal organoids were prepared from stem cell-containing crypts isolated from human ileum resections (Sato et al, 2011). The structural integrity and cellular composition of the organoids were verified by immunofluorescence staining of adherens and tight junctions and markers of various intestinal epithelial cell types (Fig 1A) (Pervolaraki et al, 2017). These organoids were readily infectable by HAstV1 as can be seen by the detection of infected cells and efficient replication of the HAstV1 genome overtime (Fig 1B–E). Infection of organoids by HAstV1 induces a strong intrinsic immune response characterized by the production of both type I IFN (IFNβ1) and type III interferon (IFNλ) at the transcriptional and protein levels (Fig 1F and G). Interestingly, this response was significantly stronger than the one generated by the transformed hIEC lines (Fig EV1), further highlighting the importance of using primary organoids when characterizing host-pathogen interactions. Pre-treatment of organoids with either type I or type III IFN significantly reduced HAstV1 infection (Fig 1H). This strong antiviral activity of IFN against HAstV1 is consistent with the previous reports using both transformed cell lines and human organoids (Kolawole et al, 2019) and with the well-described function of IFNs in controlling enteric virus infection at the intestinal epithelium (Lee & Baldridge, 2017). Figure 1. Interferons protect human intestinal organoids from HAstV1 infection Cryo-sections of human organoids were analyzed for the presence of enterocytes (E-cad), Goblet cells (Muc-2), and tight junctions (ZO-1) by indirect immunofluorescence. Nuclei are stained with DAPI. Scale bar 25 μm. Human intestinal organoids were incubated with media (mock) or infected with HAstV1. 16 hpi organoids were frozen, cryo-sectioned, and HAstV1-infected cells were visualized by indirect immunofluorescence (HAstV1 (red), nuclei (DAPI, blue). Scale bar 25 μm. Human intestinal organoids were incubated with media (mock) or infected with HAstV1. Organoids at 16 hpi were fixed, and the presence of HAstV1-infected cells (green) was visualized by indirect immunofluorescence. Apical and basolateral membranes were immunostained for actin (magenta) and Laminin (red), respectively. Nuclei are stained with DAPI (blue). Scale bar is 20 μm. Quantification of C with the percentage of infected cells determined. Human intestinal organoids were infected with HAstV1. At indicated time post-infection, the increase in viral copy number was determined by qRT–PCR. Human intestinal organoids were incubated with media (mock) or infected with HAstV1. At 24 hpi, the presence of IFNλ in the media was tested by ELISA. Dotted line indicates detection limit of the assay. Same as E but for the induced levels of either type I IFN (IFNβ1) or type III IFN (IFNλ). Human intestinal organoids were pre-treated for 24 h with 2,000 IU/ml of IFNβ1 or 300 ng/ml of IFNλ1-3. Interferons were maintained during the course of infection and the amount of HAstV1 copy numbers was assayed 24 hpi by qRT–PCR. Data information: A-G Three biological replicates were performed for each experiment. Representative immunofluorescence images are shown. Error bars indicate the standard deviation. Statistics are from unpaired t-test. Download figure Download PowerPoint Single-cell RNA-Seq profiling of human ileum biopsies To characterize the response of intestinal organoids to enteric virus infection at the single-cell level, we exploited single-cell RNA sequencing (scRNA-Seq) by using a 10× Genomics platform (Zheng et al, 2017). Over the past years, scRNA-Seq emerged as a major approach to reveal cell types, lineages, and their transcriptional programs in tissues. Yet, applying scRNA-Seq to organoids is still challenging due to the available dissociation protocols optimized predominantly for tissues, as well as due to understudied differences between tissues and organoids. In light of these challenges, prior to analysis of ileum-derived organoids, we performed scRNA-Seq analysis of human ileum biopsies with the aim to create a cell-type-annotated reference single-cell dataset of human ileum. Additionally, to increase the resolution of the study, we included in our analysis the recently published scRNA-Seq data of ileum biopsies (Wang et al, 2020). Following scRNA-Seq of human ileum biopsies, we quality controlled our scRNA-Seq data (Appendix Fig S1A–I) and we performed data integration of each of our sample and the previously reported datasets (Wang et al, 2020) and unsupervised clustering on the resulting integrated space, that revealed the presence of multiple cell subpopulations (Fig 2A). We have identified the cell types represented in the subpopulations by finding subpopulation-specific markers using differential expression of each subpopulation against all other cells (Dataset EV1) and matching them to the known marker genes of the hIEC types (Fig 2B and Appendix Fig S2A). The majority (> 87.5%) of the cells isolated from the biopsies in this study corresponded to hIECs while a small fraction (12.4%) corresponded to stromal and immune cells (Fig 2A and Appendix Fig S2B–D). This integrated dataset represents a detailed reference scRNA-Seq dataset from the human ileum containing 14 cell types (Wang et al, 2020) which is especially important for annotating cell types represented in organoids. For all cell types represented in the integrated scRNA-Seq dataset from human ileum biopsies, we selected the most differentially expressed genes as markers (Fig 2B). The pseudotime analysis (Street et al, 2018) confirmed the expected bifurcation of differentiation of stem cells along two distinct trajectories toward either enterocytes (absorptive function) or goblet cells (secretory function) (Fig 2C). Each of these two lineages are characterized by specific gradients and waves of gene expression along the differentiation trajectories such as marker genes FCBP and APOA4 (Fig 2D) among others (Fig 2E). This single-cell reference dataset contains detailed information about transcriptomics profiles of intestinal epithelial cell types in the human ileum that, as we show in the next
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