The cellular composition of barrier epithelia is essential to organismal homoeostasis. In particu... more The cellular composition of barrier epithelia is essential to organismal homoeostasis. In particular, within the small intestine, adult stem cells establish tissue cellularity, and may provide a means to control the abundance and quality of specialized epithelial cells. Yet, methods for the identification of biological targets regulating epithelial composition and function, and of small molecules modulating them, are lacking. Here we show that druggable biological targets and small-molecule regulators of intestinal stem cell differentiation can be identified via multiplexed phenotypic screening using thousands of miniaturized organoid models of intestinal stem cell differentiation into Paneth cells, and validated via longitudinal single-cell RNA-sequencing. We found that inhibitors of the nuclear exporter Exportin 1 modulate the fate of intestinal stem cells, independently of known differentiation cues, significantly increasing the abundance of Paneth cells in the organoids and in w...
Figure S4. Signaling pathways and processes associated with in vitro PC enrichment (Additional fi... more Figure S4. Signaling pathways and processes associated with in vitro PC enrichment (Additional file 9: Table S5 for reference gene lists). A Violin plot of expression contribution to a cellâ s transcriptome of Wnt pathway genes (Additional file 2: Table S2; activated by CHIR99021) across clusters as percent of transcriptome. B Violin plot of expression contribution to a cellâ s transcriptome of Notch pathway genes (Additional file 2: Table S2; inhibited by DAPT) across clusters as percent of transcriptome. C Violin plot of expression contribution to a cellâ s transcriptome of respiratory electron transport gene set (Additional file 2: Table S2) across clusters as percent of transcriptome. (PDF 888 kb)
Figure S3. Quality metrics for single-cell RNA sequencing. A Total gene number of cells maintaine... more Figure S3. Quality metrics for single-cell RNA sequencing. A Total gene number of cells maintained in analyses with a lower cutoff of n = 400 unique genes per cell. Total unique molecular identifiers (UMIs) used as the basis for cell-by-gene tables collapsed to UMI as input into Seurat with the lower bound representing n = 400 unique genes and the upper bound representing 8000 UMIs. Note: Clusters ENR + CV-3, ENR + CV-4, and ENR-1 had significantly higher levels of genes and UMIs and, intriguingly, were also the three clusters with the highest levels of Lgr5 (see Fig. 5a), indicating that stem cells may contain larger contents of RNA, as they are in a biosynthetic state before differentiation and maturation. B Violin plot of expression contribution to a cell's transcriptome of mitochondrial and ribosomal genes across identified subsets. (PDF 1153 kb)
Figure S2. Proteomic pipeline, sample-to-sample comparison, and insights from the in vitro PC pro... more Figure S2. Proteomic pipeline, sample-to-sample comparison, and insights from the in vitro PC proteome. A Schematic of proteomic analysis for samples: culture, collection, lysis, reduction and alkylation, proteolytic digestion, labeling of peptides with isobaric mass tag reagents (Tandem Mass Tags, TMT10-plex; Thermo), off-line fractionation by basic reverse phase chromatography, analysis of fractions by LC-MS/MS, identification of peptides and proteins using Spectrum Mill software (Agilent), and statistical analysis of the resulting data (moderated t test) to identify confidently differential proteins. B Proteome sample correlation between all biological (n = 2) and technical (n = 2/biological) replicates. C ENR + CD-enriched proteins are well-annotated in the gene ontology (GO) database and show robust enrichment for functions and compartments of secretory cells determined by fold enrichment vs. FDR using DAVID. D ENR-enriched proteins are well annotated in the GO database and sho...
254 Background: Prostate cancer is the second most common malignancy in men worldwide. The develo... more 254 Background: Prostate cancer is the second most common malignancy in men worldwide. The development of cancer from prostate tissue involves complex interactions of tumor cells with surrounding epithelial and stromal cells and can occur multifocally, suggesting that prostate epithelial cells may undergo cellular state transitions towards carcinogenesis. Previous studies on localized prostate cancer molecular changes have focused on unsorted bulk tissue samples, leaving a gap in our understanding of the cellular heterogeneity in the tumor microenvironment. Single-cell analyses of tumor specimens have the potential to reveal, at unprecedented resolution, cellular composition, as well as instructive intercellular interactions. Methods: To characterize the localized prostate cancer tumor microenvironment, we performed single-cell RNA-sequencing (scRNA-seq) on prostate biopsies, radical prostatectomy specimens, and matched patient-derived organoids from localized prostate cancer patien...
This article contained errors in Fig. 1b and Supplementary Data 1. In Fig. 1b the sample P061_2 w... more This article contained errors in Fig. 1b and Supplementary Data 1. In Fig. 1b the sample P061_2 was incorrectly presented with an icon denoting "pre-treatment" status. In the "Results" section, the related sentence "We performed longitudinal high-throughput scRNA-Seq (Figure 1a) on 13 pre-treatment and 24 post-treatment low-input CSF samples" was also incorrect. The sentence should have read: "We performed longitudinal high-throughput scRNA-Seq (Figure 1a) on 12 pre-treatment and 25 post-treatment low-input CSF samples". The correct samples and treatment timing were used elsewhere in the manuscript. These errors have been corrected in the PDF and HTML versions of the Article. The original version of Supplementary Data 1 showed "days since treatment" for sample P129_1 indicated as "350"; however, the sample was obtained 15 days prior to therapy. This error has been corrected in Updated Supplementary Data 1.
Mycobacterium tuberculosis lung infection results in a complex multicellular structure, the granu... more Mycobacterium tuberculosis lung infection results in a complex multicellular structure, the granuloma. In some granulomas, immune activity promotes bacterial clearance; in others, bacteria persist and grow. We identified correlates of bacterial control in cynomolgus macaque lung granulomas by co-registering longitudinal PET-CT imaging, single-cell RNA-sequencing, and measures of bacterial clearance. We find that bacterial persistence occurs in granulomas enriched for mast, endothelial, fibroblast and plasma cells, signaling amongst themselves via Type II immunity and wound healing pathways. In contrast, these interactions are largely absent in granulomas that drive bacterial control, which are often those that form later in the course of infection; these restrictive lesions are characterized by cellular ecosystems enriched for Type1-Type17, stem-like, and cytotoxic T cells engaged in pro-inflammatory signaling networks that involve diverse myeloid and non-immune cell populations. Th...
Leptomeningeal disease (LMD) is a devastating complication of solid tumor malignancies, with dire... more Leptomeningeal disease (LMD) is a devastating complication of solid tumor malignancies, with dire prognosis and no effective systemic treatment options. Over the past decade, the incidence of LMD has steadily increased due to therapeutics that have extended the survival of cancer patients, highlighting the need for new interventions. To examine the efficacy of immune checkpoint inhibitors (ICI) in patients with LMD, we completed two phase II clinical trials. Here, we investigate the cellular and molecular features underpinning observed patient trajectories in these trials by applying single-cell RNA and cell-free DNA profiling to longitudinal cerebrospinal fluid (CSF) draws from enrolled patients. We recover immune and malignant cell types in the CSF, characterize cell behavior changes following ICI, and identify genomic features associated with relevant clinical phenomena. Overall, our study describes the liquid LMD tumor microenvironment prior to and following ICI treatment and de...
Malaria-causing Plasmodium vivax parasites can linger in the human liver for weeks to years, and ... more Malaria-causing Plasmodium vivax parasites can linger in the human liver for weeks to years, and then reactivate to cause recurrent blood-stage infection. While an important target for malaria eradication, little is known about the molecular features of the replicative and non-replicative states of intracellular P. vivax parasites, or the human host-cell dependencies and responses to them. Here, we leverage a bioengineered human microliver platform to culture patient-derived P. vivax parasites in primary human hepatocytes and conduct transcriptional profiling. By coupling enrichment strategies with bulk and single-cell analyses, we captured both parasite and host transcripts in individual hepatocytes throughout the infection course. We defined host- and state-dependent transcriptional signatures and identified previously unappreciated populations of replicative and non-replicative parasites, sharing features with sexual transmissive forms. We found that infection suppresses transcription of key hepatocyte function genes, and that P. vivax elicits an innate immune response that can be manipulated to control infection. Our work provides an extendible framework and resource for understanding host-parasite interactions and reveals new insights into the biology of malaria dormancy and transmission.
The terminal steps of red blood cell (RBC) generation involve extensive cellular remodeling. This... more The terminal steps of red blood cell (RBC) generation involve extensive cellular remodeling. This encompasses alterations of cellular content through five erythroblast stages that result in the expulsion of the nucleus (enucleation) followed by loss of mitochondria and all other organelles, and a transition to anaerobic glycolysis. Whether there is any link between the erythroid removal of the nucleus and that of any other organelle including mitochondria remains unknown. Here we show that mitochondria are essential for nuclear clearance. We first demonstrate through high-throughput single-cell imaging and confocal microscopy that during mouse bone marrow erythroblast maturation, mitochondria aggregate and trail behind the nucleus as it extrudes from the cell, a prerequisite for enucleation to complete. This process is also conserved in primary human erythroblasts. We further show that active mitochondrial respiration facilitates nuclear condensation and is required for nuclear extrusion. We also show that mitochondrial activity distinguishes erythroblasts on the verge of enucleation from others at the same erythroblast stage. Surprisingly, metabolite profiling reveals that late-stage erythroblasts sustain mitochondrial metabolism and subsequent enucleation solely through extracellular pyruvate and is independent of glucose oxidation or anapleorotic reactions of amino acids. 13 C-labeled metabolite tracing further supports these findings. Thus, we provide here for the first time a link between erythroid enucleation and mitochondrial metabolism. The process described establishes an important model of mitochondrial compartmentalization within the cell for providing essential metabolites in a precise spatial and temporal manner. These findings might also be relevant to anemia associated with congenital mitochondrial diseases and/or aging.
Lung Biological Network, SARS-CoV-2 receptor ACE2 is an interferon-stimulated gene in human airwa... more Lung Biological Network, SARS-CoV-2 receptor ACE2 is an interferon-stimulated gene in human airway epithelial cells and is detected in specific cell subsets across tissues, Cell (2020), doi:
11503Background: Heterogeneity of malignant and non-malignant tumor components and their interact... more 11503Background: Heterogeneity of malignant and non-malignant tumor components and their interactions pose tremendous clinical challenges and opportunities in clinical care. Due to technical constr...
Background The molecular mechanisms underpinning the neurologic and congenital pathologies caused... more Background The molecular mechanisms underpinning the neurologic and congenital pathologies caused by Zika virus (ZIKV) infection remain poorly understood. One barrier has been the lack of relevant model systems for the developing human brain; however, thanks to advances in the stem cell field, we can now evaluate ZIKV central nervous system infections in human stem cell-derived cerebral organoids which recapitulate complex 3-dimensional neural architecture. Methods We apply Seq-Well—a simple, portable platform for massively parallel single-cell RNA sequencing—to characterize cerebral organoids infected with ZIKV. Using this sequencing method, and published transcriptional profiles, we identify multiple cellular populations in our organoids, including neuroprogenitor cells, intermediate progenitor cells, and terminally differentiated neurons. We detect and quantify host mRNA transcripts and viral RNA with single-cell resolution, defining transcriptional features of uninfected cells a...
Maintenance of pluripotency and specification towards a new cell fate are both dependent on preci... more Maintenance of pluripotency and specification towards a new cell fate are both dependent on precise interactions between extrinsic signals and transcriptional and epigenetic regulators. Directed methylation of cytosines by the de novo methyltransferases DNMT3A and DNMT3B plays an important role in facilitating proper differentiation, whereas DNMT1 is essential for maintaining global methylation levels in all cell types. Here, we generated single-cell mRNA expression data from wild-type, DNMT3A, DNMT3A/3B and DNMT1 knockout human embryonic stem cells and observed a widespread increase in cellular and transcriptional variability, even with limited changes in global methylation levels in the de novo knockouts. Furthermore, we found unexpected transcriptional repression upon either loss of the de novo methyltransferase DNMT3A or the double knockout of DNMT3A/3B that is further propagated upon differentiation to mesoderm and ectoderm. Taken together, our single-cell RNA-sequencing data p...
ABSTRACTTissue barrier dysfunction is a poorly defined feature hypothesized to drive chronic huma... more ABSTRACTTissue barrier dysfunction is a poorly defined feature hypothesized to drive chronic human inflammatory disease1,2. The epithelium of the upper respiratory tract represents one such barrier, responsible for separating inhaled agents, such as pathogens and allergens, from the underlying submucosa. Specialized epithelial subsets—including secretory, glandular, and ciliated cells—differentiate from basal progenitors to collectively realize this role3-5. Allergic inflammation in the upper airway barrier can develop from persistent activation of Type 2 immunity (T2I), resulting in the disease spectrum known as chronic rhinosinusitis (CRS), ranging from rhinitis to severe nasal polyps6-8. Whether recently identified epithelial progenitor subsets, and their differentiation trajectory, contribute to the clinical presentation and barrier dysfunction in T2I-mediated disease in humans remains unexplored3,9,10. Profiling twelve primary human samples spanning the range of clinical severi...
SUMMARYThe development of high-throughput single-cell RNA-sequencing (scRNA-Seq) methodologies ha... more SUMMARYThe development of high-throughput single-cell RNA-sequencing (scRNA-Seq) methodologies has empowered the characterization of complex biological samples by dramatically increasing the number of constituent cells that can be examined concurrently. Nevertheless, these approaches typically recover substantially less information per-cell as compared to lower-throughput microtiter plate-based strategies. To uncover critical phenotypic differences among cells and effectively link scRNA-Seq observations to legacy datasets, reliable detection of phenotype-defining transcripts – such as transcription factors, affinity receptors, and signaling molecules – by these methods is essential. Here, we describe a substantially improved massively-parallel scRNA-Seq protocol we term Seq-Well S^3 (“Second-Strand Synthesis”) that increases the efficiency of transcript capture and gene detection by up to 10- and 5-fold, respectively, relative to previous iterations, surpassing best-in-class commerc...
Proceedings of the National Academy of Sciences, 2019
Circulating tumor cells (CTCs) play a fundamental role in cancer progression. However, in mice, l... more Circulating tumor cells (CTCs) play a fundamental role in cancer progression. However, in mice, limited blood volume and the rarity of CTCs in the bloodstream preclude longitudinal, in-depth studies of these cells using existing liquid biopsy techniques. Here, we present an optofluidic system that continuously collects fluorescently labeled CTCs from a genetically engineered mouse model (GEMM) for several hours per day over multiple days or weeks. The system is based on a microfluidic cell sorting chip connected serially to an unanesthetized mouse via an implanted arteriovenous shunt. Pneumatically controlled microfluidic valves capture CTCs as they flow through the device, and CTC-depleted blood is returned back to the mouse via the shunt. To demonstrate the utility of our system, we profile CTCs isolated longitudinally from animals over 4 days of treatment with the BET inhibitor JQ1 using single-cell RNA sequencing (scRNA-Seq) and show that our approach eliminates potential biases...
The cellular composition of barrier epithelia is essential to organismal homoeostasis. In particu... more The cellular composition of barrier epithelia is essential to organismal homoeostasis. In particular, within the small intestine, adult stem cells establish tissue cellularity, and may provide a means to control the abundance and quality of specialized epithelial cells. Yet, methods for the identification of biological targets regulating epithelial composition and function, and of small molecules modulating them, are lacking. Here we show that druggable biological targets and small-molecule regulators of intestinal stem cell differentiation can be identified via multiplexed phenotypic screening using thousands of miniaturized organoid models of intestinal stem cell differentiation into Paneth cells, and validated via longitudinal single-cell RNA-sequencing. We found that inhibitors of the nuclear exporter Exportin 1 modulate the fate of intestinal stem cells, independently of known differentiation cues, significantly increasing the abundance of Paneth cells in the organoids and in w...
Figure S4. Signaling pathways and processes associated with in vitro PC enrichment (Additional fi... more Figure S4. Signaling pathways and processes associated with in vitro PC enrichment (Additional file 9: Table S5 for reference gene lists). A Violin plot of expression contribution to a cellâ s transcriptome of Wnt pathway genes (Additional file 2: Table S2; activated by CHIR99021) across clusters as percent of transcriptome. B Violin plot of expression contribution to a cellâ s transcriptome of Notch pathway genes (Additional file 2: Table S2; inhibited by DAPT) across clusters as percent of transcriptome. C Violin plot of expression contribution to a cellâ s transcriptome of respiratory electron transport gene set (Additional file 2: Table S2) across clusters as percent of transcriptome. (PDF 888 kb)
Figure S3. Quality metrics for single-cell RNA sequencing. A Total gene number of cells maintaine... more Figure S3. Quality metrics for single-cell RNA sequencing. A Total gene number of cells maintained in analyses with a lower cutoff of n = 400 unique genes per cell. Total unique molecular identifiers (UMIs) used as the basis for cell-by-gene tables collapsed to UMI as input into Seurat with the lower bound representing n = 400 unique genes and the upper bound representing 8000 UMIs. Note: Clusters ENR + CV-3, ENR + CV-4, and ENR-1 had significantly higher levels of genes and UMIs and, intriguingly, were also the three clusters with the highest levels of Lgr5 (see Fig. 5a), indicating that stem cells may contain larger contents of RNA, as they are in a biosynthetic state before differentiation and maturation. B Violin plot of expression contribution to a cell's transcriptome of mitochondrial and ribosomal genes across identified subsets. (PDF 1153 kb)
Figure S2. Proteomic pipeline, sample-to-sample comparison, and insights from the in vitro PC pro... more Figure S2. Proteomic pipeline, sample-to-sample comparison, and insights from the in vitro PC proteome. A Schematic of proteomic analysis for samples: culture, collection, lysis, reduction and alkylation, proteolytic digestion, labeling of peptides with isobaric mass tag reagents (Tandem Mass Tags, TMT10-plex; Thermo), off-line fractionation by basic reverse phase chromatography, analysis of fractions by LC-MS/MS, identification of peptides and proteins using Spectrum Mill software (Agilent), and statistical analysis of the resulting data (moderated t test) to identify confidently differential proteins. B Proteome sample correlation between all biological (n = 2) and technical (n = 2/biological) replicates. C ENR + CD-enriched proteins are well-annotated in the gene ontology (GO) database and show robust enrichment for functions and compartments of secretory cells determined by fold enrichment vs. FDR using DAVID. D ENR-enriched proteins are well annotated in the GO database and sho...
254 Background: Prostate cancer is the second most common malignancy in men worldwide. The develo... more 254 Background: Prostate cancer is the second most common malignancy in men worldwide. The development of cancer from prostate tissue involves complex interactions of tumor cells with surrounding epithelial and stromal cells and can occur multifocally, suggesting that prostate epithelial cells may undergo cellular state transitions towards carcinogenesis. Previous studies on localized prostate cancer molecular changes have focused on unsorted bulk tissue samples, leaving a gap in our understanding of the cellular heterogeneity in the tumor microenvironment. Single-cell analyses of tumor specimens have the potential to reveal, at unprecedented resolution, cellular composition, as well as instructive intercellular interactions. Methods: To characterize the localized prostate cancer tumor microenvironment, we performed single-cell RNA-sequencing (scRNA-seq) on prostate biopsies, radical prostatectomy specimens, and matched patient-derived organoids from localized prostate cancer patien...
This article contained errors in Fig. 1b and Supplementary Data 1. In Fig. 1b the sample P061_2 w... more This article contained errors in Fig. 1b and Supplementary Data 1. In Fig. 1b the sample P061_2 was incorrectly presented with an icon denoting "pre-treatment" status. In the "Results" section, the related sentence "We performed longitudinal high-throughput scRNA-Seq (Figure 1a) on 13 pre-treatment and 24 post-treatment low-input CSF samples" was also incorrect. The sentence should have read: "We performed longitudinal high-throughput scRNA-Seq (Figure 1a) on 12 pre-treatment and 25 post-treatment low-input CSF samples". The correct samples and treatment timing were used elsewhere in the manuscript. These errors have been corrected in the PDF and HTML versions of the Article. The original version of Supplementary Data 1 showed "days since treatment" for sample P129_1 indicated as "350"; however, the sample was obtained 15 days prior to therapy. This error has been corrected in Updated Supplementary Data 1.
Mycobacterium tuberculosis lung infection results in a complex multicellular structure, the granu... more Mycobacterium tuberculosis lung infection results in a complex multicellular structure, the granuloma. In some granulomas, immune activity promotes bacterial clearance; in others, bacteria persist and grow. We identified correlates of bacterial control in cynomolgus macaque lung granulomas by co-registering longitudinal PET-CT imaging, single-cell RNA-sequencing, and measures of bacterial clearance. We find that bacterial persistence occurs in granulomas enriched for mast, endothelial, fibroblast and plasma cells, signaling amongst themselves via Type II immunity and wound healing pathways. In contrast, these interactions are largely absent in granulomas that drive bacterial control, which are often those that form later in the course of infection; these restrictive lesions are characterized by cellular ecosystems enriched for Type1-Type17, stem-like, and cytotoxic T cells engaged in pro-inflammatory signaling networks that involve diverse myeloid and non-immune cell populations. Th...
Leptomeningeal disease (LMD) is a devastating complication of solid tumor malignancies, with dire... more Leptomeningeal disease (LMD) is a devastating complication of solid tumor malignancies, with dire prognosis and no effective systemic treatment options. Over the past decade, the incidence of LMD has steadily increased due to therapeutics that have extended the survival of cancer patients, highlighting the need for new interventions. To examine the efficacy of immune checkpoint inhibitors (ICI) in patients with LMD, we completed two phase II clinical trials. Here, we investigate the cellular and molecular features underpinning observed patient trajectories in these trials by applying single-cell RNA and cell-free DNA profiling to longitudinal cerebrospinal fluid (CSF) draws from enrolled patients. We recover immune and malignant cell types in the CSF, characterize cell behavior changes following ICI, and identify genomic features associated with relevant clinical phenomena. Overall, our study describes the liquid LMD tumor microenvironment prior to and following ICI treatment and de...
Malaria-causing Plasmodium vivax parasites can linger in the human liver for weeks to years, and ... more Malaria-causing Plasmodium vivax parasites can linger in the human liver for weeks to years, and then reactivate to cause recurrent blood-stage infection. While an important target for malaria eradication, little is known about the molecular features of the replicative and non-replicative states of intracellular P. vivax parasites, or the human host-cell dependencies and responses to them. Here, we leverage a bioengineered human microliver platform to culture patient-derived P. vivax parasites in primary human hepatocytes and conduct transcriptional profiling. By coupling enrichment strategies with bulk and single-cell analyses, we captured both parasite and host transcripts in individual hepatocytes throughout the infection course. We defined host- and state-dependent transcriptional signatures and identified previously unappreciated populations of replicative and non-replicative parasites, sharing features with sexual transmissive forms. We found that infection suppresses transcription of key hepatocyte function genes, and that P. vivax elicits an innate immune response that can be manipulated to control infection. Our work provides an extendible framework and resource for understanding host-parasite interactions and reveals new insights into the biology of malaria dormancy and transmission.
The terminal steps of red blood cell (RBC) generation involve extensive cellular remodeling. This... more The terminal steps of red blood cell (RBC) generation involve extensive cellular remodeling. This encompasses alterations of cellular content through five erythroblast stages that result in the expulsion of the nucleus (enucleation) followed by loss of mitochondria and all other organelles, and a transition to anaerobic glycolysis. Whether there is any link between the erythroid removal of the nucleus and that of any other organelle including mitochondria remains unknown. Here we show that mitochondria are essential for nuclear clearance. We first demonstrate through high-throughput single-cell imaging and confocal microscopy that during mouse bone marrow erythroblast maturation, mitochondria aggregate and trail behind the nucleus as it extrudes from the cell, a prerequisite for enucleation to complete. This process is also conserved in primary human erythroblasts. We further show that active mitochondrial respiration facilitates nuclear condensation and is required for nuclear extrusion. We also show that mitochondrial activity distinguishes erythroblasts on the verge of enucleation from others at the same erythroblast stage. Surprisingly, metabolite profiling reveals that late-stage erythroblasts sustain mitochondrial metabolism and subsequent enucleation solely through extracellular pyruvate and is independent of glucose oxidation or anapleorotic reactions of amino acids. 13 C-labeled metabolite tracing further supports these findings. Thus, we provide here for the first time a link between erythroid enucleation and mitochondrial metabolism. The process described establishes an important model of mitochondrial compartmentalization within the cell for providing essential metabolites in a precise spatial and temporal manner. These findings might also be relevant to anemia associated with congenital mitochondrial diseases and/or aging.
Lung Biological Network, SARS-CoV-2 receptor ACE2 is an interferon-stimulated gene in human airwa... more Lung Biological Network, SARS-CoV-2 receptor ACE2 is an interferon-stimulated gene in human airway epithelial cells and is detected in specific cell subsets across tissues, Cell (2020), doi:
11503Background: Heterogeneity of malignant and non-malignant tumor components and their interact... more 11503Background: Heterogeneity of malignant and non-malignant tumor components and their interactions pose tremendous clinical challenges and opportunities in clinical care. Due to technical constr...
Background The molecular mechanisms underpinning the neurologic and congenital pathologies caused... more Background The molecular mechanisms underpinning the neurologic and congenital pathologies caused by Zika virus (ZIKV) infection remain poorly understood. One barrier has been the lack of relevant model systems for the developing human brain; however, thanks to advances in the stem cell field, we can now evaluate ZIKV central nervous system infections in human stem cell-derived cerebral organoids which recapitulate complex 3-dimensional neural architecture. Methods We apply Seq-Well—a simple, portable platform for massively parallel single-cell RNA sequencing—to characterize cerebral organoids infected with ZIKV. Using this sequencing method, and published transcriptional profiles, we identify multiple cellular populations in our organoids, including neuroprogenitor cells, intermediate progenitor cells, and terminally differentiated neurons. We detect and quantify host mRNA transcripts and viral RNA with single-cell resolution, defining transcriptional features of uninfected cells a...
Maintenance of pluripotency and specification towards a new cell fate are both dependent on preci... more Maintenance of pluripotency and specification towards a new cell fate are both dependent on precise interactions between extrinsic signals and transcriptional and epigenetic regulators. Directed methylation of cytosines by the de novo methyltransferases DNMT3A and DNMT3B plays an important role in facilitating proper differentiation, whereas DNMT1 is essential for maintaining global methylation levels in all cell types. Here, we generated single-cell mRNA expression data from wild-type, DNMT3A, DNMT3A/3B and DNMT1 knockout human embryonic stem cells and observed a widespread increase in cellular and transcriptional variability, even with limited changes in global methylation levels in the de novo knockouts. Furthermore, we found unexpected transcriptional repression upon either loss of the de novo methyltransferase DNMT3A or the double knockout of DNMT3A/3B that is further propagated upon differentiation to mesoderm and ectoderm. Taken together, our single-cell RNA-sequencing data p...
ABSTRACTTissue barrier dysfunction is a poorly defined feature hypothesized to drive chronic huma... more ABSTRACTTissue barrier dysfunction is a poorly defined feature hypothesized to drive chronic human inflammatory disease1,2. The epithelium of the upper respiratory tract represents one such barrier, responsible for separating inhaled agents, such as pathogens and allergens, from the underlying submucosa. Specialized epithelial subsets—including secretory, glandular, and ciliated cells—differentiate from basal progenitors to collectively realize this role3-5. Allergic inflammation in the upper airway barrier can develop from persistent activation of Type 2 immunity (T2I), resulting in the disease spectrum known as chronic rhinosinusitis (CRS), ranging from rhinitis to severe nasal polyps6-8. Whether recently identified epithelial progenitor subsets, and their differentiation trajectory, contribute to the clinical presentation and barrier dysfunction in T2I-mediated disease in humans remains unexplored3,9,10. Profiling twelve primary human samples spanning the range of clinical severi...
SUMMARYThe development of high-throughput single-cell RNA-sequencing (scRNA-Seq) methodologies ha... more SUMMARYThe development of high-throughput single-cell RNA-sequencing (scRNA-Seq) methodologies has empowered the characterization of complex biological samples by dramatically increasing the number of constituent cells that can be examined concurrently. Nevertheless, these approaches typically recover substantially less information per-cell as compared to lower-throughput microtiter plate-based strategies. To uncover critical phenotypic differences among cells and effectively link scRNA-Seq observations to legacy datasets, reliable detection of phenotype-defining transcripts – such as transcription factors, affinity receptors, and signaling molecules – by these methods is essential. Here, we describe a substantially improved massively-parallel scRNA-Seq protocol we term Seq-Well S^3 (“Second-Strand Synthesis”) that increases the efficiency of transcript capture and gene detection by up to 10- and 5-fold, respectively, relative to previous iterations, surpassing best-in-class commerc...
Proceedings of the National Academy of Sciences, 2019
Circulating tumor cells (CTCs) play a fundamental role in cancer progression. However, in mice, l... more Circulating tumor cells (CTCs) play a fundamental role in cancer progression. However, in mice, limited blood volume and the rarity of CTCs in the bloodstream preclude longitudinal, in-depth studies of these cells using existing liquid biopsy techniques. Here, we present an optofluidic system that continuously collects fluorescently labeled CTCs from a genetically engineered mouse model (GEMM) for several hours per day over multiple days or weeks. The system is based on a microfluidic cell sorting chip connected serially to an unanesthetized mouse via an implanted arteriovenous shunt. Pneumatically controlled microfluidic valves capture CTCs as they flow through the device, and CTC-depleted blood is returned back to the mouse via the shunt. To demonstrate the utility of our system, we profile CTCs isolated longitudinally from animals over 4 days of treatment with the BET inhibitor JQ1 using single-cell RNA sequencing (scRNA-Seq) and show that our approach eliminates potential biases...
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Papers by Alex Shalek