From Tissue to Single Cell: Models to Characterize Immune Memory in Barrier Tissues
Abstract: The role of epithelial and stromal cells in driving inflammation, together with immune cells, is being increasingly appreciated. However, we currently lack tissue-scale frameworks to define and test how inflammation collectively impacts epithelial, stromal, and immune cells. This poses a fundamental challenge for understanding the cellular mechanisms that drive or sustain health and disease within and across barrier tissues such as the skin, airway, and intestine. Our lab is addressing this challenge by quantifying and controlling how inflammation changes the ecology of barrier tissues with single-cell resolution. About Speaker: José Ordovas-Montañes attended Tufts University, followed by graduate studies in the Harvard Immunology program. As an undergrad, José worked in human immunology labs at Biogen Idec, University College London, and Boston Children’s Hospital, where he focused on monogenic immune deficiencies. For his PhD work, José trained with Uli von Andrian, studying how the nervous system and the immune system function together as the principal sensory interfaces between the internal and external environments. As a Damon Runyon Postdoctoral Fellow in the Laboratory of Alex K. Shalek at MIT, the Broad Institute, and the Ragon Institute, he began charting maps of human gut and airway, and discovered how human stem cells can be shaped by, and remember, inflammation. José started his group in 2019 at Boston Children’s Hospital and is supported by a talented team undergraduate students, graduate students, technicians and colleagues.
Preparing Samples for Single Cell Methods
Single cell sequencing is a relatively recent approach that enables scientists to study biological pathways at unprecedented resolution. When preparing single cell suspensions for downstream experiments such as single cell RNA sequencing (scRNA-seq), researchers must optimize steps from cell dissociation through library preparation to get quality results. In this Technique Talk brought to you by The Scientist, Arpita Kulkarni from Harvard Medical School’s Single Cell Core will share a practical guide on how to design a successful scRNA-seq experiment from the wet bench perspective. About Arpita Kulkarni: Arpita Kulkarni is associate director of the Single Cell Core at Harvard Medical School. As a cell and developmental biologist, Kulkarni uncovers the diverse mechanisms in which normal developmental pathways go awry in disease, elucidating disease etiology at single cell resolution. She leverages cell biology, genomics, medical engineering, and single cell and spatial technologies for translational research applications, including therapeutic development in oncology and neuro-psychiatry. Learning Objectives: Types of single cell sequencing and different -omics that scientists can study at single cell resolution• Understanding the basics of scRNA-seq, how it differs from bulk RNA-seq, and when to choose this method over other approaches• Key sample preparation considerations and best practices for designing and carrying out a scRNA-seq workflow• Choosing scRNAseq platforms and combining single cell sequencing with other technologies• Analyzing single cell data
Unlocking BioMarker Discovery: Transforming Sample Challenges Into Research Milestones
In this webinar, explore how Levitation Technology revolutionizes biomarker discovery, offering a lifeline to researchers grappling with the complexities of low-quality samples, About Dr. Camila Egidio: Dr. Camila Egidio is a Molecular Biology Scientist with extensive experience in developing new products and systems to support biomarker discovery and patient testing in the biotechnology industry. In her 14 years of professional experience in the Bay Area, Dr. Egidio has led the development of over 10 products for RUO, IVD and LDT use using high throughput methods at single cell and single molecule sensitivity. In her current role, Dr. Egidio and team reveal how magnetic levitation can segregate finite viable cells present in low quality samples while preserving intact transcriptional signatures for accurate molecular characterization and biological discoveries. Applications: Single-Cell, Oncology Key takeaways: • Learn about single-cell RNA sequencing as a relevant tool for drug discovery• See data showing how magnetic levitation enriches sparse, healthy, viable cells in low-quality samples and maintains native state for downstream biomarker discovery.
Elucidating the Leptomeningeal Immune Response to Cancer With Single-Cell Proteogenomic Profiling
Metastases to the central nervous system from solid primary tumors are a frequent and fatal complication of cancer with limited therapeutic options. Despite the advent of immunotherapy, it remains largely unknown whether and how metastatic cells in the brain and surrounding cerebrospinal fluid (CSF)-filled leptomeninges interact with the immune system. To approach these interactions in an unbiased manner, Jan Remsik and his colleagues profiled an unexpectedly heterogeneous population of cell types in human and mouse CSF with single-cell analytics. These samples were inherently challenging to process due to a relatively low number of cells from each human subject or mouse collection, extreme cell fragility, and contamination by myelin and other unwanted debris in the case of the mouse samples. Since FACS-sorted samples suffered an unbearable amount of stress that prevented their processing, the team used the LeviCell 1.0 system to deplete the debris and enrich for viable cells in stress-free conditions. Incorporating the LeviCell run into their pipeline resulted in the capture of sufficient numbers of high-quality cells for CITE-seq. Such a proteogenomic approach, combining more than 100 cell surface signals with whole transcriptome sequencing, allowed Remsik and his colleagues to elucidate the response of the CSF immune system to cancer at an unprecedented resolution. About Dr. Jan Remsik: Jan Remsik is a Postdoctoral Fellow at the Adrienne Boire Lab at Memorial Sloan Kettering Cancer Center. Intrigued by the ability of cancer cells to colonize the immune-populated cerebrospinal fluid, Jan Remsik generated a series of syngeneic animal models of leptomeningeal metastasis (LM) and accompanying single-cell atlases of human and mouse LM. Using these tools, he studies the mechanistic nature of cancer-immune cell interactions to develop alternative immune-therapeutic approaches against metastasis within this space. Applications: Multi-omic single cell analysis, scRNAseq, CITEseq, PDX studies, cancer, brain metastasis, cell viability enrichment, cell subtype enrichment, immune cell phenotyping Key takeaways: • LeviCell rescues fragile and low quality samples at high yield with a fast and easy to use workflow obtaining samples at high viability and with native state preserved• Improved sample quality generates high quality multi-omic single cell data with high % of cells passing filter and retained in the analysis• Cell subtype representation is preserved in samples processed with LeviCell, even the most fragile cell subtypes such as dendritic cells and neutrophils
RNA-Seq: Enhancing Single-Cell Data and Cell Recovery Rates for Complex Tissue Samples
In this GEN webinar, Dr. Agne Antanaviciute and Stefan Zaharievski, describe how a novel alternative to traditional cell sorting and labeling—LeviCell—can be used to significantly improve single-cell data quality and cell recoveries in complex clinical tissue samples. Additionally, they will detail how they have observed considerable increases in cell recoveries per reaction and overall improvements in data quality while preserving the cell type composition of the samples. Finally, our presenters will show how the LeviCell platform is compatible with CITE-Seq and cell hashing protocols for high throughput processing of multiplexed samples in one single cell reaction. About Dr. Agne Antanaviciute: Agne Antanaviciute is a computational biologist working at the MRC Human Immunology Unit at MRC Weatherall Institute of Molecular Medicine, University of Oxford. She has worked in the field of single-cell and spatial biology since 2017, focusing on tissue immunology, development and GI inflammatory and infectious diseases. About Mr. Stefan Zaharievski: Stefan Zaharievski is a Field Application Scientist – EMEA for LevitasBio. He is a graduate of biotechnology and biofabrication. After obtaining his MSc from Utrecht University, the Netherlands, he has since enjoyed working in expanding the adoption of innovative technology and its use in scientific research. Applications: Single-Cell, Organoids Key takeaways: Learn about significant improvements in single-cell data quality and cell recoveries for complex clinical samples that achieve the following: • 5x increases in cell recoveries• Overall improvements in data quality• Preservation of cell type composition of samples
Common challenges to building a single-cell sample cohort
In this webinar, Ronan Chaligné, director of the Single Cell Research Initiative at MSK, shares common challenges that he has encountered when building a clinical sample cohort. He speaks to different technologies that have been adopted over the years to serve the needs of the Single Cell Research Initiative, including the use of levitation technology as an alternative to fluorescence-activated cell sorting (FACS). About Dr. Ronan Chaligné: Director, Single Cell Research Initiative & Assistant Lab Member,Sloan Kettering Institute Ronan Chaligné is the director of the Single Cell Research Initiative and an assistant member of the Pe’er lab at Memorial Sloan Kettering Cancer Center (MSK). Chaligné received his Master of Science degree in cell biology at Université Val d’Essonne in Évry, France, and his doctorate at Institut Gustave Roussy in Villejuif, France. He completed postdoctoral work at Institut Curie in Paris and Weill Cornell Medicine in New York City. His research interests include the pathways and epigenetic factors involved in the initiation and propagation of malignancies. In his current position at MSK, he aims to dissect genetic and epigenetic mechanisms of cancer and development at the single-cell level. Applications: Single-Cell, Oncology Key takeaways: • Hear about the challenges associated with building a clinical sample cohort• Overview of various technologies adopted to serve the needs of the Single Cell Research Initiative• Use of Levitation Technology as an alternative to fluorescence-activated cell sorting (FACS)
Interrogating Cancer Drivers via CRISPR: Mutant TP53 as a Therapeutic Target in Hypodiploid B-cell Acute Lymphoblastic Leukemia
Watch this webinar to learn how Dr. Flores was able to go from 1%–2% lentiviral transduction efficiency to >95% in hard-to-transduce hypodiploid cell lines and primary cells. Dr. Flores presents how he was able to quickly and gently enrich transduced clones to allow such high transduction efficiency and the generation of knockouts for further studies. About: Ernesto Diaz-Flores, Ph.D. University of California, San FranciscoSan Francisco, CA As a leukemia researcher, Dr. Flores’ ultimate goal is to find new cures for high-risk leukemia through an understanding of the mechanisms mediating leukemogenesis. During his time as assistant professor at the University of California, San Francisco, he has identified B-cell lymphoma 2 (BCL-2) as a promising therapeutic target and validated venetoclax as an effective drug against this leukemia. These studies contributed to the first clinical trial of venetoclax in pediatric acute lymphoblastic leukemia (ALL). In follow-up work, Dr. Flores provides evidence for a highly effective combinatorial therapy (Pariury et al., submitted) as well as biomarkers or response to inotuzumab immunotherapy (Benhert et al., submitted). His current work focuses on studying pharmacological and immunotherapeutic opportunities for targeting mutant p53 in the context of acute leukemia. This work represents a new paradigm in the field of p53 and hypodiploid leukemia and is expected to bring novel treatments to the clinic. Applications: Oncology, Cell Line Development Key takeaways: • Learn how 1%-2% lentiviral transduction efficiency was successfully increased to >95% in hard-to-transduce cell lines and primary cells• See data demonstrating the successful generation of isogenic hypodiploid cell lines that were p53 deficient with knock-in p53 wildtype or mutant alleles.
Broadening Drug Discovery: Use of Unperturbed, Native State Cells to Uncover True Biological Targets at Single-Cell Level
In this webinar, we will present how single-cell RNA sequencing has progressed the field of drug discovery and how upstream sample preparation methods that preserve the native state of cells are critical in identifying relevant biomarkers for drug discovery and development. About Dr. Camila Egidio: Dr. Camila Egidio is a Molecular Biology Scientist with extensive experience in developing new products and systems to support biomarker discovery and patient testing in the biotechnology industry. In her 14 years of professional experience in the Bay Area, Dr. Egidio has led the development of over 10 products for RUO, IVD and LDT use using high throughput methods at single cell and single molecule sensitivity. In her current role, Dr. Egidio and team reveal how magnetic levitation can segregate finite viable cells present in low quality samples while preserving intact transcriptional signatures for accurate molecular characterization and biological discoveries. Applications: Single-Cell, Oncology Key takeaways: • Learn about single-cell RNA sequencing as a relevant tool for drug discovery• See data showing how magnetic levitation enriches sparse, healthy, viable cells in low-quality samples and maintains native state for downstream biomarker discovery.
Reduce scRNA-seq Sparsity with the LeviCell System
In this webinar, Dr. Debia Wakhloo discusses how the LeviCell system reduces data sparsity, improves the quality of scRNA sequencing, and maximizes the biological information that can be gleaned from single cells. About Dr. Debia Wakhloo: Dr. Debia Wakhloo is a Product Application Scientist at LevitasBio. She is a neuroscientist by training, focusing on cognition and neurodegenerative diseases. She has implemented tools like single-cell RNA sequencing and Drop-seq for her research since 2017. Application: Single-Cell Key takeaways: • Learn about data sparsity in single-cell sequencing and the production of non-biological zeroes that skew downstream analysis• Importance of artificial vs. truly biological zeroes• Learn how the LeviCell reduces data sparsity and improves the quality of scRNA-seq to maximize the biological information gleaned from single cells.