Cell Biology 2020

There are many methods of nucleic acid isolation. Each technology offers different approaches of purification of the template. However, it is important to control carry over of “process contaminants”. This can manifest with altered sample quantitation and/or unusual downstream reaction results. Being mindful of the upstream contributing factors helps with understanding the template for downstream applications inherent to each unique technology.

Learning Objectives:

1. Molecular biotechnology techniques: nucleic acid sample preparation and quantitation

For more than a century, breakthroughs in biological sciences have relied on the ability to study cells outside of respective organisms.  While majority of cell culturing is still performed using conventional tissue culture treated plastic surfaces, there are growing emphasis and need for non-conventional culture-wares utilizing microporous membranes and microfluidics to create more physiologically relevant in vitro systems.  Here we describe the novel microfluidic-based dynamic live cell imaging platform CellASIC ONIX2, where physiologically relevant microenvironmental parameters such as the perfusion of media/reagent, temperature, and gas compositions can be precisely controlled on-demand via software during the entire imaging experiment.  Unlike most live imaging platforms merely aiming to maintain temperature and gas at certain levels, CellASIC® ONIX2 platform’s ability to control fluidics and perfusion offers true on-demand control of cellular micro-environment during live cell imaging, and its standard plate form factor can instantly upgrade any existing inverted microscopes into a fully capable live cell imaging platform.  Overview of applications covering hypoxia, apoptosis, migration, and suspension immune cell imaging will be presented with appropriate emphasis coupled to microporous membrane based tissue models and assays. Specific attention will be given to microfluidic designs targeted for use with different cell types and fluorescent probes for live cell imaging.  Any scientists interested in creating membrane-based tissue models, as well as imaging scientists wanting to broaden their applications will benefit from this webinar.

Learning Objectives:

1. Learn how the ability to control fluidics and perfusion can provide true, on-demand control of cellular micro-environment during live cell imaging

2. Learn about specific microfluidic designs targeted for use with different cell types and fluorescent probes for live cell imaging

Extracellular vesicles (EVs) are lipid bilayer-delimited pieces of cells that are released from the plasma membrane as "ectosomes" and from the endosomal system as "exosomes." EVs serve both to dispose of toxic materials and to facilitate intercellular communication, so they have many roles to play in disease, including neurodegenerative diseases such as Alzheimer's. Although the long-distance, inter-organ actions of EVs are an exciting topic of research, EVs in solid tissues are most likely to act locally, near the site of release. Our group has adapted a rigorous method for EV separation from tissues. Here, we discuss this method and some of the challenges and opportunities it presents. We then examine molecular profiling of EVs from brain of Alzheimer's disease patients and matched controls and assess possible contributions of APOE genotype. We review the implications of our findings for peripheral biomarkers of disease, using single-particle characterization technologies. All results are shared in the context of the "Minimal Information for Studies of EVs" (MISEV), the consensus document on best practices in the field that was issued by the International Society for Extracellular Vesicles and most recently updated as MISEV2018 (Théry and Witwer, et al, J Extracell Vesicles, 2018). We expect that rigorous and reproducible EV science will continue to develop and contribute to personalized medicine in the coming years.

Learning Objectives:

1. Explain the challenges and opportunities in studying extracellular vesicles in tissue

2. Identify associations between Alzheimer's disease and EV cargo

3. Introduce rigorous approaches to EV separation and characterization

Extracellular ligands bind to receptors on the cell surface leading to receptor internalization. Once internalized into small vesicles, the vesicles fuse with an organelle known as the sorting endosome (SE). The SE is a sorting station that directs receptors to different pathways, in part by sorting nexin (SNX) proteins. Notably, SNX17 interacts with a multitude of receptors in a sequence-specific manner to regulate their recycling. However, the mechanisms by which SNX17-labeled vesicles that contain sorted receptors bud and undergo vesicular fission from the SE remain elusive. Recent studies suggest that a dynamin-homolog, EHD1, catalyzes fission and releases endosome-derived vesicles for recycling to the plasma membrane. However, the mechanism by which EHD1 is coupled to various receptors and regulates their recycling remains unknown. Herein, we seek to characterize the mechanism by which EHD1 couples with SNX17 to regulate the recycling of SNX17-interacting receptors. Our central hypothesis is that SNX17 couples receptors to the EHD1 fission machinery. Co-immunoprecipitations and in vitro assays provide evidence that EHD1 and SNX17 directly interact. We also found that inducing internalization of a SNX17 cargo receptor, Low Density Lipoprotein Receptor Related Protein 1  (LRP1) led to recruitment of cytoplasmic EHD1 to endosomal membranes.  In addition, 3D surface rendering and quantification of overlap volumes demonstrate that SNX17 and EHD1 partially co-localize on endosomes, and this overlap was further increased upon LRP1 internalization. Moreover, SNX17-containing endosomes were larger in EHD1-depleted cells compared to wild-type cells, suggesting that EHD1 depletion impairs SNX17-endosomal fission. This study helps clarify our current understanding of endocytic trafficking, by providing significant new insight into the process of endosomal fission and connecting the sorting and fission machineries.

Learning Objectives:

1. Define the process by which endocytic trafficking occurs

2. Explain the mechanism by which endosomal fission is mediated

Intestinal organoids are self-organizing, 3D structures derived from either pluripotent stem cells or from primary tissues with the abiltiy to recapitulate some of the spatial architecture and in vivo function of the organ.  They are increasingly used to model intestinal development and diseases including cancer, celiac disease, inflammatory bowel diseases and host microbiome interactions. In this recent pandemic caused by SARS-CoV-2, studies have shown that this virus can also infect the gut epithelial cells that express ACE2 and TMPRSS2 receptors. Our patient-derived duodenum organoids express both of these markers.  Thus, intestinal organoid models can be a useful tool for researchers studying the basic biology of SARS-CoV-2 and other coronavirses. In this webinar, we describe two model systems for gastrointestinal research: 1) 3dGRO™ Human iPSC Derived Colon Organoids are derived from integration-free human iPS cells that were differentiated in a multi-step process to colon organoids. The organoids express colon-specific markers, can be propagated long-term and functionally swells in response to forskolin. 2) Gastrointestinal Organoid Biobank was developed by researchers at the University of Michigan Center for Gastrointestinal Research (UMCGR). The biobank contains over 50 highly characterized intestinal organoids from both normal and diseased patients derived from multiple regions of the digestive systems including small intestine (duodenum, ileum), stomach, rectum and colon. RNA-Seq data are available for both model systems

Learning Objectives:

1. How to differentiate human iPSC to colon organoids

2. How to culture and maintain intestinal organoids

3. How to use intestinal organoids in a screening or functional assay

Extracellular vesicles (EVs) are nanosized vesicles containing active proteins, lipids, and different types of genetic material such as non coding RNA species, related to the characteristics of the originating cell. They possess a distinctive capacity to communicate over long distances. EVs have been involved in the modulation of several pathophysiological conditions and, more importantly, stem cell-derived EVs appear as a new promising therapeutic option. Convincing evidence of the regenerative potential of EVs released by stem cells and, in particular, mesenchymal stromal cells has been shown in different injury models. My presentation will focus on the potential of stem cells-EVs in acute and chronic renal injury, describing mechanisms of action and active cargo. Besides, the clinical application of stem cell-EVs will be discussed.

Learning Objectives:

1. Rationale of the application of Stem Cell-EVs for regeneration

2. Role of EV cargo for Regenerative medicine

3. EV in vivo Biodistribution

4. Limits and promises for clinical application

Cell culture continues to be a critical tool for most life science research and applications.  From HeLa to iPSCs and 3D to organoids, culture methods have become more advanced and techniques further refined.  During this presentation we will discuss different model systems and current trends in cell culture, sharing tips and tricks for optimizing your culture conditions.  Troubleshooting problematic cell cultures with our experts ensures reproducible data and faster generation of impactful science.

Learning Objectives:

1. Basic techniques such as thawing cells, passaging cells, transfection, and cryopreservation

2. Tips on how to prevent/overcome some of the most common issues in cell culture such as contamination and cell line misidentification

MicroRNAs (miRs) are small non-coding RNAs whose expression is altered in several types of human cancers. Recent evidence supports their inter-cellular transfer through extracellular vesicles (EVs) secreted both by the cancer cells and by the surrounding cells of the Tumor Microenvironment (TME). These findings have suggested that the behavior of cancer cells can be significantly modified by the cross-talk with the surrounding TME as mediated by the exchange of EV-miRs. This lecture will focus on some of the most relevant implications of vesicular miRs in the biology of cancer cells and its surrounding TME.

Learning Objectives:

1. Explain how to classify different types of Extracellular Vesicles

2. Identify different possible outcomes of miR transfer to and from cancer cells

3. Learn how to exploit this knowledge to identify new therapeutic targets

Extracellular vesicles (EV) from many cell types have demonstrated therapeutic potential against many different diseases. Inhibiting progress in this area is the capability to produce EVs in sufficient quantity to deploy them in in vivo research applications and ultimately the clinic.  Current methods can utilize large numbers of flasks and serum starvation in a batch mode process, or large volume conventional bioreactor systems. It is clear that culture conditions affect the concentration, composition and therapeutic activity of secreted extracellular vesicles. 3-D capillary bioreactors represent the most in vivo like way to culture cells. They can support the culture of large numbers of cells at high densities, 1-2X10e8 cells/ml, 2X10e10 total cells.  Cells are bound to a porous support with a 20kDa molecular weight cut off (MWCO) so cell passaging is not required and EVs cannot cross the fiber in either direction. Secreted EVs are concentrated, free from contaminating serum EVs, show reduced membrane fragment contamination and in some cases have demonstrated enhanced bioactivity. The functional principles of 3-D capillary bioreactors will be shown, and examples of the current and potential future of clinical EV scale-up will be discussed.

Learning Objectives:

1. What are the three fundamental differences between a 3-D hollow fiber bioreactor and a T flask?

2. What is the definition of a Dalton?

3. In what ways is a 3-d hollow fiber bioreactor more in vivo like?

Studying exosomes is extremely challenging, due to enormous technical difficulties in defining and isolating such small and molecularly diverse nanovesicles. The progress in the biomedical field has been rather slow because of the lack of effective isolation approach. Extracellular vesicles (EVs) shed by cells are a highly complex population with a size range of 10-1000 nm, which is mainly composed of exosomes and microvesicles that differ in cell origin and biogenesis. Due to the substantial size overlap among these membrane vesicles, confusion on the origin and nomenclature of EVs has spread through the field. This talk reviews both historic and cutting edge exosome isolation technologies and platforms along with exosome methods of production, isolation and purification which have been shown to have significant effects on yield, viability and function. Further, exosome subtypes are critical to future research, biomarker identification and therapeutic development. Currently, the most-recognized isolation approach is ultracentrifugation, which has been shown to exhibit marked heterogeneity in isolated exosomes with variable sizes and protein compositions, indicating the inability to differentiate exosome subpopulations and other vesicle types (e.g., microvesicles and apoptotic blebs). Moreover, the isolation efficiency of ultracentrifugation is rather low (~5-25%) and involves multiple time-consuming steps for repetitive centrifugation and filtration (>10 hrs). Size-exclusion methods are prone to dilution, contaminations, and pressure-caused damage. To date, there are no well-defined methods for exosome isolation in high-efficiency and high-throughput, which severely hindered the broad applications of exosomes in the biomedical field. We close by introducing Clara ExoRelease Beads, a new purification method called “immuno-isolation and release” that offers standardization, reproducibility, and subtyping while preserving exosome functionality.

Learning Objectives:

1. Understand methods of isolating and enriching exosomes along with their pros/cons of the different techniques

2. What do we mean when we talk about subtyping, and what is its importance?

I will present our new computer vision algorithm, ST-Net, which can computationally synthesize spatially resolved transcriptomics directly from H&E histology images (He et al. Nature Biomedical Engineering 2020). I will demonstrate ST-Net on breast cancer data, and show how it characterizes tumor spatial heterogeneity and quantifies tumor-immune interactions. I will then discuss how similar algorithms can capture complex morphological changes over time of individual cells.

Learning Objectives:

1. Understand how deep learning can integrate imaging with genomics

2. Understand how to characterize spatial and temporal variation in cells and connect it with human diseases

The extracellular matrix (ECM) is a three-dimensional structure that provides physical support for tissues/organs and biochemical/biomechanical cues for tissue morphogenesis, differentiation, and homeostasis. The continual remodeling (degradation/new synthesis) of the ECM is crucial for tissue homeostasis, development, and injury processes and contributes/regulates tumor malignancy, growth, and patient response to cancer treatment. Recent evidence shows that Wilms’ tumor (WT), pediatric cancer that accounts for 95% of the renal malignancies in children, could be considered an aberrant nephrogenesis, where nephron progenitors (NP) expressing SIX2 and CITED1  proliferate uncontrollably, hampering terminal differentiation into functional mature nephron structures. We have characterized via transcriptomic analysis and immunohistochemistry the composition of the ECM within the WT-NP and determine its role in cancer development, and identified how modulation of ECM proteins and their receptors in the NP niche might favor self-renewal vs. differentiation. We show that by altering the NP niche, we could promote NP self-renewal maintenance and hampered differentiation. Our data suggest that ECM secreted by cancerous NP promotes tumor development and growth, thus targeting ECM remodeling processes might be essential for the development of innovative treatment specifically designed to halt WT progression, particularly in patients with severe prognosis or recurrences.

Learning Objectives:

1. Identify the ECM composition in Wilms’ tumor

2. Identify how variation in the Wilms’ tumor nephron progenitor niche may promote continuous self-renewal and hamper differentiation

Chronic kidney disease (CKD) is a major public health threat, affecting 11-15% of the U.S. population alone. Currently, there are no effective therapies to cure CKD. Drug therapies are not potent, and directed only at slowing deterioration of renal function and treating metabolic consequences of CKD. With end-stage renal disease (ESRD) the only options are dialysis or transplantation.  To address the large unmet need for CKD therapies we identified potent renal protective effects of amniotic fluid stem cell derived extracellular vesicles (AFSC-EVs) in preclinical studies. AFSC-EVs are uniquely suited and represent an innovative therapeutic strategy for treatment of CKD. Using Alport syndrome (AS) as a model of CKD, we have previously shown that administration of amniotic fluid stem cells (AFSC) provides therapeutic value, improves renal function and prolongs animal survival. We established that the beneficial effect of AFSC is mainly attributed to secretion of extacellular vesicles (EVs) that contribute to the paracrine activity of AFSC in the kidney, and that administration of EVs alone recapitulate the protective effects of AFSC providing functional and survival benefits. AFSC-EVs are characterized by an array of exosomal and mesenchymal surface markers and miRNA cargo typical to the cell of their origin. In light of potential translation to treat CKD, in addition to mouse lines we characterized EVs derived from human AFSC. We have established that hAFSC are genotypically stable, can be easily scaled up and are high producers of EV. We have reduced EV heterogeneity by use of human clonal cell lines of AFSC, which can produce EVs with comparable expression profile and efficacy in vivo in our animal model of CKD, suggesting high reproducibility. We developed specific identity and potency assays thus guaranteeing the generation of lots of EVs with similar characteristics for therapeutic application. We performed extensive characterization of clonally derived EVs by proteomic and miRNA-seq analysis and using hollow fiber bioreactor system we are also able to increase EV production to clinical scale without losing characteristic purity, potency and efficacy. In order to advance the translation of hEVs and to explore their safety, targeting and effectiveness, we also developed a state of the art in vivo monitoring capabilities using novel magnetic nanoparticle agent for labeling EVs to study EV biodistribution by means of MRI technology. Our biodistribution studies showed that when injected in vivo into AS mice, hAFSC-EVs localized in the kidney, corrected proteinuria and prolonged the life-span of treated mice. In conclusion, we strongly believe that our findings will significantly enhance the development of EVs for paracrine regulation of glomerular diseases, with potential implications for patients affected by CKD.

Learning Objectives:

1. To understand the structure and function of the glomerulus, development of chronic kidney disease  and the pathophysiology of Alport syndrome

2. To understand the characterization of functional extracellular vesicles using in vitro and in vivo models, and potential imaging strategies for live tracking of EVs in vivo

GeoMx Digital Spatial Profiling Technology! This leading digital technology offers multiplexed measurement of protein and nucleic acids with an unparalleled level of precision. Quantify 10's-10,000's of targets (RNA or protein) on a fresh frozen or formalin-fixed, paraffin-embedded (FFPE) tissue section in a completely non-destructive manner. The newly launched GeoMx Cancer Transcriptome Atlas (CTA) is designed for comprehensive profiling of the tumor biology, tumor microenvironment, and the immune response. Profile the RNA expression over 1,800 genes simultaneously with spatial resolution in any region of interest from a single tissue section using the GeoMx Digital Spatial Profiler.

Learning Objectives:

1. Define spatial genomics and it's advantage over gene expression approaches

2. Understand the GeoMx workflow and resulting data

3. Explain its application to immune oncology

Reactive oxygen species (ROS) are well-known for their detrimental effects leading to oxidative stress, cell death, aging, and degenerative disorders. However, there is increasing evidence that ROS are also specifically produced to regulate cellular processes including cell proliferation, differentiation, and migration. Our group has recently demonstrated that ROS are also critical for neurite outgrowth and actin-dependent growth cone motility in cultured Aplysia neurons. In order to investigate the role of ROS derived from NADPH oxidase (Nox), a major ROS source, we moved our research into a new model system, developing zebrafish embryos. Using pharmacological inhibition of Nox as well as zebrafish that are deficient in specific Nox isoforms, we found that ROS produced by Nox2 are critical for the development of retinotectal connections and response of retinal ganglion cell growth cones to guidance cues such as slit2. Thus, our results suggest that Nox-derived ROS play a critical role in neuronal development, especially in axonal growth and guidance.

Learning Objectives:

1. Recognize ROS as signaling molecules in nervous system development

2. Explain the value of Aplysia and zebrafish as model systems for in vitro and in vivo studies of axonal growth and guidance

Chronic kidney disease (CKD) is a global health threat, affecting over 10% of the world population, including an estimated 37 million Americans. Importantly, glomerular diseases account for 70% of all the CKD. To date, no effective treatment for CKD exists, except that of dialysis or transplantation, which are associated with comorbidities, complications, and poor quality of life. Both of these treatment options present an enormous economic burden on the healthcare system, not to mention that transplantation is not even readily available due to a shortage of donor organs. Therefore, new breakthrough knowledge to generate more potent therapies for CKD is needed.

In the glomerulus, podocytes and endothelial cells together with the glomerular basement membrane form the glomerular filtration apparatus, and damage to any one of its components leads to kidney disease. Many different theories of disease progression have emerged over time, ranging from podocyte depletion hypothesis to recent evidence for metabolic dysfunction. However, none of these claims have been able to clearly elucidate the exact path of progression, which has ultimately resulted in failed attempts for treatment.

To better understand disease mechanisms and the integrated function of the glomerular and tubular cell types we applied spatial transcriptome analysis tools to investigate patterns of gene expression in kidney biopsies from healthy patient and patients affected with Alport syndrome (AS), focal segmental glomerulosclerosis (FSGS), and membranous nephropathy (MN). Single glomeruli and tubular and interstitial regions were selected and served as areas of interest for the generation of the Whole Transcriptome Atlas.

Results showed distinct separation between tubular and glomerular regions both in healthy as well as in diseased kidneys. Transcripts typical of glomerular cells were highly enriched in the glomeruli and not in the tubules. Marked differences were observed between glomeruli from AS, FSGS, MN and healthy control samples. Measurable differences in gene expression were also evident between the different etiologies possibly highlighting the mechanistic differences amongst them.

In conclusion, this strategy enables us to answer unresolved questions about glomerular and tubular damage during CKD and repair mechanisms and will help in the development of new therapies to halt disease progression. The knowledge gained from these studies might be applicable to other forms of CKD.

Learning Objectives:

1. To understand the basic principles of special transcriptomics

2. To learn the basic principles for interpreting spatial transcriptomic data