This course provides life scientists with an introduction to the principles and practices of cell culture facilitating their ability to develop the use of in vitro systems. The course is predicated on the application of the most rigorous principles of quality control.
From Donor to Cell Lines: Comparison of Methods, Endothelial Cells from Aorta, Chick Embryo Culture, Human Skin Fibroblasts, Hepatic Cells; Explants and Cell Cultures; Contrasting Properties of Normal and Transformed Cells: Finite Life Span; Senescence and Telomeres; Transfection with Telomerase Gene; Mutagenesis and Carcinogenesis Assays; The in vitro environment: Sterility; Judicious use of Antibiotics, Mycoplasma Effects and Testing, The 2005 Consensus Document on Good Tissue Culture Practices; Low Density and Clonal Growth: Methods and use in Evaluation of Nutrients, Cell Proliferation, and Cytotoxicity; Cell Hybridization, Somatic Cell Genetics and Gene Mapping to Chromosomes; Cell Line Identification and Authentication: Karyology, Isoenzymes, Short Tandem Repeats. Cross-Contamination: Causes and Prevention; Karyotyping: Solid and G-banding. Chromosome Aberrations; Cell Culture and Biotechnology Interface: Transfection and Selection, Use of Genetically-Engineered Cells for Medical/Commercial Applications, Suspension and Mass Culture Techniques; Cell Culture Repositories: Starting and Maintaining a Cell Bank; Immortalized Cells.
Subculturing 3 Different Cell Lines: Cell Counting and Cell Viability; Chick Embryo Culture; Gross Cytology of Cells: Staining, Morphological Changes During the Cell Cycle; Colchicine Synchronization; Metaphase Spreads; Solid Staining and G-banding; Cloning: Cloning by Limit Dilution and Plating efficiency; Transformed cells: growth patterns, growth in soft agar, and aggregate Formation; Chemoluminescent Assays: for Cell proliferation and Evaluation of Toxins; Transfection of Cells. Telomerase Assay; Apoptosis; Isoenzyme Assay for Detection of Interspecies Cross-Contamination
Laboratories using cultured mammalian cells run the risk of cross-contamination and misidentification of their cell lines. It has been estimated that as many as 20 percent of the cell lines being used for research are not what they are purported to be. Furthermore, the problem continues to grow. Reliable procedures for cell line identification and authentication exist and should be among the important quality control measures practiced by those responsible for the integrity of the research.
After a review of well documented cases and preventative measures, this 3-day lecture and “hands-on” laboratory course will focus on the three most useful methods for authentication and detection of intra- and inter-species cross-contamination.
Also, cell type identification by intermediate filament immunocytochemistry and proper design of a cell bank will be discussed.
This lecture and laboratory course will provide an introduction to proteomics technology. Both principles and advanced methodologies will be discussed with an emphasis on protein identification tools, shotgun sequencing and bioinformatics technologies.
Introduction to Cryobiology: Biophysical and Biochemical Principles Involved in Cryobiology; Ice Formation; Seeding Temperature; Freezing Curves; Introduction to Gamete Freezing Methods; To Freeze or Not to Freeze; Equipment Description: Description of Different Cryogenic Vessels and Freezers, Vials and Straws, Inventory Systems; Embryo Slow Freezing Method; Two Step Freezing and Vitrification Methods; Introduction to Rapid, Ultra-Rapid and Vitrification Methods; Sperm Freezing; Introduction to Mouse Sperm Freezing and IVF; Ovary Freezing: Introduction to Ovary Freezing and Surgical Implantation.
Induced pluripotent stem cells (iPSC) represent enormous potential in that they are capable of differentiating into virtually any cell type in the human body. Directing this differentiation into specific cell types in a consistent and efficient manner enables researchers to investigate new therapy and screening approaches in patient-derived cells. This 5 day hands-on training workshop will provide participants with the training and knowledge to help the researcher bring iPSC technology to the laboratory. Students will gain practical knowledge for developing new cell lines from different cell types. Lectures will discuss the expression of genes required for inducing pluripotency and methods of making (virus, RNA, plasmid) and maintaining iPS cells. Lectures on conditions needed for differentiating iPSC to neural, epithelial, and hematopoietic lineages will also be discussed.
The emphasis of the course is on practical information that will help the investigator in the laboratory. Emphasis will be placed on deriving iPSC and differentiation to the neural lineage. Laboratory exercises are intense and will be directed by experts with a working knowledge of the techniques. Labs will cover methods for making iPSC and picking iPSC colonies. In situ analysis of pluripotency on live cells will also be conducted. Laboratory exercise on neural stem cells will include cryopreservation techniques and immunocytochemical analysis of pluripotent marker (Oct4) and neural markers (Sox1, Nestin and Sox2).
Overview; Differentiation of Human Pluripotent Stem Cells into NSC; Differentiation Methods; Cell Culture Methods; Introduction to Mouse and Human Induced Pluripotent Stem Cells; Cellular Reprogramming Methodologies; mRNA Reprogramming of Patient Samples; Optimizing mRNA Reprogramming Efficiency; Differentiating IPSC to Retina Pigment Epithelium; Epigenetic Understanding of Pluripotency; Differentiating IPSC from CD34+ cells; Differentiating IPSC to the Hematopoietic Lineage
Differentiation of IPS to Neutrophils; Preparation of Cells From Embryoid Bodies To Study Differentiation; Differentiating IPSC to Neurons; Techniques Used for neural differentiation; Neural Induction and Neural Expansion; NSC Staining for Pluripotent Marker (Oct4) and Neural Markers (Sox1, Nestin and Sox2); Cryopreservation of Neural Stem Cells
Often late stage clinical trials are terminated due to cardiotoxicity. There is great need to develop proper screens that are predictive of human clinical response to medications. This course will cover numerous applications using cardiomyocytes. The lectures will cover cardiac development and cardiac diseases which provides the necessary background for this course in appreciating how stem cells can be differentiated from iPSCs and be used to develop disease in a dish models as well as screens to monitor specific cardiac phenotypes such as arrhythmia and cardiac toxicity. Lectures will also cover the methodology to drive differentiation of iPSCs toward cardiac lineages and the development of cardiac reporter lines that will be useful for screening applications.
The laboratory exercises will include basic handling of cardiomyocytes and then delve into discovery techniques that focus on disease modeling and phenotypic screening for small molecule therapeutics. Lab exercises will conclude with providing exposure to transfection techniques as well as assays for proarrhythmia and toxicity.
Lectures: Cardiac Development and Disease; iPS and ESC: Methodology for Differentiation of Cardiomyoctyes from ESC and iPSC ; iPS and ESC: Overview on Benefits and Limitations of iPSC/ESC-derived Cardiomyocytes; Characterisation and Validation of hESC Derived Cardiomyocytes; High Content Screening for Cardiotoxicity of Anti-Cancer Drugs in hESC Derived Cardiomyocytes; Bioenergetic Modulation of Kinase Inhibitor Cardiotoxicity in hESC Derived Cardiomyocytes; Cardiac angiogenesis and vascular biology; Cardiac Physiology; Cardiac Electrophysiology and Functional Assays; Functional Improvement Following Cell Therapy for Ischemic Myocardium.
Laboratory Topics: Cardiac differentiation from iPSCs ; Basic Handling: Thawing and Plating iPSC-derived Cardiomyoctyes; Discovery Techniques; MEA Demonstration; xCelligance Demonstration; PART I (Transfecting iCell Cardiomyoctyes with one or a Combination of the Following: (a) Fluorescent Protein Marker and Subsequent Analysis via Flow for Transfection Efficiency, Luciferase Reporter, Most Likely CRE-Luciferase and Then Induction and Read-out of the System, (c) siRNA to Knock Down Housekeeping Gene and then Quantify Via Flow or HCl or Knock Down Ion Channels and Provide a Functional Readout); Compound dose response curves; Transfection and reporter assays; Organelle Toxicity; Discovery Techniques PART II: Disease Modeling and Phenotypic Screening for Small Molecule Therapeutics (Hypertrophy With Protein Based HCl Readout and a Phenotypic Screen with 6 Compounds to Look for Amelioration of the Pathology); Reporter Assays; GE Cytell DemonstratioN: Imaging (GE Lab); Organelle Toxicity; Field Trip to NCATS, Lecture on High Content Screening Methods
Recent advances in generating iPSCs now allow for their derivation from blood. This recent advance enables basic and clinical researchers to reprogram a blood cell into an iPSC and then further differentiate into any cell type. This capability allows researchers to develop “disease in a dish” paradigms to investigate disease and therapy mechanisms.
In this one week workshop, participants will learn how to generate iPSC from blood samples using a non-integrating approach. Due to the length of this procedure (iPSC generation ~3-4 weeks etc.) starting material (CD34+ cells or mononuclear cells) will be provided for each investigator and only critical stages will actually be performed during the laboratory portion of the workshop.
In addition to learning how to culture cells and reprogram blood cells into iPSCs, we will also present some of the latest methodologies for directing differentiation of these iPSCs into different lineages (e.g. hepatocytes or cardiomyocytes). Therefore, this course will package together the essential methodology to take a CD34+ cell isolated from blood, reprogram this cell, and then direct differentiation into multiple different lineages.
Lecture Topics: Introduction to Human Induced Pluripotent Stem Cells; Cellular Reprogramming Methodologies, Reprogramming from Blood; Specialized Media and Tools for Isolation and Enrichment of Blood-Derived Cells; Reprogramming using Cytotune Sendai Virus and Episomal Vectors; Differentiating IPSC to MSCs; Differentiating IPSC to RPEs; Overview of Validation Tests for Pluripotency; Discussion With Experts: Troubleshooting and Question Session, Planning Your Experiments the Right Way, Pam Robey, Ph.D (NIDCR), Rupa Shevde, Ph.D (Life Technologies), Barbara Mallon, Ph.D (Stem Cell Unit), Guokai Chen, Ph.D (NHLBI), Kapil Bharti, Ph.D (NEI).
Laboratory Topics: Observation of Monday’s cells and Cryopreservation; Maintenance of human iPSCs and Differentiation of iPSCs via Embryoid Bodies or Monolayer Platforms; iPS colony identification from TeSR-E7 Reprogramming Experiments; Making Embryoid Bodies Using Aggrewell; Neural Rosette Identification and Scoring; Reprogramming Using Cytotune Virus; Progression of Colony Formation via Fixed Dishes; Picking of Reprogrammed Colonies; Neon Electroporation, Transfection; Validation Tests for Pluripotency; RT-QPCR Assay for iPSC Characterization
The emphasis of the course is on practical information that will help the researcher bring iPS technology to the laboratory. The staff conducting the course includes experts from academia and industry.
Lectures will discuss the expression of genes required for inducing pluripotency, methods of making (virus, RNA, plasmid) and maintaining iPS cells, growth conditions for differentiation (emphasis on hematopoietic and cardiac), and contrasts with embryonic stem cells.
Labs will cover techniques for staining for alkaline phosphatase and immunofluorescent markers of pluripotency on live cells. Furthermore, methods for picking iPS colonies and DNA transfections will also be included. Although the techniques will be conducted mostly on mouse cells, their applicability to human cells will be discussed.