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
The emergence of stem cells as important tools for biomedical research prompts this offering. Lectures cover importance, origin, and fate of diverse stem cells (hematopoietic, muscle, nerve, skin and embryonal) and the factors which control their differentiation. Special emphasis will be on isolation, identification, culture, and use of stem cells and their progeny.
Topics: Bone Marrow Stem Cell Plasticity; Hematopoietic Stem Cells; Cell Lines as Models of as Stem Cells; Flow Cytometry and Stem Cell Isolation and Characterization; Murine embryonic Stem Cells; Mesenchymal Stem Cells; Differentiation of Stem Cells into Bone; Isolation of Cells from Bone Marrow Collection; Histology Marrow vs. Peripheral Blood; Lever Stem Cells; Stem Cells in CNS; Differentiation of Hemotopoietic Cells; Stem Cell Transplantation: Re-engineering the Immune System; Regulatory Perspective Regarding Stem Cells and Gene Therapy.
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
Super Resolution Microscopy represents a group of recently developed light microscopic techniques that are able to exceed diffraction-limited resolution (less than 200um). This course will focus on three types of Super Resolution Microscopy, Structured Illumination Microscopy (SIM), Stochastic Optical Reconstruction Microscopy (STORM) and Stimulated Emission Sepletion (STED).
The course is designed for cell biologists with prior experience in light microscopy who wish to add super resolution microscopy to their research program. Participants will acquire both a theoretical understanding of super resolution microscopy and practical experience using state-of-the-art super resolution microscopes.
Lectures: Introduction to Super Resolution Microscopy; Advances in Super Resolution Microscopy; Theoretical Background of SIM, PALM/dSTORM, and STED Imaging; Advances in Fluorescent Protein and Organic Dye Technology; Applications of Super Resolution Microscopy using SIM and dSTORM; Challenges Associated with Obtaining Good SIM, STORM and STED Images; Potential Artifacts Common to Super Resolution Imaging.
Laboratory Topics: Working in groups of five, students will rotate through six work stations (Leica gSTED, GE- SIM, Nikon STORM, Zeiss PALM, Shroff Lab – Instant SIM, Shroff Lab – Dual View Plane Illumination Microscope). During the laboratory sessions, the following topics will be covered: Introduction and Feature Highlights of the Instrument; Imaging Acquisition Procedures; Sample Preparation Requirements and Recommendations (Dye Choices and Imaging Solutions); Imaging Data Analysis and Presentation; Trouble-shoot Common Problems in SIM Imaging; Acquisition of 2D and 3D STORM Images.