Lecture Summaries

Course lecture summaries.
WEEK #	TOPICS	LECTURE SUMMARIES
1	Introduction to the Course	The course will begin with both the students and the instructors introducing themselves and getting to know one another and ourscientific interests. The instructors will then review the syllabus andexpectations of the course. The end of the class period will be devoted to providing the students with information and helpful instruction to ease them into the primary scientific literature, such as advice on methods for searching for and reading biomedical scientific literature and a basic introduction to the focus of the course: stem cell biology. The purpose of this class session is to shape the remainder of the course to fit the interests of the students, to help students to feel comfortable engaging with one another and the instructors, and to provide adequate background and tips for the students to enable them to succeed in the course.
2	Embryonic Stem Cells, Gene Targeting and Transgenic Animals	We will begin the session with a discussion of findings concerning pluripotent embryonic stem (ES) cells: cells with the ability to generate different cell types of the body. The first paper presents the establishment of pluripotent cells from mice. This discovery helped greatly to enhance knowledge of early mammalian development. The study of pluripotent cells and the genome led to the ability of scientists to manipulate genes using gene-targeting strategies, which brought about key insights in biomedical research. The genome can undergo homologous recombination, a mechanism of DNA repair in which there are exchanges of nucleotides between two similar strands of DNA. Scientists can use homologous recombination to delete a gene or part of it, add a gene, or introduce mutations that are known for specific human diseases. The second paper focuses on an early gene-targeting strategy and how it was used to create genetically modified animals, known as transgenic animals. Transgenic animals were first developed in the early 1980s, and researchers can use gene targeting in pluripotent stem cells to generate animal models with mutations in or deletions of genes that are similar to those found in human diseases. Optional ResearchChannel. "Understanding Embryonic Stem Cells." September 16, 2008. YouTube. https://www.youtube.com/watch?v=nYNBNZJ8Xck iBioMagazine. "Mario Capecchi: The Birth of Gene Targeting." March 13, 2011. YouTube. https://www.youtube.com/watch?v=WQr6ZeNe-vE
3	Transgenic Animals in Complex Disease Modeling	In the previous session, students learned about gene targeting and how it can be used in pluripotent stem cells to create model systems with genetic mutations, knock-ins or knock-outs of genes that can mimic the genotypes of various human diseases. Today, the first paper presents the use of gene targeting in mice to model the phenotype of human disease, in particular diabetes, and its limitation when gene deletion causes lethality. The second paper presents the next step in gene-targeting: a conditional system known as Cre / loxP. The Cre / loxP system, from the bacterial virus P1, was established to create tissue-specific gene targeting events, for gene mutations or deletions that cause embryonic lethality when expressed ubiquitously in transgenic animals. The Cre/loxP system led to new knowledge of complex diseases that was not previously possible using previous transgenic models.
4	Regenerative Medicine: Human Embryonic Stem Cells	Previous sessions have highlighted the importance of pluripotent stem cells in animal model systems and the importance of using gene targeting strategies to create systemic or tissue-specific phenotypes that model human diseases. The first paper presents the establishment of pluripotent stem cells from human origins, providing a cell culture system that overcomes species-specific boundaries for the study of human disease. We will discuss the strategy for human embryonic stem cells. The second paper presents the use of these cells for regenerative medicine. Regenerative medicine is a field of research that focuses on replacing or engineering human cells or tissues to correct a disease phenotype and restore the cell or tissue to a normal functioning ability. The advancement of science to develop human pluripotent cells allowed for the generation of many human-specific cells and tissues that could be used for the regenerative medicine field and in potential future clinical trials for human diseases in which the diseased cell or tissue could be replaced by a healthy human cell or tissue.
5	Nuclear Transfer and Cellular Reprogramming	In the previous session, students learned that the use of pluripotent stem cells in regenerative medicine has potential limitations, one of which is the possibility of host rejection of the foreign cells or tissues. In this class session, we will discuss methods to overcome this limitation. The first paper presents nuclear transfer, a method of cloning that removes the DNA from an unfertilized egg and injects new DNA, which can then form a normal cell that undergoes divisions and replicates the injected DNA. These cells can be used to form a cloned organism, such as the well-known Dolly the sheep with a genome derived from the injected DNA. Since the DNA is injected directly into the cell and the cell undergoes its normal replication, it is not recognized as a foreign object to be removed by the host immune system. Another method to overcome host rejection is to use reprogramming, which converts one cell type to another, more specifically from a terminally differentiated cell state to an embryonic- like pluripotent cell state. Thus, the pluripotent cells are not genetically foreign. The second paper presents a technique in which human fibroblasts can be reprogrammed into a pluripotent state using a specific cocktail of four reprogramming factors, known as the Yamanaka factors. These pluripotent cells, called induced pluripotent stem (iPS) cells, are patient-specific and overcome potential host rejection. Optional Tuxillaplanet. "2012-09-29 NHK The Future of iPS Cells, Shinya Yamanaka & Ian Wilmut in Discussion." October 8, 2012. YouTube. https://www.youtube.com/watch?v=bhfRYTJ2p3E
6	Induced Pluripotent Stem Cells and Disease Modeling	In the previous session, we discussed the ability to reprogram fibroblast cells from a human into induced pluripotent stem cells. In this session, we will examine disease models using patient-specific induced pluripotent stem cells. Both papers discuss the use of induced pluripotent stem cells from patients with specific mutations to model and study complex diseases, specifically sickle cell anemia (paper 1) and Alzheimer's disease (paper 2).
7	Large Transgenic Mammalian Model Systems	In one early session, we discussed the creation of small animal transgenic model systems for the study of human disease. In this session, we will look at large animal transgenic model systems and the ways in which they have advanced our knowledge of human disorders. Although they are more difficult to study, larger mammalian models might be more evolutionarily similar to the human species and therefore provide further insights into the mechanisms of disease. The first paper presents a transgenic pig that allows for the study of retinitis pigmentosa, a blinding disorder caused by the loss of the rod photoreceptor cells of the eye. The second paper presents transgenic cattle that are used to improve the quality of milk for human consumption and also as a tool to generate biopharmaceuticals.
8	Oral Presentations	Oral presentations will occur in this session. All students will give their oral presentations as described in the Assignments section of this course.
9	Ethical Concerns with Stem Cell Biology	The instructors will begin the session with a brief overview of the course topics that have been discussed. We will then engage in a discussion of ethical concerns in stem cell biology. Stem cell research has been highly controversial since its development. This stem cell controversy has led to the loss of funding from the government during one stage of stem cell biology research, and at other times has led to an increase in funding, along with the development of specific legislation regarding the use of stem cells in research. This class session is not focused on primary biomedical literature but instead will be used to engage in a conversation about the controversy in stem cell research, as it is a subject that will arise during times of funding and stem cell-related research breakthroughs and has shaped the work that has been completed to this day in the research field.
10	ZFN and TALEN Strategies for Genome Editing and Disease Modeling	We have discussed the establishment of embryonic stem cells and patient-specific induced pluripotent stem cells and the ways in which gene-targeting strategies have been developed to create transgenic animal models and study human diseases. This session will begin our discussion of more recent gene-modification strategies for human disease modeling using stem cells. The first paper presents zinc finger nucleases, which can take advantage of the endogenous DNA repair machinery of a cell to target unique sequences for a more precise alteration of the genome. The second paper presents another targeting system that can be engineered more rapidly to bind to target DNA sequences, called transcription activator-like effector nucleases (TALENs). These engineered nucleases allowed for genome editing in situ in a cell. The class will discuss two examples of using these techniques to correct or create models for human disease.
11	CRISPR/Cas9 Strategies for Genome Editing and Disease Modeling	In the previous session, students learned about genome editing using engineered nucleases to create or correct models of human disease. In 2013, a gene editing strategy called CRISPR/Cas9 was developed based on bacterial innate immunity and repeats in bacterial sequences, and this simplified the process of gene deletion, addition, and modification. The first paper will provide insight into the development of CRISPR/Cas9. We will address the use of CRISPR/Cas9 as a more rapid way to generate transgenic mice. The second paper presents the use of this gene editing strategy in a clinically relevant cell type: human hematopoietic stem cells. Optional Lynn Marquis. "Game Changing Therapeutic Technology." August 4, 2014. YouTube. https://www.youtube.com/watch?v=Egf7vyxe3dQ McGovern Institute for Brain Research at MIT. "Genome-Editing with CRISPR/Cas9." November 5, 2014. YouTube. https://www.youtube.com/watch?v=2pp17E4E-O8
12	3D Culture Systems	Earlier in the course, we discussed regenerative medicine and the importance of pluripotent stem cells to provide healthy cells or tissues. However, this work was limited to a single cell type grown in a dish. Organoids, three-dimensional organ-buds that can be grown in laboratory culture systems, have been highly influential in understanding complex tissues. The first paper presents the use of human embryonic stem cells to grow optic cup organoids, leading to an enhanced understanding of the neural retina. The second paper presents the growth of intestinal organoids from cystic fibrosis patient-specific stem cells and the use of CRISPR/Cas9 gene targeting to repair the diseased tissue.
13	Trans differentiation	In previous sessions, we discussed the use of cellular reprogramming to convert one terminally differentiated cell type into an embryonic-like pluripotent state. Scientists have also been working to directly reprogram one terminally differentiated cell type into another, a process called transdifferentiation. In this session, we will discuss two examples of transdifferentiation from one cell type to another, the methods used for reprograming, and the limitations that still exist with this approach. The first paper presents an early study of transdifferentiation, in which terminally differentiated cell types were converted into myoblasts and myotubes. The second paper presents the reprogramming of fibroblasts into cardiomyocytes.
14	Student Topic of Choice	In this class session, we will discuss a topic related to pluripotent stem cells and genome engineering for modeling human disease. We have left this topic open as a student topic of choice, so that any subject in which the students hold a particular interest can be discussed further. Potential topics include a deeper critique and discussion of any of the previous class sessions, as well as other categories listed below. At the end of this class, students and instructors together will discuss the course, what would and could have been better, and also consider the future of stem cell biology. Potential Topics Any class session topic discussed previously in the course Nuclear transfer and preimplantation genetic diagnosis: In this class, we would discuss methods for reprogramming somatic cells from diseased patients into a pluripotent cell state (e.g. somatic cell nuclear transfer) to obtain patient-matched cell lines for the study of human disease and the testing of potential therapeutic agents. We will then look into the use of preimplantation genetic diagnosis (PGD), a form of genetic profiling that is used for embryos prior to implantation. With this method scientists can screen for specific genetic diseases to identify embryos at risk. We will discuss using PGD to select human pluripotent stem cells at risk for Fragile X Syndrome, and the ability to model this human disease in vitro. Naïve and primed states of pluripotency: The ability of a stem cell to form multiple cell types occurs during natural mammalian development through formation of the blastocyst. In previous sessions, we discussed mouse and human embryonic stem cells, along with new techniques that have been developed to induce pluripotency in mammalian cells. However, differences between human and mouse pluripotency have been found. In this session, we would discuss the epigenetic (cellular and physiological traits inherited by the daughter cells) and pluripotent cell state changes found after induction of pluripotency and differentiation. Current status and ethics of clinical trials using human embryonic stem cells: Human embryonic stem cells and genome engineering have greatly enhanced our ability to model human diseases and find potential therapeutic agents to treat or cure these diseases. However, the ethical controversy discussed earlier in the course, along with limitations in our understanding of the pluripotent state of these cells, have limited the number of clinical trials using these cell types. In this session, we would discuss one ongoing human clinical trial using embryonic stem cells to halt macular degeneration of the eye. We would also discuss the hurdles for these clinical trials and the ways in which scientific advancements might overcome such limitations and lead to treatments of human disease.