Lecture Notes

1 Introduction Introductory presentation by the instructor about the main areas of the class, including the history of nuclear transfer, embryonic stem cells etc. Overview of class material and personal introductions. Instructions about how to locate and read scientific literature (NCBI PubMed searches).
2 Nuclear transfer, part 1 Nuclear transfer was developed in the 1950s in frogs to test whether a differentiated cell maintains the potential (and the genetic material) to generate an entire organism. This approach ultimately led to the cloning of Dolly, the sheep. In the first paper we will discuss the generation of Dolly. The second paper shows that pluripotent embryonic stem cells can be derived from adult cells through nuclear transplantation. This finding indicates the usefulness of the technology to generate patient-specific embryonic stem cells, a topic of major importance to regenerative medicine, which aims at replacing lost cells in diseases such as Parkinson's or Diabetes.
3 Nuclear transfer, part 2 After discussing the technology and derivation of nuclear transfer ES cells last week, the first paper today demonstrates the next step: The therapeutic application of nuclear transfer derived ES cells in a disease model. One limitation to the nuclear transfer technology is the need for unfertilized oocytes as recipients. Based on a number of experiments it was believed for the past 25 years that zygotes (fertilized eggs), which can be more easily obtained, cannot be used for nuclear transfer. The second paper corrects this view and shows that zygotes can be efficiently used as recipients in nuclear transfer experiments.
4 Embryonic stem cells and adult stem cells That ES cells can be grown for an unlimited time in culture while maintaining the potential to differentiate into all cell types of the body raises the question of the mechanism involved. The first paper describes the identification of Nanog, a key transcription factor, involved in embryonic stem cell maintenance and pluripotency. It has been found that many tissues contain stem cells (tissue-specific or adult stem cells). Their potential to generate other cell types outside of the tissue/lineage (transdifferentiation) is controversial. The second paper addresses this issue of adult stem cell plasticity and transdifferentiation.
5 Epigenetics - DNA methylation DNA methylation is the only known covalent modification of mammalian DNA. Methylation does not change the coding function of the DNA but rather adds regulatory information to the sequence. DNA methylation patterns are established and maintained by three different enzymes. The first paper shows the role of the DNA methyltransferases Dnmt3a/b in de novo methylation and development. The second paper describes stage- and lineage-specific DNA methylation during development.
6 Epigenetics - Histone modifications The DNA is wrapped around an octamer of histone proteins (H3, H4, H2A and H2B). In addition to direct modification of the DNA (DNA methylation) the histone N-terminal tails are themselves subject to modifications that can influence gene expression. The first paper describes how differential packaging of the same gene in different cell types or states either allows or restricts access of the gene to the transcriptional machinery, providing a mechanism for differential gene regulation. One of the histone modifications is lysine 27 (K27) methylation of the H3 histone. This repressive mark is established by proteins of the polycomb-group family, which was first discovered in flies. The second paper shows how the H3K27-polycomb system sets up developmental states to allow for appropriate differentiation.
7 Epigenetics - Reading the epigenome In the past years many single regions have been studied for the presence of DNA methylation and histone marks. Using techniques that we will discuss today, it has been possible to scale these gene-specific or locus-specific approaches to allow more comprehensive studies. These papers describe approaches for the analysis of DNA methylation and histone methylation on a genome-wide scale.
8 Field trip and written assignment Novartis Institute for Biomedical Research, Epigenetics Program
Working in industry: Discussion with Dr. Francois Gaudet (Group leader-Novartis Epigenetics Program).
9 Oral presentations Oral presentations: Each student has to prepare and present a Microsoft® PowerPoint® presentation based on his/her proposal.
10 Reprogramming by defined factors, part 1 The first paper describes the strategy used to isolate MyoD, the classic master regulatory gene which has the ability to transdifferentiate a variety of cell and tissue types into muscle. The second paper shows that differentiated B cells can be reprogrammed into macrophages by the forced expression of specific transcription factors.
11 Reprogramming by defined factors, part 2 The previous papers showed that transcription factors can change cell fates when expressed in certain cells. One ultimate goal of stem cell research and nuclear transfer experiments is to understand the reprogramming process, with the hope of accomplishing the de-differentiation of a somatic cell to an embryonic state using defined factors (rather than using the oocyte to drive this process). These papers demonstrate that fibroblasts can be directly reprogrammed to an embryonic state by introducing a set of defined transcription factors.
12 Ethics discussion Despite the promise of embryonic stem cells for both research and therapeutic applications this topic remains highly controversial because embryonic stem cells involve the destruction of an early-stage embryo (the blastocyst). In this last class we will discuss some of the ethical issues surrounding nuclear transfer and embryonic stem cell research. As a part of this discussion we will consider the 2007 White House report about stem cell research.