DEV3032 2024 Workshop 1 Answers PDF

DEV3032 Workshop #1 NOTE: the questions associated with these workshops are designed to consolidate major concepts of the Unit. While these are not directly assessed, active preparation and participation will be of great benefit to your understanding of concepts that will be examined....

DEV3032 Workshop #1 NOTE: the questions associated with these workshops are designed to consolidate major concepts of the Unit. While these are not directly assessed, active preparation and participation will be of great benefit to your understanding of concepts that will be examined. DEV3032 Workshop 1 Workshop objectives At the end of the Workshop, students will have: 1) Reviewed basic theory on embryonic and induced pluripotent stem cells 2) Identified and worked through technical aspects of iPSC derivation and differentiation 3) Discussed a set of ethical questions relating to iPSCs 4) Had the opportunity to engage in peer-to-peer scientific Discussions, and to work together to generate a consensus group response for a range of questions DEV3032 Workshop 1 Logistics (how the Workshop will run) Instructions 1) I will present a short overall Introduction (this!) 2) I will then introduce a segment of the Workshop that we will work through 3) We will work through the questions as a group – so be prepared to speak up (or add to the chat NOTE: For later study – use these MCQs as study tools - identify the right answer, and briefly suggest why each answer is right/wrong All the answers will be presented as we go through the Workshop, and available for you later DEV3032 Workshop 1 DEV3032 Workshop 1 Pluripotent stem cells 1) Theory 2) Techniques 3) Ethics DEV3032 Workshop 1 Workshop objectives Pluripotent stem cells 1) Theory 2) Techniques 3) Ethics DEV3032 Workshop 1 Manuscript readings Embryonic stem cells (ESCs) Murine embryonic stem cells were first cultured by the Kaufman and Martin groups in 1981, followed (a considerable time later!) by the first human embryonic stem cell culture techniques reported in 1998. Follow the links below to the first 2 murine ESC papers: Evans and Kaufman: http://www.nature.com/nature/journal/v292/n5819/abs/292154a0.html Martin: http://www.pnas.org/content/78/12/7634 DEV3032 Workshop 1 Manuscript readings Embryonic stem cells (ESCs) DEV3032 Workshop 1 Manuscript readings Embryonic stem cells (ESCs) DEV3032 Workshop 1 Manuscript readings Embryonic stem cells (ESCs) DEV3032 Workshop 1 Theory Section 1: Group discussion DEV3032 Workshop 1 Theory 1) Theory Embryonic stem cells (ESCs) Question 1: From which cells of an embryo are embryonic stem cell lines derived? A) Teratocarcinomas B) Germ stem cells C) Inner cell mass cells D) Trophectoderm DEV3032 Workshop 1 Theory 1) Theory Embryonic stem cells (ESCs) Question 1: From which cells of an embryo are embryonic stem cell lines derived? A) Teratocarcinomas B) Germ stem cells C) Inner cell mass cells D) Trophectoderm DEV3032 Workshop 1 Theory Embryonic stem cells (ESCs) Question 2: Which of the following regions of this early mouse embryo is the origin of cells that are cultured as embryonic stem cells?? A) A B) B C) C D) D E) E E A Question 3: What is the structure to which each arrow is pointing? D C A) B) B C) D) E) DEV3032 Workshop 1 Theory Embryonic stem cells (ESCs) Question 2: A) A B) B C) C D) D E) E Question 3: E What is the embryonic structure to which A each arrow is pointing? D A) Blastocyst C B) Zona pellucida C) Inner cell mass B D) Trophoblast E) Blastocoel DEV3032 Workshop 1 Theory Embryonic stem cells (ESCs) Question 4: In one of the original ESC papers, Martin cultured her embryonic stem cells in media “conditioned” by teratocarcinoma cells. This helped to keep her stem cells pluripotent. What do you think might be the growth factor that the teratocarcinoma cell line was releasing into the media that kept the cells in this pluripotent state? NOTE: This information was not known at the time this paper was published – it was only identified later! A) BMP4 B) ESC1 C) PSA-1 D) DME E) LIF DEV3032 Workshop 1 Theory Embryonic stem cells (ESCs) Question 4: In one of the original ESC papers, Martin cultured her embryonic stem cells in media “conditioned” by teratocarcinoma cells. This helped to keep her stem cells pluripotent. What do you think might be the growth factor that the teratocarcinoma cell line was releasing into the media that kept the cells in this pluripotent state? NOTE: This information was not known at the time this paper was published – it was only identified later! A) BMP4 B) ESC1 C) PSA-1 D) DME E) LIF DEV3032 Workshop 1 Theory Embryonic stem cells (ESCs) (this article will help - http://www.nature.com/nrg/journal/v7/n4/full/nrg1827.html#B86) Question 5 (short answer): Briefly outline 3 abilities that are used to define a cell line as “pluripotent. Answer: 1) Ability 1: 2) Ability 2: 3) Ability 3: DEV3032 Workshop 1 Theory Embryonic stem cells (ESCs) (this article will help - http://www.nature.com/nrg/journal/v7/n4/full/nrg1827.html#B86) Question 4 (short answer): Briefly outline 3 abilities that are used to define a cell line as “pluripotent. Answer: 1) Ability to contribute to embryo (blastocyst injection) 2) Ability to contribute to the germline 3) Ability to form teratocarcinoma when injected into 4) Ability to form derivatives of all 3 germ layers in vitro 5) etc DEV3032 Workshop 1 Manuscript readings Embryonic stem cells (ESCs) Follow this link to the first human ESC paper: Thomson et al http://science.sciencemag.org/content/282/5391/1145 The vast potential of embryonic stem cells led to an explosion in research into these cell lines, as well as the realisation that there were limitations inherent in these cells that needed to be resolved before the real goal of personalised stem cell-based medicine could be realised. DEV3032 Workshop 1 Manuscript readings Human embryonic stem cells (hESCs) Science 06 Nov 1998: Vol. 282, Issue 5391, pp. 1145-1147 DOI: 10.1126/science.282.5391.1145 DEV3032 Workshop 1 Manuscript readings Embryonic stem cells (ESCs) Question 5 (general discussion): Why did it take so long (from the first mouse line published in 1981 until 1998) for the first human ESC line to be developed? ANSWER: DEV3032 Workshop 1 Manuscript readings Embryonic stem cells (ESCs) Question 5 (short answer): Why did it take so long (from the first mouse line published in 1981 until 1998) for the first human ESC line to be developed? ANSWER: There are many reasons for this lag time, but some of the major reasons include the fact that there are fundamental biological differences between human and mouse ESCs, such as the utility of LIF to maintain pluripotency (doesn’t work the same way in human and mouse ESCs!). The other critical issues was, of course, the ethical implications inherent in destroying a human embryo in order to produce a research cell line. DEV3032 Workshop 1 Manuscript readings Induced pluripotent stem cells (iPSCs) In 2006, the Yamanaka group from Japan published the first report of the derivation of de-differentiated adult cells – the first “Induced Pluripotent Stem Cells (iPS cells)”. Follow this link below to the first iPS paper http://www.cell.com/cell/abstract/S0092-8674(06)00976- 7?_returnURL=http%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS00928 67406009767%3Fshowall%3Dtrue …and the world changed!! DEV3032 Workshop 1 Manuscript readings Induced pluripotent stem cells (iPSCs) Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors Kazutoshi Takahashi,1 Koji Tanabe,1 Mari Ohnuki,1 Megumi Narita,1,2 Tomoko Ichisaka,1,2 Kiichiro Tomoda,3 and Shinya Yamanaka1,2,3,4,* 1Department of Stem Cell Biology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan 2CREST, Japan Science and Technology Agency, Kawaguchi 332-0012, Japan 3Gladstone Institute of Cardiovascular Disease, San Francisco, CA 94158, USA 4Institute for Integrated Cell-Material Sciences, Kyoto University, Kyoto 606-8507, Japan *Correspondence: [email protected] DOI 10.1016/j.cell.2007.11.019 SUMMARY issues is to induce pluripotent status in somatic cells by direct reprogramming (Yamanaka, 2007). Successful reprogramming of differentiated hu- We showed that induced pluripotent stem (iPS) cells man somatic cells into a pluripotent state would DEV3032 Workshop 1 can be generated from mouse embryonic fibroblasts allow creation of patient- and disease-specific (MEF) and adult mouse tail-tip fibroblasts by the retrovi- DEV3032 Workshop 1 Pluripotent stem cells 1) Theory II 2) Techniques 3) Ethics DEV3032 Workshop 1 Theory Section 2: Group discussion DEV3032 Workshop 1 Theory 1) Theory Induced pluripotent stem cells (iPSCs) Question 1: What 4 factors did Yamanaka’s group identify as being able to drive iPS cell derivation? A) OSKM B) ENTS C) KLTS D) OTKN With the correct answer you have chosen, what do these letters stand for? DEV3032 Workshop 1 Theory 1) Theory Induced pluripotent stem cells (iPSCs) Question 1: What 4 factors did Yamanaka’s group identify as being able to drive iPS cell derivation? A) OSKM B) ENTS C) KLTS D) OTKN With the correct answer you have chosen, what do these letters stand for? Oct3/4, Sox2, Klf4, c-Myc DEV3032 Workshop 1 Theory Induced pluripotent stem cells (iPSCs) Question 2: Which mouse cell type did Yamanaka first use to produce iPS cells? A) Foreskin fibroblasts B) Embryonic fibroblasts C) Tail tip fibroblasts D) Skin fibroblasts DEV3032 Workshop 1 Theory Induced pluripotent stem cells (iPSCs) Question 2: Which mouse cell type did Yamanaka first use to produce iPS cells? A) Foreskin fibroblasts B) Embryonic fibroblasts C) Tail tip fibroblasts D) Skin fibroblasts DEV3032 Workshop 1 Theory Induced pluripotent stem cells (iPSCs) Question 3: The epigenetic status of which 2 pluripotency network endogenous genes was assessed by Yamanaka as supportive evidence of pluripotency acquisition? A) OCT3/4 and KLF4 B) SOX2 and KLF4 C) TCL1 and FBX15 D) OCT3/4 and NANOG DEV3032 Workshop 1  During reprogramming Theory Induced pluripotent stem cells (iPSCs) Question 3: The epigenetic status of which 2 pluripotency network endogenous genes was assessed by Yamanaka as supportive evidence of pluripotency acquisition? A) OCT3/4 and KLF4 B) SOX2 and KLF4 DNA methylation DNA C) TCL1 and FBX15 status methylation D) OCT3/4 and NANOG DEV3032 Workshop 1 Theory Induced pluripotent stem cells (iPSCs) Question 4: What is the known function of each of the 4 “Yamanaka” factors? A) B) C) D) DEV3032 Workshop 1 Theory Induced pluripotent stem cells (iPSCs) Question 4: What is the known function of each of the 4 “Yamanaka” factors? Answer: A. OCT3/4 – pluripotency factor/transcription factor B. SOX2 – pluripotency factor/transcription factor C. C-MYC - oncogene/transcription factor D. KLF4 – oncogene/transcription factor DEV3032 Workshop 1 Theory Induced pluripotent stem cells (iPSCs) Question 5: Somatic cells and pluripotent cells have the same genetic information. What is the status of their epigenetic information? A) Pluripotent cells undergo significant epigenetic changes during differentiation B) Somatic cells and pluripotent cells have the same epigenetic modifications C) Somatic cells do not have epigenetic modifications D) Pluripotent cells do not have epigenetic modifications DEV3032 Workshop 1 Theory Induced pluripotent stem cells (iPSCs) Question 5: Somatic cells and pluripotent cells have the same genetic information. What is the status of their epigenetic information? A) Pluripotent cells undergo significant epigenetic changes during differentiation B) Somatic cells and pluripotent cells have the same epigenetic modifications C) Somatic cells do not have epigenetic modifications D) Pluripotent cells do not have epigenetic modifications DEV3032 Workshop 1 Theory Induced pluripotent stem cells (iPSCs) Question 6: You are a researcher involved in reprogramming cells. Which delivery mode for your reprogramming factors is considered the safest, but least efficient? Choose the CORRECT option below: A) Lentivirus B) Protein C) Transposon D) MMLV-derived retrovirus DEV3032 Workshop 1 Theory Induced pluripotent stem cells (iPSCs) Question 6: You are a researcher involved in reprogramming cells. Which delivery mode for your reprogramming factors is considered the safest, but least efficient? A) Lentivirus B) Protein C) Transposon D) MMLV-derived retrovirus DEV3032 Workshop 1 Theory Induced pluripotent stem cells (iPSCs) Question 7: Which of the delivery modes is considered the least safe, but most efficient? A) Lentivirus B) Protein C) Transposon D) MMLV-derived retrovirus Why is your choice not considered safe? ANSWER: DEV3032 Workshop 1 Theory Induced pluripotent stem cells (iPSCs) Question 7: Which of the delivery modes is considered the least safe, but most efficient? Gonzales et al., 2 A) Lentivirus B) Protein C) Transposon D) MMLV-derived retrovirus Why is your choice not considered safe? ANSWER: The retroviruses integrate into the genome, with unknown impacts on the regions of integration. It is not possible to remove them. The transduced factors may also continue to be expressed, reducing the ability of the cells to differentiate. DEV3032 Workshop 1  Theory Induced pluripotent stem cells (iPSCs) KARAGIANNIS ET AL. Table 1. Different reprogramming methods Method Type Efficiency Preparation of Materials Delivery Procedure Removal of Exogenous Factors Referenc Gonzales et al., 2 Retro/lentivirus Virus !!! Easy Easy Difficult 232, 33 Sendai virus Virus !!! Difficult Easy Easy 85 Adenovirus Virus ! Difficult Moderate Moderate 318 Plasmid DNA ! Easy Difficult Moderate 256 Episomal plasmid DNA !! Easy Easy Moderate 394 PiggyBac transposon DNA !! Difficult Easy Moderate 138, 37 Minicircle DNA DNA ! Difficult Difficult Moderate 133 Synthetic RNA RNA !!! Difficult Difficult Easy 361 Recombinant protein Protein ! Difficult Difficult Easy 151 genome. Lentivirus-based vectors have similar properties mune response through the interferon pathway, r but are reported to have higher reprogramming efficiency nant B18R protein of the Vaccinia virus is used to m and less variability (368, 395). The stable genomic integra- this negative effect.DEV3032 This approach is viewed Workshop 1 to h tion achieved by these viral systems is beneficial for high mutagenic risk than DNA-based approaches includ DEV3032 Workshop 1 Pluripotent stem cells 1) Theory 2) Techniques 3) Ethics DEV3032 Workshop 1 Presentations by the experts Induced pluripotent stem cells (iPSCs) Listen to one of the foremost leaders in the stem cell field – this is a scientist who has changed the world of stem cell research (and the world!)! Shinya Yamanaka delivered his Nobel Lecture on 7 December 2012 at Karolinska Institutet in Stockholm. Follow this link to listen (listen to the 46 minute version when you’re free!): https://www.nobelprize.org/nobel_prizes/medicine/laureates/2012/yamanaka- lecture.html DEV3032 Workshop 1 Presentations by the experts Induced pluripotent stem cells (iPSCs) https://www.nobelprize.org/nobel_prizes/medicine/laureates/2012/yamanaka-lecture.html DEV3032 Workshop 1 Techniques Research scenario #1 iPSCs This DEV3032 research Group represents a leading research group of stem cell scientists. You are the young scientists of the Research Group, and you are driving new innovations in your research field. Within this larger Group, consider your chosen disease based on your different germ layer/line Prac Groups, and consider the series of research questions presented. DEV3032 Workshop 1 Research scenario #2 Questions With your Group disease in mind, and based on your knowledge of iPSC derivation and differentiation, address the following questions: 1. What would be an appropriate source population of your iPS cells, and why? 2. What is your target population of cells – ie what is the ultimate differentiated cell type you wish to produce from your iPS cells in order to treat your disease? 3. Outline briefly how each of these 4 steps is achieved: a) Generation of pluripotency: b) Confirmation of pluripotency: c) Differentiation into the required lineage: Information can be sourced from d) Confirmation of the target cell population: the lecture material, textbooks, internet sites, or the published 4. Write the title of your manuscript! literature. DEV3032 Workshop 1 DEV3032 Workshop 1 Pluripotent stem cells 1) Theory 2) Techniques 3) Ethics DEV3032 Workshop 1 Techniques Ethics scenario #1 iPSCs Your group of young scientists have successfully derived the cell types you were aiming for and have published your research in Cell Stem Cell – congratulations!! As you drink some champagne (or other appropriate celebratory beverage), you start to discuss the next stage in your research. And as the evening goes on, your discussions begin to be a little more philosophical… DEV3032 Workshop 1 Ethics Ethics scenario #1 iPSCs Unintended consequences and scenarios… As Jeff Goldblum (as Dr Ian Malcolm) said in “Jurassic Park”: “Yeah, yeah, but your scientists were so preoccupied with whether or not they could that they didn't stop to think if they should”. DEV3032 Workshop 1 Ethics Ethics scenario #1 iPSCs You have made perfect iPS cells to treat the disease of your original patient. Those cells might also be used for other, HLA-matched recipients. - Would this be ok? You’ve put in the work to make the cells. Are they now yours to control? Who holds the rights to the cells? DEV3032 Workshop 1  https://www.immunology.org/hela-cells-1951 Ethics Ethics scenario #1 Example Hela: an immortal cervical cancer cell line derived in 1951 from Henrietta Lacks, a young African American mother of 5, who died soon after from her cancer. She was never asked nor gave consent, HOWEVER bioethics at the time differed from now Hela cells - around 74,000 published studies, critical reagent in polio vaccine development (Salk) and early HIV isolation. Two Nobel prizes (HPV and telomerases). Considered critical for the “omics” revolution ie genomics, transcriptomics, and proteomics. Her children were given the opportunity many years later to view her cells under a microscope… Read also: “The immortal life of Henrietta Lacks”, a novel written by Rebecca Skloot 2010. DEV3032 Workshop 1  Ethics Ethics scenario #2 iPSCs Consider the questions below - we will discuss some answers together IPS cells hold the genetic information of a patient/individual – can you sequence them? What privacy implications does that have? Can you pass them on to your collaborators? Can you genetically modify them – are you modifying someone’s genome without their consent? DEV3032 Workshop 1  Ethics Ethics scenario iPSCs There are no right answers here, but as scientists, it is incumbent upon us to consider what we are doing, and imagine the possible benefits, and costs, of our work As he witnessed the first detonation of a nuclear weapon on July 16, 1945, a piece of Hindu scripture ran through the mind of Robert Oppenheimer: “Now I am become Death, the destroyer of worlds”. DEV3032 Workshop 1 Pluripotent stem cells - end Workshop #1 - DEV3032 Workshop 1 Next workshop: Stem cells in reproductive biology (some really interesting knowledge..and techniques…and ethics (!) DEV3032 Workshop 1