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Blastocyst The stage at which embryonic and extra-embryonic tissues are segregated (about 5 days post fertilization). Trophoblast ICM Consists of: 1. The Inner Cell Mass (ICM) which will give rise to the embryo 2. The trophoblast which will give rise to the placenta Culturing ES cells Human ES cell grown on a monolayer of mouse embryonic fibroblast cells known as “feeder cells”. Feeder Cells are slowly being replaced by culture media with specific factors required for maintaining ES cell fate: Leukemic Inhibitory Factor (LIF) Bone Morphogenetic Protein (BMP) Both are secreted by feeder cells and/or are present in serum used in media. Lodish et al., Molecular Cell Biology How can you be sure they are really ES cells on the plate? 1) Allow the cells to differentiate spontaneously on a Petri plate. They will do this spontaneously when they get crowded. 2) In liquid suspension, allow the cells to spontaneously form embryoid bodies with three germ layers. 3) Inject the cells into a mouse (or other mammal) with a suppressed immune system and observe the formation of teratomas. ES Cell Differentiation (neurons) Embryoid Bodies Teratoma Growth factors induce stem cell differentiation Research into… factors that promote/repress differentiation …greatly increases the potential for successful stem cell therapy. Gilbert, Tyler, Zackin: Bioethics (2005) Embryonic Stem Cells (ES cells) ICM Embryonic Stem Cells (ES cells) The Knockout Mouse Transgenic Technology using ES Cells TLR5 knockout prone to obesity WT Gene Modification in Mice -orKnockout Mouse Technology First transgenic mouse: 1977 First Knockout mouse: 1989 The Knockout Mouse – targeted gene inactivation Tsai et al., 2007 Targeted Gene Inactivation (Knockout) Homologous Recombination with Positive/Negative Selection neor confers resistance to an antibiotic (Geneticin, aka G418). Tsai et al., 2007 tk (Herpes Simplex Virus thymidine kinase) phosphorylates ganciclovir and blocks DNA synthesis. G418 Griffiths et al., An Introduction to Genetic Analysis Introduction of Transgene into ES cells in Culture Transduction: use of a viral vector to insert DNA (complications from virus) Transfection: use calcium phosphate precipitate of DNA and add to cells in a monolayer, some take up DNA Lipofection: use liposomes as a vector to introduce DNA, fusion at cell membrane Electroporation: use electric current to transiently create small pores in cell membrane that DNA can pass through Cloning Gun for electroporation Tritech Research Chimeric Mice Archeology Museum Florence Chimera vs. Mosaic The offspring will be… Strain: 129 (Brown, A/A) Mate with Males (Black a/a) Strain: C57B10/6J (Black, a/a) Genotype? Chimera Lodish et al., Molecular Cell Biology Chimeric mice are mated to Black mice. ES cell derived progeny are Brown (Brown is dominant to Black). Not all Brown progeny carry the KO allele. Screen with…? How long to get a homozygote? Lodish et al., Molecular Cell Biology Knockin Mouse Tis21::GFP Knockin Southern Blot GFP expression in Central Nervous System Haubensak et al., 2003, PNAS Somatic Cell Nuclear Transfer (SCNT) Early experiments in the 1960’s showed some success with cell reprogramming with amphibians. Wilmut et al., 1997 First successful SCNT involving adult mammalian differentiated donor cells “Dolly” “Dolly” SCNT – somatic cell was fused with an enucleated oocyte. Egg donor and nucleus donor were from different types of sheep (Blackface and Dorset). Gilbert, Tyler, Zackin: Bioethics (2005) Cloning Anne McLaren, 2001 (UK): One week after the news that a sheep had been cloned, a poll was conducted . 90% of people thought that human cloning should be banned 67% of people thought that cloning animals was unacceptable 56% of people said they would not eat the meat of a cloned animal Reasons for Cloning 1. Reproductive cloning: creating a genetically identical individual 2. Therapeutic cloning: creating genetically identical ES cells for medical purposes Cloning Humans Hwang Woo-suk and Moon Shin-yong of Seoul National University have developed a line of human Embryonic Stem Cells (ES cells) by cloning adult human somatic cells (Hwang et al., 2004). RETRACTED – he fabricated data Many other labs have reported success with SCNT for therapeutic purposes only. Gilbert, Tyler, Zackin: Bioethics (2005) Stem cell therapy Lewis, Human Genetics March 9, 2009 - Obama repeals policy preventing federal tax money from funding embryonic stem cell research. "In recent years when it comes to stem cell research, rather than furthering discovery, our government has forced what I believe is a false choice between sound science and moral values... In this case, I believe the two are not inconsistent. As a person of faith, I believe we are called to care for each other and work to ease human suffering. I believe we have been given the capacity and will to pursue this research — and the humanity and conscience to do so responsibly.” - Obama, 3.9.09 At present, research on human SCNT may not be conducted with federal funds but there is no federal ban on privately funded research in this direction. Some states have issued a ban on human SCNT for reproduction and others have issued the ban for reproduction or therapy. Stem Cells for Treating Disease Drugs to treat symptoms Existing cell lines, aborted embryos, IVF “leftovers” ES cells differentiate into functional neurons Parkinson’s Disease Patient SCNT to create ES cells to differentiate into functional neurons Gene therapy: correct genetic defect with various strategies iPS cells differentiate into functional neurons FDA Approved ES Cell Therapy Clinical Trials As of Spring 2012….. 2010 Starsgardt and Age-Related Macular Degeneration (cause blindness) Advanced Cell Technology 12 patients 2009 Spinal Cord Injuries Geron Corporation (since has dropped stem cell division of company) 10 patients