Cloning and Stem Cells A wood panel painting (~1500A.D.) depicting the limb transplantation miracle by Saints Cosmos and Damian. According to legend Cosmos and Damian were physicians who successfully transplanted the limb of a dead Moor onto a patient whose leg required amputation. Can we regenerate parts of the body? • Red Deer replace their antlers every year. It takes ~3 months. • We replace our blood cells. ~100 million /hour • If part of the human liver is removed, it will re-grow • Salamanders and Newts can re-grow their limbs •Video – newt limb regeneration In general: regeneration in vertebrates doesn’t work very well Limbs and most organs aren’t replaced Can cells be reprogrammed? Experimental Example: Amphibian tadpole: Preprogrammed Muscle cells were transplanted into gut region of cells in the embryo but they stayed as muscle cells. Cells are programmed and “know’ what they will become if the cells stay intact. How? “Central Dogma” DNA RNA protein DNA in the nucleus expresses genes→ makes RNA → RNA makes proteins. Some of that protein goes back into the nucleus and programs the cells to keep making more of their kind. What is a Stem cell? A cell of an embryo • Can reproduce itself Or • Can enter a differentiation pathway whereby one of its daughters is differentiated and the other stays an undifferentiated stem cell (totipotent) Totipotent: Having the ability to differentiate into ALL cell types. For example, the zygote and early embryonic cells are totipotent since they can differentiate into any cell type during development. Pluripotent: Having the ability to differentiate into many cell types. In Mammals, embryonic stem cells are pluripotent, and came from totipotent cells. They can differentiate into various fetal or adult cells. Where do we get stem cells? Stem cells can be found naturally in 1) A Fertilized Egg (embryonic stem cells (ES)): 150 cells called (blastocyst) contains two types of cells: 1) trophoblast 2) inner cell mass Embryonic stem cells are obtained from the inner cell mass. Video: (human emb. development) A reliable stem cell reference: http://www.isscr.org/science/faq.htm Other ways to get stem cells: 2. Cancers can form a variety of cell types (connective tissue, cartilage, blood cells) can be transplanted into host Ovarian cancer stem cells animals and in some cases turn into non-cancerous normal cells 3. (Since 1981) Start w/ an embryo. Blastocyst cells can be put in culture and grow indefinitely. When put into a host they will differentiate into a variety of cells. Stem cells are found in small numbers in various tissues in the fetal and adult body Examples: • blood stem cells are found in bone marrow → give rise to all specialized blood cell types. • newborn baby: umbilical cord, possibly baby teeth and amniotic fluid These adult stem cells are multipotent – able to differentiate into a limited number of cell types. Stem cells tend to make a heterogeneous collection of differentiated cells Video: creating lines We haven’t figured out how to make stems cells differentiate into every single type of tissue. …..Yet. Advantages of Embryonic Stem cells • Capacity for indefinite proliferation in culture • Can be differentiated into a variety of cells including germ cells • Can be maintained as frozen stocks •Problems: –Undergo spontaneous genetic changes –Hard to make into certain cell types (e.g. pancreas) (nerve tissue is fairly easy to make, however) –Immuno-rejection unless patient is immuno-suppressed. (not ideal). Nuclear reprogramming and Therapeutic cloning • Can we make rejuvenated embryonic cells from adult differentiated cells (of the same individual)? • This would avoid the problem of immuno- rejection Somatic Cell Nuclear transfer 1. Start with egg (frog, mouse, human) 2. Use UV to kill pronucleus (egg chromosome)-left w/ cytoplasm 3. Pick up another cell in a way such that the cell wall is broken but intact nucleus (using a special micropipette) 4. Inject the intact nucleus into the cytoplasm-only egg cell. The cell thinks It’s been fertilized. It starts to grow into an embryo and then an adult. •This cloning is inappropriate in humans (called reproductive cloning) video: SCNT http://www.dnalc.org/cloning.html Problems w/ SCNT (somatic cell nuclear transfer) • Some transplanted nuclei show a long- term memory of their original gene state so they won’t allow reprogramming. • only about 1/3 of nuclear transplants work However, we’re making progress! Legal Issues In the United States, as of July 2011: • it is illegal to use federal funding for research on human embryos derived from SCNT. • It is legal to use federal funding for research on embryos derived from IVF, if those embryos are not going to be used for anything else (like making a baby). Long-term Goal Reprogram cells without having them go through a nuclear transfer to an egg In the news: Sept. 2007 “Researchers Isolate Adult Stem Cells for First Time in Tendon tissue” (adult fibroblasts) Maybe a cure for Alzheimer's disease, Parkinson's disease, and spinal cord injury, Lots of stories out there…. Induced Plueripotent Stem cells (iPS) also called Adult Stem cells iPS cells from adult human cells Developed by two independent research teams: James Thomson and colleagues at University of Wisconsin-Madison Shinya Yamanaka and colleagues at Kyoto University, Japan 5 Common Ethical Objections • Slippery slope – where do you draw the line between frogs & mice, and humans • Cloning is unnatural. (Antibiotics are unnatural also) • Killing of a potential life. “An embryo = a human being” Actually, an embryo would never progress until it is put into a womb. • God intends humans to suffer – we need to have diseases. • It’s good for us to see how others tolerate suffering. (2)Transfect stem cell- (3)Harvest and culture cells associated genes into the cells according to ES cell culture, using by viral vectors. Red cells are mitotically inactivated feeder cells successes! (lightgray) (4)A small subset of the transfected cells become (1)Isolate and culture donor cells. iPS cells and generate ES- like colonies.
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