stem cells, cloning.pdf

STEM CELLS A fertilized egg cell develops into a complete organism That egg cell has the capability to • Replicate • differentiate into different kinds of specialized cells Embryogenesis is often accompanied with a progressive loss of developmental capacity from a totipotent zygote Stem cells:...

STEM CELLS A fertilized egg cell develops into a complete organism That egg cell has the capability to • Replicate • differentiate into different kinds of specialized cells Embryogenesis is often accompanied with a progressive loss of developmental capacity from a totipotent zygote Stem cells: undifferentiated cells with potential! Stem cells have ability to self-renew or to differentiate into various cell types in response to appropriate signals Stem cell SELF-RENEWAL (copying) Identical stem cells Stem cell DIFFERENTIATION (specializing) Specialized cells Why self-renew AND differentiate? In general specialized cells can no longer divide; eg, skin, RBCs, gut cells) cannot undergo mitosis ❑Need stem cells to renew ❑Some exceptions: liver cells or T-cells 1 stem cell Self renewal - maintains the stem cell pool 1 stem cell 4 specialized cells Differentiation - replaces dead or damaged cells throughout your life Classification of embryonic stem cells Where are stem cells found? embryonic stem cells blastocyst - a very early embryo tissue stem cells fetus, baby and throughout life Embryonic stem (ES) cells are pluripotent Blastocyst: very early stage embryo cells inside = ‘inner cell mass’ outer layer of cells = ‘trophectoderm’ embryonic stem cells taken from the inner cell mass fluid with nutrients culture in the lab to grow more cells differentiation Gene expression: transcripts for generic markers of pluripotent stem cells All possible types of specialized cells! ES cells give rise to disorganized growths called teratomas • do not display axis formation or segmentation • Unlike embryos, ES cells on their own are incapable of generating the body plan • Cartilage, bone, skin, nerves, gut and respiratory lining form when ES cells are injected into host animals Stem cell niches Niche stem cell Microenvironment around stem cells that provides support and signals regulating self-renewal and differentiation Direct contact Soluble factors niche Intermediate cell Understanding the microenvironment around stem cells is as important as understanding stem cells themselves Embryonic stem cells: routes to differentiation Cell types derived from human ES cells in vitro: • Nerve, astrocyte, oligodendrocyte • Hematopoietic stem cells • Insulin producing cells • Cardiomyocytes • Hepatocytes • Endothelial cells Embryonic stem (ES) cells: Challenges skin neurons embryonic stem cells What controls the process so that the stem cells make the right amount of each cell type, at the right time? Do the same molecules mediate fate decisions in ES cell culture? blood ? liver Tissue stem cells: Principles of renewing tissues Stem cell stem cell: ❑ ❑ ❑ ❑ self renew divide rarely high potency rare committed progenitors: ❑ ❑ ❑ ❑ “transient amplifying cells” multipotent divide rapidly no self-renewal specialized cells: ❑ work ❑ no division Embryonic stem cells have important applications in biomedical research • Basic studies of early human development and its disorders: birth defects, childhood cancers • Functional genomics in human cells • Discovery of novel factors controlling tissue regeneration and repair • In vitro models for drug discovery, treatment and toxicology ✓ ✓ ✓ ✓ ✓ ✓ Severe immune deficiency Diabetes Parkinson’s disease Spinal injury Demyelination Myocardial infarction • Source of tissue for transplantation medicine Uses of stem cells • In the laboratory, to create an infinite variety of living human cells to better test the disease-fighting ability of new drugs in test tubes • As patch kits, with cells injected to repair injured body parts, such as spinal cord or heart muscle cell • To create transplants that could be used to replace body parts. Adult stem cells can ONLY make the kinds of cell found in the tissue they belong to e.g., blood stem cells can only make the different kinds of cell found in the blood; brain stem cells can only make different types of brain cell Haematopoietic stem cells (HSCs) NK cell T cell B cell dendritic cell megakaryocyte HSC platelets erythrocytes macrophage neutrophil bone marrow eosinophil basophil committed progenitors specialized cells Tissue stem cells: Neural stem cells (NSCs) Neurons Interneurons Oligodendrocytes NSC Type 2 Astrocytes Type 1 Astrocytes brain committed progenitors specialized cells Tissue stem cells: Gut stem cells (GSCs) Paneth cells Goblet cells GSC Endocrine cells Columnar cells Small intestine committed progenitors specialized cells Tissue stem cells: Mesenchymal stem cells (MSCs) Bone (osteoblasts) Cartilage (chondrocytes) MSC bone marrow Fat (adipocytes) committed progenitors specialized cells Use of human adult stem cell therapy 1968: human adult stem cells used in first successful bone marrow transplant ❑process includes irradiating bone marrow to destroy the faulty stem cells, replacing them with normal bone marrow stem cells from a healthy and immune-compatible donor Today, bone marrow is transplanted routinely to treat a variety of blood and bone marrow diseases, blood cancers, and immune disorders Blood stem cell transplantation May be used to treat diseases such as: ❑diseases of the blood (e.g., cancers, blood cell disorders) ❑bone marrow failure diseases ❑certain immunodeficiencies (diseases that result from missing or dysfunctional immune cells) A successful blood stem cell transplantation can potentially cure patients of some diseases by replacing their blood stem cells Whether blood stem cells are used from cord blood, bone marrow, or peripheral blood depends on ✓patient’s age ✓disease ✓availability of appropriate donor Heart muscle repair with adult stem cells in mice Cord blood stem cells Cord blood = blood that remains in placenta and umbilical cord after baby’s birth; rich in stem cells, which can ❑Grow robustly in vitro w/o differentiating ❑Give rise to mesoderm, neuroectoderm and endoderm ❑in vivo: differentiation into neural cells, bone and cartilage, blood, myocardial and hepatic cells Cord blood as source of stem cells for transplantations Advantages Disadvantages • lower risk of carrying blood borne infectious diseases, or of causing potentially fatal immune response, graftversus-host disease • Fewer blood stem cells available from umbilical cord sample • offers matched source of stem cells for patients who cannot find an immunological match in bone marrow donor registries • immune system recovers more slowly after a cord blood transplantation, putting the recipient at greater risk for certain infections • More cord blood sample cannot be obtained after initial collection Limitations to using adult stem cells Currently only type of stem cell commonly used to treat human diseases; use limited by the ff: 1. Multipotent, not totipotent 2. present in only minute quantities ➢ difficult to isolate and purify. 3. Limited capacity to multiply 4. May contain more DNA abnormalities—caused by sunlight, toxins, and errors in making more DNA copies Cancer stem cells A small subpopulation of cells within tumors with capabilities of self-renewal, differentiation, and tumorigenicity when transplanted into an animal host • Not all the cells within a tumor can maintain tumor growth ❑most cancers are not clonal By identifying the stem cells in tumors, it could possibly be shown that only the cancer stem cells propagate the tumor Cancer Stem Cells (CSCs) Cells with stem-like behaviors have been found in the following cancers and others as well: Breast Cancer; Colon Cancer; Leukemia; Prostate Cancer; Melanoma; Pancreatic Cancer & Some Malignant Brain Tumors Implications for therapy of cancer may require elimination of the minority cancer stem * Cure cell population of the tumor as well as the non-CSC majority of cancer cells. "It's like dandelions in the back yard: You can cut the leaves off all you want, but unless you kill the root, it will keep growing back.“ John Dick, leader of the team that discovered colon and leukemia CSCs possibility that different therapies may be needed to eliminate * The cancer stem cells complicates the search for definitive cures. For example, some CSCs appear to be more resistant to radiation than other cells of the tumor Obstacles in the use of embryonic stem cells in therapy Pluripotent stem cells, while having great therapeutic potential, face formidable technical challenges: ❑Scientists must learn how to control their development into all the different types of cells in the body ❑The cells now available for research are likely to be rejected by a patient's immune system. ❑Ethical considerations of using stem cells from human embryos or human fetal tissue Induced pluripotent stem cells (iPS cells) ‘genetic reprogramming’ = add certain genes to the cell cell from the body induced pluripotent stem (iPS) cell behaves like an embryonic stem cell differentiation culture iPS cells in the lab Advantage: no need for embryos! all possible types of specialized cells Induced pluripotent stem cells (iPS cells) genetic reprogramming pluripotent stem cell (iPS) cell from the body (skin) differentiation Method of the Year 2009: iPS cells iPSC: applications • iPSC can be prodded into becoming beta islet cells to treat diabetes, blood cells to create new blood free of cancer cells for a leukemia patient, or neurons to treat neurological disorders • Can reprogram skin cells into active motor neurons, egg and sperm precursors, liver cells, bone precursors, blood cells • patients with untreatable diseases such as, ALS, Rett Syndrome, Lesch-Nyhan Disease, and Duchenne's Muscular Dystrophy donate skin cells iPSC reprogramming research Question: Do you expect iPSC cells to have exactly the same characteristics as ESCs? But expression of some genes may be better indicators than others Mice from iPS cells Organoids 3D structure grown from stem cells ❑ consisting of organ-specific cell types through cell sorting, spatially restricted lineage commitment ❑ shows some evidence of organ function Causes for concern: Pluripotent/totipotent stem cells could theoretically be used to grow a human embryo in vitro. Therefore, if anyone wants to do research in that area, we need to know. This is currently not possible. Cells or organoids with neural (brain) potential could be generated. This is no problem, unless an investigator wants to implant those in an animal brain. Chance of animal with human cognition is very low, but we need to know and follow up. Organoids: brain organoids in vitro are not a problem. But what if we could achieve some form of cognition? Science fiction right now, but we need to know. Human embryos: can be grown to some extent in vitro. How far can this go? Currently 14 days or until certain developmental milestones are reached, whichever comes first Chimeras Preliminary studies allowed, carrying to term not Allowed with monitoring in place Chimeras Why might we be concerned: • If the human cells contribute to the pig brain: Is this still a pig? Does it need special protections? Or should it be destroyed right away because it breaches species boundaries? • Or if the pig develops more human facial features? • Or if the human cells contribute to the germline, i.e. to oocytes or sperm cells? Even if data are provided that might justify carrying to term, breeding such animals is not allowed (because of risk of inadvertent germline transmission) Pre-implantation genetic diagnosis and selection • • allows couples to have a child that does not carry the genetic disease about which they are concerned instead of seeking to change the genes in unhealthy embryos, can select the healthy embryos themselves for implantation in the mother Types of cloning Recombinant DNA technology: DNA/ molecular/ gene cloning gene 1 gene 2 Reproductive cloning: uses cloning procedure to produce a clonal embryo which is implanted in a woman's womb with intent to create a fully formed living child--a clone Therapeutic cloning: uses cloning procedure to produce a clonal embryo, but instead of being implanted in a womb and brought to term, it is used to generate stem cells Is cloning an organism the same as cloning a gene? • Cloning an animal refers to making an exact genetic copy of that organism. • Cloning a gene means isolating an exact copy of a single gene from the entire genome of an organism. ➢Usually involves copying the DNA sequence of that gene into a smaller, more accessible piece of DNA, such as a plasmid Reproductive cloning Defined as the deliberate production of genetically identical individuals ➢asexual form of reproduction; ➢all the progeny’s genes would come from a body cell of a single individual Technology used generates an animal that has the same nuclear DNA as another currently or previously existing animal Gurdon's* experiment: cloning a frog via nuclear transfer *The Nobel Prize in Physiology or Medicine 2012 was awarded jointly to Sir John B. Gurdon and Shinya Yamanaka "for the discovery that mature cells can be reprogrammed to become pluripotent" Cloning of Dolly the sheep, the first cloned mammal Dolly was shown to be genetically identical to the white-faced sheep and not to the blackface ewe, which clearly demonstrated that she was a successful clone Why Clone? • To mass produce organisms with desired qualities, • e.g., a prize-winning orchid or a genetically engineered animal • To replace lost or deceased family pets and repopulating endangered or even extinct species • To create genetically modified organisms, e.g., pigs, from which organs suitable for human transplants could be harvested • xenotransplantation : transplant of organs and tissues from animals to humans Idaho Gem, the world's first cloned mule. He is an identical genetic copy of his brother, a champion racing mule called Taz, and the first clone to be born in the equine family. Five cloned piglets, born in Virginia, USA on March 5, 2000. The world's first cloned piglets were produced by PPL Therapeutics from an adult sow using a slightly different technique from the one that produced Dolly. Reproductive cloning: environmental applications • In 2001, Noah, a clone of an endangered wild ox was born • BUT died from an infection about 48 hours after its birth. • Other endangered species that are potential candidates for cloning: ❑ African bongo antelope ❑ Sumatran tiger ❑ giant panda Noah, the first endangered animal to be cloned Celebrity Sheep Died at Age 6 Dolly, the first mammal to be cloned from adult DNA, was put down by lethal injection Feb. 14, 2003. Prior to her death, Dolly had been suffering from lung cancer and crippling arthritis. Although most Finn Dorset sheep live to be 11 to 12 years of age, postmortem examination of Dolly seemed to indicate that, other than her cancer and arthritis, she appeared to be quite normal. HOWEVER, cloning extinct animals a much greater challenge Egg, surrogate needed to create the cloned embryo would be of a species different from the clone e.g., wooly mammoth to be cloned using an elephant as surrogate (Feb. 2011) Human reproductive cloning: how it might work Misconception #1: Instant Clones! A common misconception is that a clone, if created, would magically appear at the same age as the original. Cloning = alternative way to create an embryo, not a fullgrown individual. Misconception #2: Carbon Copies! Differences in the environment, development may significantly affect the phenotype of the clone CC, the first cat to be cloned, and Rainbow, the donor of CC's genetic material. Why don't they look exactly alike? Risks of cloning In 2002, researchers at the Whitehead Institute for Biomedical Research in Cambridge, Massachusetts, reported that the genomes of cloned mice are compromised ❑In >10,000 liver and placenta cells of cloned mice, ~ 4% of genes function abnormally ❑Abnormalities do not arise from mutations but from changes in the normal activation or expression of certain genes Early successes in human cloning 2001 – First cloned human embryos (only to 6-cell stage) created by Advanced Cell Technology (USA) 2004* – Claim of first human cloned blastocyst created and a cell line established (Korea) – later proved to be fraudulent *Hwang, W.S., et al. 2004. Evidence of a Pluripotent Human Embryonic Stem Cell Line Derived from a Cloned Blastocyst. Science 303: 1669-1674. Should humans be cloned? • Only 1 - 2 viable clones produced for every 100 experiments • ~30% of clones born alive are affected with "large offspring syndrome" , other debilitating conditions • Several cloned animals have died prematurely from infections and other complications The same problems would be expected in human cloning! Should humans be cloned? Also, scientists do not know how cloning could impact mental development ❑While factors such as intellect and mood may not be as important for a cow or a mouse, they are crucial for the development of healthy humans Any attempt to clone humans at this time is considered potentially dangerous and ethically irresponsible