* Stem Cells, A Molecular Biology and Pharmaceutical Biotechnology Spring 2009 Audrey Minden, Ph.D. Department of Chemical Biology Ernest Mario School of Pharmacy Susan Lehman Cullman Laboratory for Cancer Research, Room 205 (732)-445-3400 (X 253) email@example.com * Stem Cells: Part I. Introduction to stem cells and stem cell research Part II. Use of stem cells in human disease treatment Part III. Techniques for generating embryonic stem cells Part IV. Challenges with studying embryonic stem cells * Part I. Introduction to stem cells and stem cell research Characteristics of Stem cells Some important concepts in stem cell research a. Differentiation vs. proliferation b. Cell replication and cell cycle c. Why stem cells hold promise for disease treatment 3. Four major types of stem cells a. Embryonic stem cells b. Fetal tissue stem cells c. Umbilical cord stem cells d. Adult stem cells 4. Potency of stem cells a. Totipotent b. Pluripotent c. Multipotent d. Unipotent Which of the following is a characteristic of stem cells? They are terminally diferentiate b they can divide indefinitely c. They are found mostly in the nervous system. Answer c * What are stem cells? - Stem cells are cells that serve as a normal reservoir for new cells needed to replace damaged or dying cells. - Stem cells have a lasting ability to multiply when called upon. 1. Characteristics of Stem cells Part 1 * Three characteristics of stem cells: - Not terminally differentiated -- to be discussed further - Can divide without limit - When a stem cell divides, each daughter cell has two choices: - remain a stem cell - begin a differentiation process - at least one has to remain a stem cell * Stem cells can replicate and produce two daughter cells An important characteristic of stem cells is that only one, but not both, daughter cells go on to differentiate. The other must remain an undifferentiated stem cell to maintain the reservoir. * Proliferation In culture, they look round, And don?t stick to surface Differentiation- Becoming a certain types of cell 2. Some important concepts in stem cell research a. Differentiation vs. proliferation * The Cell- All individuals are made of cells. Cells arise only from other cells. * Cells in different layers of the skin Tissues and organs are made up of individual cells with specific functions arranged in a specific order. Skin, for ex, is comprised of several thin layers of cells that are nourished by small blood vessels. [a fig?]. These are differentiated cells. Increasing diffentiating as you go up. Is terminally proliferative * b. Cell replication and the cell cycle Cells replicate by going through a cell cycle and dividing into two cells: The cell cycle: The cell is capable of completely replicating itself [Fig p. 15 - Kiessling]. All cells contain all the genes that make up the entire organism, and these are formed into chromosomes. All the DNA replicates when the cell replicates. * Phases of the cell cycle: M, G1, S, G2 2n 4n 2n 4n 4n 2n * Cells that undergo terminal differentiation usually do not replicate any more Differentiation versus proliferation: Once a cell replicates, it often undergoes additional changes that allow it to become a specialized cell and carry out specific tasks. This is terminal differentiation. It is usually accomplished in response to a specific factor that impacts the new daughter cell. F or example, NGF can cause a cell to become a nerve cell. Cells that undergo differentiation usually do not divide any more. T hey exit the cell cycle and enter a mitotically quiescent phase termed G0. [My cell cycle fig p. 6, modify title]. Most cells that enter G0 do not ever replicate again, but some cells, like stem cells, can be recruited from G0 to re-enter G1. Stem cells are not necessarily constantly dividing, but they can respond to a signal and exit G0 to go through G1. To be considered a stem cell the cell must (1)be able to exit G0 and enter the cell cycle, (2) must be undifferentiated, (3) must be able to produce daughter cells that are also undifferentiated, t (4) o renew the stem cell population, and (5) must be able to produce cells that will differentiate into a specific type. M G 1 S G 2 i n t e r p h a s e G o restriction point * Adult diseases can be broadly divided into three problems at the cellular level: - Too much cell division can lead to tumors - Too little cell division can lead to inability to repair damaged tissue - Defective cell function diseases such as hemophelia Stem cells are a self-renewing population of cells that have the potential for use in treating human diseases Adult diseases can be broadly divided into three problems at the cellular level: 1) too much cell division Can lead to tumors 2) too little cell division Can result in the inability to repair damaged tissues and organs, such as severed spinal cord nerves or weak heart muscles 3) defective cell function Can result in diseases such as hemophilia (lack of synthesis of blood clotting factors by liver cells). c. Stem cells and disease treatment: * Treatment for human diseases: - Surgery or pharmacological intervention - Renewal of damaged tissue (Stem cell therapy). Historically, treatment for adult diseases has been surgery or pharmacological intervention Example - heart transplants to replace weakened hearts, drugs to correct abnormal heart rhythms. A new concept is encouraging the growth of new cells, for ex, repairing weak heart muscle by encouraging the growth of new muscle cells. This new way of thinking of curing diseases brings STEM CELLS to the forefront. * a. Embryonic stem cells (ES cells) b. Fetal stem cells- an older embryo c. Umbilical cord stem cells d. Adult stem cells- ex hematopoitetic stem cells 3. Four major types of stem cells * 4. Potency of stem cells: a. Totipotent- example fertilized egg b. Pluripotent c. Multipotent d. Unipotent (progenitor)- proliferating, not Differntiaing, programmed to become one type of cell Totipotent stem cells would be taken from an early fertilized eggs that only has about 8 cells or less. They are totiptent because they can give rise to all tissues, including extraembryonic tissue (including things like the placenta). An egg is also totipotent. Most ES cells are pluripotent. These are taken from an early embryo. They are taken from the inner part of the embryo (ICM). They can give rise to all tissues but not to extraembryonic tissue, thus they are called pluripotent, rather than totipotent. Most adult stem cells are multipotent. They can give rise to a number of different cell types, but not to all tissue types. Example - adult hematopoietic stem cells, these can beomce many types of blood cells, But no a neuron or skin cell * a. Embryonic stem cells: (pluripotent) 3. Four major types of stem cells * Totipotent here Inner ball: Where embryonic stem cells (pluripotent) Outer part is extraembyonic tissue, yolk sack, etc Don?t need to remember names of lineages. ICM is important here to remember, this is where we isolate embryonic stem cells from * Most of the existing human ES cell lines in the world were derived from unused embryos created for couples seeking IVF treatment. They are derived from a blastocyst. A very early stage embryo. Less than a week after a human egg is fertilized, the developing embryo contains about 100-150 cells that have not yet begun to differentiate. The embryo at this stage is a hollow ball, called a blastocyst. The blastocyst consists of an outer cell mass which would later become the placenta, and an inner cell mass (ICM), which would become the fetus. Inside the womb these ICM cells would continue multiplying beginning to specialize by the 3rd week. The embryo, called a gastrula at this stage, would contain three distinctive germ layers (endoderm, mesoderm, ectoderm) whose descendants would ultimately form hundreds of different tissue types in the body. Stem cells are from the ICM before it differentiates into the three tissue types. They are thus pluripotent, and can form any cell type in the body. To make the cells, researchers remove the ICM from the blastocyst. The ICM is placed on a plate containing feeder cells, to which it will attach. In a few days, cells grow out of the ICM and form colonies. These cells are called ES cells if they meet two criteria: 1) they display markers known to characterize ES cells, 2) they undergo several generations of cell division, demonstrating that they constitute a stable, or immortalized, cell line. ---- These ES cells can differentiate into assorted cell types in a culture dish * Generation of Embryonic Stem (ES) cells Review of early embryogenesis. - The egg - Meiosis - Fertilization - Cleavage - Blastocyst and Gastrula stages - ES cells are made from blastocyst stage cells - Implantation * The egg is totipotent The egg is a large single cells with a large nucleus They are difficult to study because they do not grow in culture The only source of eggs in the lab is surgical removal Understanding the development of embryonic stem cells requires some understanding of the egg, and how it develops into an embryo. The egg: The egg is a large single cell, with a large nucleus. The egg has a unique cell cycle. Given the importance of reproduction of the species, there must be multiple safeguards to ensure an adequate supply of eggs with the potential to give rise to new offspring. Eggs are hard to study though because unlike many other cells, the egg cell cycle can not be supported in culture. This is a big road block to nuclear transplant stem cell scientists. In the absence of a laboratory culture system to generate oocytes, the sole source of human eggs is surgical recovery from the ovaries of normal healthy adult women. Review of early embryogenesis. The egg: * Meiosis, and the formation of polar bodies 2N 4N 2N 1N 2N 4N 2N * Oocytes arrest at two stages Developing eggs are called oocytes. Vertebrate oocytes (developing eggs). Meiosais is arrested twice. The first time it is arrested at the diplotene stage of meiosis, during which oocytes grow to a large size. Oocytes then resume meiosis in response to hormonal stimulation each month, and complete the first stage of meiotic division. Asymmetric cytokinesis gives rise to a small polar body. Most vertebrate oocytes are then arrested again at metaphase II. (already discussed) * Meiosis, and the formation of polar bodies 2N 4N 2N 1N 2N 4N 2N Haploid cells at the end with one copy of each chromosome * Fertilization induces completion of meiosis and emission of a second polar body Fertilization induces the transition from metaphase II to anaphase II, leading to completion of meiosis and emission of a second polar body Fertilization * Meiosis, and the formation of polar bodies 2N 4N 2N 1N 2N 4N 2N At the end of meiosis, cells is splitting but one in reality is polar body. Instead of ending up with 4 cells, you end up with 1 plus 3 polar bodies in egg cells * The zygote and cleavage: The first cleavage 4 cell to 16 cell stage - formation of morula Cleavage * After fertilization by sperm: Zygote: The first thing to form is the zygote [Fig 5.1]: They zygote is the unique stage in which the pronuclei lie adjacent to each other within the egg cytoplasm - the pronuclear egg stage. DNA replication occurs in each pronucleus. ---- Cleavage: Once activated, the egg initiates a series of unique cell cycles, each of which results in identical daughter cells with smaller volumes. The reduction in size is why this is termed cleavages, rather than divisions. At the two cell stage the cells are called blastomeres. The two cell stage marks the first co-mingling of maternal and paternal genes. The third cleavage leads to the onset of the morula stage. This starts at the 16 cell stage, at about day 3. By the 32 cell stage the cells need to grow in size as well as just divide. Each cell is still totipotent at this stage. Most embryos enter the uterus at the morula stage. ----- By the 16 cell stage, some cells are different from other cells. At the 8 cell stage, each blastomere (cell) has contact with other blastomeres and the outside of the cell cluster. But by the 16 cell stage, one or two blastomeres is completely trapped inside the cell mass and have no contact with the outside world [Fig p.96].Thus you start to have cells in different conditions, and come to an end of the time when all cells are equal and totipotent. (But still pluripotent). Note cells change in terms of what their enviroment is like In 4 cells stage, every cell is in same enviroment, touching outside and each other But in the 16 stage, some are touching the outside and some don?t. * Blastocyst stage, gastrula stage, implantation * At about day 5 a cavity forms and a blastocyst forms. It has started to attach itself to the inside wall of the uterus. The enclosed cells inside the blastocyst signal the an early embryonic commitment event. The cells on the outside become committed to giving rise to the placenta, those inside become the embryo. The outside cells are called trophoblast cells, the inside cells are the inner cell mass cells (ICM). The decision to become either ICM or trophoblast cell is characteristic of the type of commitment decisions that occur throughout embryonic development. One of two paths is chosen and once chosen, back differentiation does not occur. Only those cells that have not taken the next step of differentiation remain available for the full spectrum of differentiation events. These are also the basic properties of stem cells. ICM and trophoblast cells develop distinct characteristics with respect to cell cycle times and gene expression. ES cells are derived from the ICM of the blastocyst. Next a gastrula would form. Cells of the ICM have the potential to become several tissue types?. * What can embryonic stem cells do? Stem cells can differentiate into many different types of cells. Stem cell differentiation Into dopamine neurons If u grow cells in culture, you can cause them To diffentiate into many different types of Cells Don?t memorize this note outcome, just notice can differentiate into many types of cells * Cells derived from: Cell types they give rise to: brain Good source of dopamine neurons developing retina neural crest cells melanocytes, neurons, glial cells, bone, cartilage, smooth muscle, blood hematopoietic cells lymphocytes, monocytes, neutrophils, rbcs, megakaryocytes liver, pancreas, gut organs derived from endodermal cells Fetal stem cells (multipotent) have been isolated from numerous tissues: Fetal stem cells have been isolated from fetal nervous tissue. Stem cells have been identified from the developing retina. This led to discovery that adult retinas also contain a small portion of self renewing stem cells. The neural crest cell is a neural stem cell. Its interesting because it is capable of differentiating into several different types of cells. They are highly migratory and give rise to many cell types including pigment producing cells (melanocytes), neurons, glial cells, bone, cartilage, smooth muscle, and blood cells. Thus - they have remarkable plasticity. They have a remarkable pluripotent nature. Hematopoietic stem cells have been isolated successfully from fetal and adult animals. They give rise to blood and immune cells. Fetal stem cells found in organs deriving from endodermal embryonic cells - liver, pancreas, and gut. Interestingly, however, stem cells are also found in these organs in the adult. * c. Umbilical cord blood stem cells (multipotent)- less ethical dilemma, less rejection d. Adult stem cells usu considered multipotent (unipotent (progenitor), multipotent, some may be pluripotent) Considered multipotent stem cells because although they naturally become blood cells and immune system cells, they have the potential to become many different types of cells. Cord blood is rich in stem cells. They are desirable because they cause less rejection problems because they have not yet developed antigens that can be recognized by the immune system. And umbilical cord blood does not have mature immune system cells that can attack the recipient (graft versus host disease). (But they have high potency) * Some examples of adult stem cells: - Blood vessel cells - Sperm - Bone marrow cells - Skin cells Some examples, 1) blood vessel cells can regenerate when vessel is damaged. 2) spermatogenesis. Fetal testes is populated with stem cells that are not stimulated to produce more cells until puberty. Once sperm production is initiated, it continues throughout life. Billions of new sperm are produced daily. 3) bone marrow. Adults maintain a population of blood stem cells that also produce billions of new blood cells each days. 4) Skin ?. A fundamental characteristic of all adult stem cells is that their developmental potential of adult stem cells is restricted.. Only some adult tissues contain stem cells at all. * Hematopoietic stem cells Bone graft Multipotential stem cell Hematopoietic stem cell Platelets Macrophage Erythrocytes Eosinophil Neutrophil Megakaryocyte Mast cell Basophil T lymphocyte Natural killer cell Dendritic cell B lymphocyte Lymphoid progenitor cell Myeloid progenitor cell Monocyte Marrow Bone Bone marrow is home to the most studied model of adult stem cells, haematopoietic stem cells, which give rise to progenitors of the blood and immune cell families. All cellular elements of the blood, including cells of the immune system, arise from multipotent hematopoietic stem cells in the bone marrow. These multipotent stem cells divide to produce two types of stem cells - a common lymphoid progenitor that gives rise to cells of the acquired immune system, and a common myeloid progenitor that gives rise to cells of the innate immune system. Once differentiated, the cells have limited life spans, ranging from less than a day to a few months. They are continually replenished by the division of a common stem cell, the multipotent stem cell. The maintenance of the differentiated blood cell population is dependent on the continual proliferation of the stem cell, because the differentiated cells themselves can no longer proliferate. This is the major difference between the bone marrow stem cells and differentiated blood cells - the loss of ability to divide again. Hematopoietic stem cells are especially interesting because bone marrow transplants are a type of stem cell therapy. In fact, they represent one of the first examples of stem cell therapy. Used in blood diseases such as leukemia * Skin stem cells: The skin is a good example of a tissue made up of different types of cells, some of which are differentiated, and replenished by stem cells. Skin is made up of many cell types, but the defining component is the epidermis. The epidermis is frequently damaged, and thus needs an efficient repair and renewal mechanism. The epidermis is multilayered (stratified) epithelium composed largely of cells called keratinocytes. They are named keratinocyte because their specialized function is to produce keratins, an intermediate filament protein which gives the epidermis its toughness. These cells are arranged in layers [Figure p. 1261, Alberts]. The innermost layer contains the basal cells. These are usually the only cells that divide. Above this layer are several layers of larger prickle cells, the granular cells. Here the cells are sealed together in a water proof barrier, which is a major and essential function of the skin. The outermost layer consists of dead cells whose intracellular organelles have disappeared. They are reduced to flattened scales, or squames, filled with densely packed keratin. Stem cells aren?t necessarily constantly dividing, but they can respond to a signal and exit G0 into G1: Skin is wounded. A cascade of emergency signals is released by the damaged cells. A blood clot forms. Underlying skin cells (the dermis) knit together to seal the wound against fluid loss and infection. They turn on synthesis of keratins, to become skin cells. The stem cells beneath the dermis receive signals to begin to divide to replace the epidermal cells that are moving up to become dermis. During this process the skin stem cells that have been resting at G0 must awaken and enter G1. The stem cells will continue to produce daughter cells until the damaged area is repopulated. Skin has more stem cells than other tissues. * Part II. Stem cells in human disease treatment A. Neurodegenerative diseases. i. Parkinson?s disease ii. Alzheimer?s disease iii. Disease involving lack of myelin a. Multiple sclerosis b. Krabbe?s disease c. Leukodystrophy iv. Lou Gehrig?s disease (ALS) v. Spinal cord injury B. Tissue system failures i. Diabetes ii. Cardiomyopathy iii. Liver failure Different types of stem cells that may be used in disease treatment Diseases for which stem cell therapy holds promise: 4. Embryonic versus adult stem cells * What types of stem cells can be used in treatment of disease? Embryonic stem cells (ES cells) Fetal stem cells Adult stem cells 1. Different types of stem cells that may be used in disease treatment * Embryonic Stem (ES) cells: These cells have the most potential for treatment of disease because they are pluripotent * Embryonic stem cells can differentiate into many different types of cells. Stem cell differentiation Into dopamine neurons * ES cells can form tumors if used directly Therefore an important part of stem cell research is developing ways to start the differentiation process in culture Researchers are studying ways to trigger ES cells to differentiate into many types of cells ES cells that have already started along the differentiation process are the best ES cells to use for stem cell therapy * Differentiation of Embryonic stem cells into dopamine neurons. * Fetal stem cells in disease treatment: Stem cells from embryonic brain - Parkinson?s disease treatment Use of adult stem cells in disease treatment: The use of adult stem cells in therapy would be ideal for many reasons: - Ethical reasons - Easier to obtain than fetal and embryonic cells - Can come from the patient and thereby eliminate rejection problems * A. Neurodegenerative diseases i. Parkinson?s disease ii. Alzheimer?s disease iii. Diseases that involve lack of myelin a. Multiple sclerosis (MS), b. krabbe?s disase, c. leukodystrophy iv. Lou Gehrigs Disease (ALS) v. Spinal cord injury 2. Diseases for which stem cell therapy holds promise Until the 1990s a central dogma of neuroscience was that adult mammalian nerve cells could not regenerate and damaged nerves could not repair. This differed from amphibians and other lower animals that could regenerate nerves as well as limbs. This changed, as scientists began to find ways to observe proliferating cells, and found that there is some proliferation on neuronal cells in adult brain. Later, renewal of neurons in some situations could be observed, and most recently, neuronal stem cells have been identified, which can differentiate into neuronal cells in culture. Nevertheless, growth of new neurons is extremely inefficient, and it is not clear why stem cells do not renew them efficiently, as they do in some other tissues. The challenge now is to learn to stimulate neuronal stem cells to multiply and differentiate when needed to repair damaged nerves. One promising possibility is the possibility of injecting stem cells of some sort into a damaged brain to speed up the repair process. This possibility holds promise for several diseases. [SLIDE] * i. Parkinson?s Disease Degeneration of dopamine producing nerve cells in the substantia nigra This part of the midbrain helps control movement These cells produce dopamine, a neurotransmitter Once they die the cells don?t grow back Patients have loss of muscle control, tremors, stiffness Parkinson?s results from degeneration of dopamine-producing nerve cells in the substantia nigra, a tiny area of the midbrain that helps control movement [Fig.p.159]. Once these nerve cells die they do not seem to grow back, at least not at a significant rate. The cells of the substantia nigra release a chemical called dopamine, which is one of a family of neurotransmitters, molecules that elicit a response from nerve cells. Substantia nigra neurons communicate with nerve cells in the striatum, which play a major role in controlling body movements. The result of loss of substantia nigra neurons is loss of muscle control, tremors, and stiffness. The cause of the loss of this specific type of brain cell is not known. * Treatments for Parkinson?s disease: Traditional methods rely on the use of L-dopa L-dopa is converted to dopamine Regulating the dose is difficult Fetal cells This area is under investigation Transplanted adult nerve cells do not establish the right connections, but there are promising results with fetal nerve cells (from 7-9 wk fetuses) These have been done on human patients and monkeys Some success, but also many failures Embryonic stem cells Researchers have developed neural precursors from ES cells in culture Current treatment focuses on the administration of a chemical, L-dopa, which is converted in the brain to dopamine and helps make up for the loss of dopamine production by substantia nigra neurons. A big problem with L-dopa is regulating the dose. Too much can lad to involunatary twitching, and repetitive muscle movements, or too little leads to difficultiy moving. And after a time, the body no longer responds to L-dopa and the symptoms worsen. Memory can also become impaired. Use of fetal stem cells in treating Parkinson?s disease is a new treatment under investigation: Transplanted adult nerve cells do not establish the appropriate connections, but there have been promising results using fetal nerve cells, from 7-9 wk old fetuses. Studies have been carried out in human patients as well as in monkeys Some improvements have been seen There have also been many failures however, and rejected tissues. This leads to the third and possibly most promising potential treatment which is the use of embryonic stem cells. ES cells can not be used directly because of the development of teratomas Instead, highly purified neuronal precursors need to first be developed in culture * Five stage method for inducing cultured ES cells to differentiate into neurons (dopamine and serotonin) These cells were grafted into brains of rat models of Parkinsons Rats showed remarkable improvement Use of ES cells in Parkinsons treatment Researchers reported a 5-stage method of inducing ES cell cultures to differentiate into neurons [Fig.p.161]. Using chemicals, the ES cells were first used to differentiate into pluripotential neurons. Next, two types of neurons were developed, those that produce serotonin and those that produce dopamine. After the growth factor was removed from the cells, the neurons matured into fully functional nerve cells. These researchers reported a transcription factor called nuclear receptor related - 1 (Nurr1) that plays a role in the differentiation of dopamine neurons. They reasoned that if they could cause that transcription factor to be expressed in the neurons developed from ES cells, they could enrich the population of precursors for dopamine neurons. They used transfection, to ensure expression of Nurr1 by some of the ES cells. The cells now expressed Nurr1 under a strong promoter. These are now called transgenic ES cells. They took these cells and used the 5 step method. Now the dopa cells express Nurr1. It worked - they enriched the population of dopa cell precursors. They needed to test their cell lines in an animal model. Rats that have been chemically treated to kill dopamine neurons are a good model for Parkinson?s. After treatment, the rats have impaired motion, like Parkinson?s patients. They grafted a half million of the new neurons transgenic for Nurr1 into the rats midbrain. These cells were amazingly integrated properly into the mouse brains. They saw remarkable improvement in the grafted rats. No tumors were found. Thus, ES cells can provide an unlimited source of controlled cells that can integrate into a host without causing tumors. To avoid rejection, the ideal therapy would be stem cells derived from the patients themselves. * ii. Alzheimer?s 4 million Americans have Alzheimer?s today This number is expected to triple in 4 decades Nerves in the hippocampus and frontal lobes are affected first All parts of the brain are eventually affected The normal repair mechanism is not sufficient Stem cells could provide the boost needed to balance the destruction Unlike Parkinson?s stem cells would need to migrate to many parts of the brain Alzheimer?s Alzheimer?s was discovered when Dr Alois Alzheimer in 1906 noticed tangles and clumps called plaque in a patient with dementia. 4 million Americans have Alzheimer?s today. This is expected to triple in the next 4 decades. The hippocampus, where memories are laid down, and the frontal lobes, the seat of logical thought, are affected first. The affected nerves produce a neurotransmitter called acetylcholine. These cells send out long axons to distant parts of the brain. All areas of the brain are eventually affected. For stem cells to work they will need to do more than take up residence in just one area (as in the substantia nigra in parkinson?s). They will need to migrate to all the various structures of the brain that are damaged and integrate into the existing neurons. In Alzheimers, the normal repair mechanism is not fast enough to compensate for the disease. Stem cells could provide the boost needed to balance the destruction. Research has recently found a genetic compontent to alzheimers. A specific allele of the apolipoprotein E gene predisposes a person towards Alzheimers. Maybe stem cells could be engineered to eliminate this allele, and they might halt the onslaught and even reverse damage. * Stem cell research and Alzheimer?s Neural cells were generated from human ES cells These were injected into aged memory impaired rats. Rats showed considerable improvement. There have been no trials in humans yet, but research with rodents is encouraging. Aged memory impaired rats were injected with neural stem cells Stem cells spread throughout the brain Existing cells rejuvenated Treated rats did better than control in water mazes In some cases they even did better than younger rats Human ES cells had been the source) * iii. Diseases that involve lack of myelin: a. Multiple sclerosis b. Krabbe?s disease c. Leukodystrophy Oligodendrocytes are a cell type which send out processes called myelin, which insulates the axons of nerves Other brain syndromes: Another important cell type in the brain is oligodendrocytes. They send out processes to the nerve axons and insulate them from the surrounding cytoplasm. The insulation is called myelin and without it, nerve impulses damp out, slowing, or halting [Fig.p.165]. Diseases that involve lack of myelin include multiple sclerosis, krabbe?s disease, and leukodystrophy. Would stem cells be able to penetrate all the spaces necessary to myelinate axons? * Stem cell research and myelin diseases. Would stem cells be able to penetrate all the space necessary to myelinate axons? Use of mouse models deficient in myelin - These mice develop tremors, called shiver or twicher mice Researchers used human fetal stem cells or neural stem cells derived from ES cells These cells are injected into the brains The stem cells differentiated into oligodendrocytes and wrapped naked axons with myelin They migrated to the correct places In 60% of the mice, the tremors disappeared * MS clinical trial MS is a disease where the body?s immune system attacks the central nervous system -WBCs attack the protective myelin sheaths surrounding nerve fibers in the brain and spinal cord -In the trial, patients? immune cells were destroyed. -Patients were then injected with bone marrow stem cells -17 of 21 patients improved, suffering fewer problems with balance and vision, none of them declined over 2-4 years * iv. Lou Gehrigs Disease (ALS): Characterized by death of motor neurons Stem cells have held promise in mouse models: - Mouse models make use of a virus that destroys lower motor neurons. - These mice drag their hindquarters as they walk - Stem cells were injected into the spines. - Stem cells migrated to the damaged area in about 60% of the mice, and differentiated into neurons. - About half the mice recovered control of their hindquarters. Lou Gehrigs Disease (ALS) is characterized by death of motor neurons. Mouse models of ALS have been generated. Used a virus called Sinbis which destroys the lower motor neurons of the mice, causing them to drag their haindquarters when they walk. The neural death is a good model for ALS, which is characterized by death of motor neurons. They injected stem cells into spines of the paralyzed mice and gave them time to migrate. They found that stem cells migrated to the damaged area and that about 6% of them differentiated into neurons. Half of the mice recovered control of their hindquarters. --- Using these techniques, scientists hope to use stem cells to treat many other disorders of the brain. Animal trials are being carried out for Hungton?s disease, Alzheimer?s, strokes, Tay-Sachs, and alcohol impairment. * v. Spinal cord injury: Stem cells have shown promise in mouse models: - Hindquarters were paralyzed from a spinal cord injury - 9 days later, mice were injected with neuronal precursors derived from ES cells that were treated with retinoic acid - Used cyclosporine to inhibit immune response and rejection - A few weeks later, scanned brains and spinal cords - Some new projections formed, newly sprouted axons were visible - After a month, several mice recovered some mobility. Spinal cord injury: Studies cited before indicate that new nerve cells can be encouraged to grow and integrate with a host brain. But what about a damaged spinal cord? And more than just nerve cells is required. After damage, a cyst called a syrinx is formed, and the nerves at the injury site become demyelinated, so oligodendrocytes are also needed to infiltrate and insulate the nerve cells. Mouse models whose hindquarters were paralyzed from a spinal injury. 9 days after injury the mice were injected with neuronal precursors that had been derived from ES cells treated with retinoic acid. They were injected into the syrinx. The mice were given cyclosporine to inhibit immune response and prevent rejection. A few weeks later some mice were killed and their brains were scanned for new cells. Some new projections formred, newly sprouted axons were visible, after a month, several of the mice recovered some mobility and coordination. None developed tumors. * v. Spinal cord injury: Studies using marmosets (primates): - Use of fetal stem cells, amplified to over a million cells - The fetal stem cells were obtained from aborted fetuses. - Unlike ES cells, fetal cells can not be immortalized in culture. Marmoset (primates) whose arms were paralyzed due to spinal cord injury showed improvements after the transplant. They used fetal stem cells, amplified to over a million cells. This was more affective if the stem cells were given at the right time frame after trauma - they had a better chance of differentiating into neurons. Fetal neural stem cells had been obtained from aborted fetuses for these studies. The fetal cells were cultured and expanded. Unlike ES cells, however, fetal neural stem cells have do not provide an immortal cell line in culture. * B. Diseases caused by tissue system failures i. Diabetes ii. Cardiomyopathy iii. Liver failure * i. Diabetes: Inability to manufacture or use insulin Insulin is a hormone that regulates uptake of glucose into cells - Insulin is normally secreted by specialized cells in the pancreas in islets of langerhorn region Without insulin, undigested sugars build up in the blood plasma instead of entering the glucose pathway in cells Since glucose metabolism is a principal energy source, the cells and tissues are energy deprived - Over time, there is widespread damage to tissues and organs Diabetes affects 170 million people worldwide. Its characterized by the inability to either manufacture or use insulin, the protein hormone that regulates the uptake of glucose into cells. Insulin is secreted by specialized cells within the pancreas that are organized into regions called islets of langerhans. Without insulin, undigested sugars build up in the blood plasma instead of entering the glucose pathways within cells. Since glucose metabolism is a principal energy source, the cells and tissues are energy deprived. Over time this causes widespread damage to tissues and organs. * - Type 1 diabetes: Juvenile onset: absence of insulin absence of islet cells in the pancreas Type 2 diabetes: usually affects adults nearly normal levels of circulating insulin The insulin does not stimulate normal glucose uptake This type of diabetes can often be controlled by diet and exercise Type 1 diabetes can be treated with islets transplanted from donated organs, but requires large amounts of donated pancreases -Type 1: Type 1 diabetes involves cell loss, and is therefore a likely candidate for stem cell treatment. Can be cured with islets transplanted from donated organs. But this requires large amounts of donated pancreases. Stem cell therapy would involve differentiating stem cells into islet cells or islet cell precursors, and then transplanting them into the patient. This raises the need to isolate pancreatic stem cells and develop methods to differentiate them into islet precursors. -Pancreatic stem cells from adult organs have been hard to identify and isolate. Pancreatic stem cells also appear to be fleeting in the developing fetus, so researchers are turning to embryonic stem cells. It would be ideal to derive pancreatic stem cells from the diabetic?s own cells, in order to avoid an immune response. -Mouse research: Researchers have developed a way to cause ES cells to differentiate into Insulin secreting cells. These cells were injected into the spleens of mice with chemically induced diabetes. The mice were soon producing their own insulin and normalizing their glucose levels. Some of the mice were essentially cured. Indicating that stem cells had successfully integrated into the bodies of the host mice. These results are promising, but still very inefficient, and researchers are working on improving this. -Later, primate ES cells were shown to be able to spontaneously differentiate into pancreatic cells. This was also done so with human ES cells but can not evaluate them further due to funding laws on ES cells. -Another approach involves regulating the immune response. Type 1 diabetes is an autoimmune disorder, where the diabetic?s own immune system targets the islet cells for destruction. If they are replenished, would they just be destroyed again? It might be possible to genetically engineer the cell cultures so they won?t be attacked by the immune system. The genes that encode the cell surface proteins that target the islet cells could be excised. ----- -Type 2 diabetes: If, as suspected, a genetic flaw lies behind type 2 diabetes, then genetic engineering could repair a deviant gene. The corrected stem cells would be transplanted back into the patient. Such systems have worked in mouse models of autoimmune diseases. * Stem cell therapy and type 1 diabetes Type 1 diabetes involves cell loss, therefore it?s a good candidate for stem cell therapy Stem cell therapy would involve differentiating stem cells into islet cells or islet cell precursors and then transplanting them into the patient Researchers have found ways to cause mouse ES cells to differentiate into insulin secreting cells. These were injected into mice with chemically induced diabetes Mice soon produced their own insulin, and some were cured, but the results are still inefficient Primate and human ES cells could also differentiate into pancreatic cells * Ideas for using stem cells to treat Type II diabetes Type II diabetes is likely caused by a genetic flaw. Researchers propose to use genetic engineering to repair the deviant gene in stem cells. The corrected stem cells would then be transplanted back into the patients. * ii. Cardiomyopathy: A disease characterized by weakened muscle fibers Diagnosed at a heart attack Cardiomyocytes: The cells that comprise the heart. These cells are capable of limited regeneration. A disease of the heart characterized by weakened muscle fibers. Affects 4.8 million people in the U.S. Usually its diagnosed at a heart attack. It has long been assumed that cardiac muscle, like neural tissue, does not regenerate in adults. After a heart attack, you don?t see regeneration. But now its thought that some limited regeneration occurs. Cardiomyocytes, the cells that comprise the pumping muscle of the heart, divide and start to repopulate the area. But the response is too little and too late. But researchers have reported that hematopoietic stem cells derived from bone marrow can, in vivo, differentiate into apparent cardiomyocyte precursors. Thus, these stem cells seem to be more ?potent? than expected?. Injecting these marrow cells into damaged rat hearts could heal the tissue. The healing included heart muscle and the vasculature that supports the heart. Injected cells were primitive enough to be able to differentiate into multiple tissue types. * Stem cell research and Cardiomyopathy: - Use of ES cells: Human ES cells can generate cardiomyocyte precursors when they are allowed to form embryoid bodies (EBs) Large amounts of these cells can be injected into the injured area Repair needs to take place immediately for good outcomes. - Use of adult stem cells: Hematopoietic stem cells derived from bone marrow can differentiate in vivo into apparent cardiomyocyte precursors Injecting these marrow cells into damaged rat hearts could heal the tissue Another group showed that human ES cells automatically generate cardiomyocyte precursors when they are allowed to form embryoid bodies. Large amounts of cardiac precursor cells can be generated in vitro and injected into the injured area. As usual, ES cells derived from the patient would be best. But the repair needs to take place immediately, longer wait times lead to less positive outcomes, but making your own cells can take a long time. Therefore it would be best for patients to have a bank of their own stem cells. In 2000 the AHA endorsed ES cell research and considered funding some studies. They said that ES cell research represents the most promising medicial and scientific research to fight cardiovascular disease. But when they announced their intentions, a mail in campaign spearheaded by a catholic archbishop convinced them to cancel their plans. There are still no federally funded studies that use human ES cells. * iii. Liver failure: - Adult stem cells hold promise for liver disease treatment: Hematopoietic stem cells may be a source of liver stem cells Researchers studied bone marrow recipients. They found that donor cells populated the liver, especially in a patient with recurrent hepatitis C - ES cells and liver disease treatment: ES were shown to give rise to hepatic cells after forming EBs. These were injected into the hepatic portal vein of mice. The cells integrated into the host liver and formed typical hepatic cells. Liver failure: In 2000 liver biopsies were examined from bone marrow recipients. They found that donor cells populated the liver, and to quite a large degree. (They looked at females with a male donor, and vv, so they could look at Y chromosome). The largest infiltration was in a patient who had recurrent hepatitis C, indicating that the infiltration was motivated by injury, and that bone marrow cells were recruited to differentiate into hepatic cells and repair damage. --- Mouse studies: ES cells can also give rise to hepatic cells. After allowing ES cells to form embryoid bodies, hepatic cells start to show up within 12 days. Some of the cells were injected into the hepatic portal vein of mice, where they integrated into the host liver and formed typical hepatic cells. * 4. Embryonic versus adult stem cells * ES cells are derived from early embryos and considered to be pluripotent. They can develop into any type of cell. Most of the existing human ES cell lines in the world were derived from unused embryos created for couples seeking IVF treatment. They are derived from a blastocyst. A very early stage embryo. Less than a week after a human egg is fertilized, the developing embryo contains about 100-150 cells that have not yet begun to differentiate. The embryo at this stage is a hollow ball, called a blastocyst. The blastocyst consists of an outer cell mass which would later become the placenta, and an inner cell mass (ICM), which would become the fetus. Inside the womb these ICM cells would continue multiplying beginning to specialize by the 3rd week. The embryo, called a gastrula at this stage, would contain three distinctive germ layers (endoderm, mesoderm, ectoderm) whose descendants would ultimately form hundreds of different tissue types in the body. Stem cells are from the ICM before it differentiates into the three tissue types. They are thus pluripotent, and can form any cell type in the body. To make the cells, researchers remove the ICM from the blastocyst. The ICM is placed on a plate containing feeder cells, to which it will attach. In a few days, cells grow out of the ICM and form colonies. These cells are called ES cells if they meet two criteria: 1) they display markers known to characterize ES cells, 2) they undergo several generations of cell division, demonstrating that they constitute a stable, or immortalized, cell line.--- These ES cells can differentiate into assorted cell types in a culture dish * Although the best cells for therapy, ES cells pose many problems. Ethical and legal problems, Problems with obtaining enough cells. Problems with rejection by the immune system * Recently, adult stem cells have also shown great promise in stem cell research Adult stem cells may be more potent than previously thought * Experiments to demonstrate the multipotent nature of adult bone marrow stem cells Injection into mouse blastocysts: In another exp, single bone marrow MAP cells were injected into mouse blastocysts to generate chimeras. Only a small percentage of the offspring were chimeras but those that were contained MAP cells in brain, retina, liver, intestine, kidney, spleen, bone marrow, and skin. [Fig. 143]. These studies suggest that the bone marrow MAPCs were intrinsically capable of contributing to tissues of different origin, or that they acquired the ability to do so during their time in culture. * Injection of bone marrow stem cells into veins of adult mice: Another test of the potential for MAPs to contribute to adult tissues: They were injected into blood veins of adult mice to determine if they could establish themselves in any tissues. After a few months, tissues of injected mice were examined. The MAPs were found in blood, bone marrow, spleen, lung, liver, and intestine [Fig.143]. These tissues are known to have their own stem cells. They were not found in skeletal muscle, heart muscle, brain, or kidney, all tissues known to be deficient in their own populations of stem cells. This suggests that there are rigorous control mechanisms for controlling stem cell populations in organs and that these may need to be overcome for stem cell therapy to be effective. Studies such as those above need to be repeated and tested further, to determine if indeed bone marrow cells from adult animals contain pluripotent stem cells capable of contributing to a wide variety of tissues. -----
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