Using Allogeneic Placenta Derived Mesenchymal Stem Cells to Treat Multiple Sclerosis

Oscar Villarreal, Arturo Zamarripa, Immanuelle Azebe-Osime, Alejandra Martinez, Shuhui Wan Abstract: In an attempt to discover more about the neurodegenerative disease that is multiple sclerosis (MS), we looked at the manner in which placenta derived mesenchymal stem cells, or commonly referred to as MSCs, helped to treat the mysterious condition that plagues many. In our experiment, mice will be induced into chronic experimental autoimmune encephalomyelitis (EAE), which simulates a disease, similar to multiple sclerosis, in mice. The brain of the mice will be looked at to see the effects of the EAE on the brain’s white matter and grey matter. MRI scans will be performed before they were infected with EAE and then they will be routine scans to see if there are any visible lesions caused by the EAE. The mice will then be administered MSCs derived from human placenta. This was done so biannually. The expected results from the placenta MSC treatment involved a regression of the EAE in the mice. The mice will be observed for a time period of one year to see the long-term effects of treatment using placenta derived MSCs. Objective: The main goal of this experiment is to test further if using placenta derived MSC could be an effective treatment for multiple sclerosis. In order to test this we will use mice with EAE and treat them with our MSCs. From the mice we should gather enough evidence, through various forms of information detailed in the methods section, to determine if the placenta derived MSCs have the same, or better, effect as bone marrow MSCs on MS. If desired results were obtained from this experiment, then placenta derived MSC have a potential to be a test on humans. And if the further clinic trials can prove the effectiveness of placenta MSC in patients with multiple sclerosis, then treatment using MSCs will be an efficient solution for multiple sclerosis. Introduction: One of the most researched diseases in the 20th and 21st century, Multiple sclerosis, as the name suggests, is the scarring of the axon’s myelin sheath. The neuron is made up of nucleus, dendrites, and the axon. For the nerves to send signals to the brain, action potential, a kind of electric charge, has to be sent through the axon. Some nerves have very long axon so the information doesn’t need to be passed from one neuron to another but for most of the neurons the action potential has to trigger synapses that secrete vesicles to other neurons, which then in turn triggers action potential until it reaches the brain. All of this happens in less than a blink of an eye. For example, when someone steps on a nail, the action potential is sent through on of the longest axons in the body to the brain so that the person jumps and removes their foot from the nail. With multiple sclerosis, the action potential cannot be effectively transmitted

4 Research has been done on MSC with these cells. but mostly bone marrow MSC because they are most accessible since they can be collected from the patient themselves. cartilage.between the neurons in the body or even passed along one single neuron because the myelin sheath. fat. placenta etc. Some of these cells go to the bloodstream in the brain. the light flickers when the lamp is on or it doesn’t turn on at all. But research has been done that shows that placenta-obtained MSCs are better than the bone marrow MSC for treating multiple sclerosis because the MSC can be cultured to different types of cells but the bone marrow’s is mainly bone.5 MSC has two ways it can help with multiple sclerosis. the axon cannot handle the change in membrane capacitance so the body overheats. Although the specifics are not know. myelin sheath in neuron cells resembles the insulator of a lamp. 1 So when a multiple sclerosis patient steps on a nail. which come from adults.2 Apparently the T cells don’t recognize the myelin sheath as part of the body and attack it and the glial cells that normally produce myelin for the neurons (oligodendrocyte). If the insulator is damaged. a protective sheath of the axon that acts like an insulator. demyelination of the axons is the pathological symptom of multiple sclerosis.2 Like the infamous cancer. they might feel it if the myelin of that axon is not damaged. which are cells from fertilized embryos. progressive relapsing.4 Therefore the t cells would stop attacking. The brain quickly fixes it by fixing the barrier but the cells are trapped. This type of stem cells is not controversial like embryonic stem cells.3 As discussed in previous paragraphs. is damaged. secondary progressive. multiple sclerosis has many types. It can either help with just remyelination or through its immunosuppressive ability to stop the t-cells from attacking the neurons. A type of white blood cells called the t cells somehow breakthrough the blood brain barrier and enters the brain. there are relapsing remitting. it is known that with its immunomodulatory properties. might feel pain after a longer time than usual or they might not feel it at all and keep on hurting themselves. the immune system fights against the substance by releasing white blood cells. According to the national multiple sclerosis society. Wharton’s jelly in the umbilical cord. MSC can be gotten from different places like the bone marrow. These MSCs are also “newer” than the bone marrow stem cells. and primary progressive and each have different characteristics. The best guess of cause of demyelination states that when a foreign substance entering the body. What generate the action potential now? The neurons were always reproducing only they would not be able to work properly. umbilical cord blood.2 With advancing research it has been discovered that mesenchymal stem cells could be the solution to the problem.6 Based on all the previous data collected from so many others working in the field of regenerative medicine. MSC can inhibit the t-cells and other cells in the immune system that are attacking the oligodendrocytes and the axon myelin. muscle etc. Therefore the damaging is not only done to the myelin sheath but also cells that could generate myelin sheath. According to the University of California San Francisco Multiple Sclerosis center. we plan to take MSCs and apply them in a slightly different . The MSC has reparative mechanism so that they can save the damaged cells. The difference between the bone marrow MSC and placenta MSC is that the placenta MSC have markers that show it can grow neurogenic cells. The neurons also give random signals to the brain when there is a change in temperature like exercise or hot baths (called Uhthoff’s syndrome) because of the partial or almost non-existent myelin sheath.

About 245 female mice.7 Once the mice have all been treated. and thus we have to use placentas to derive these stem cells. The mesenchymal stem cells will then be prepared for use according to the procedure done by Yonit Fisher-Shoval. Induction of EAE On Mice and Placenta MSCs Transplantation: In order for the experiment to be conducted. have proved to be a good source of MSCs. Every week 2 mice are sacrificed in order remove their brains and see how the disease is being affected by the MSC treatment. The mice are then continued to be observed for any signs of disease progression or any improvements. the sample size in this experiment is quite large relative to other case studies that have been performed. not much work has been done with them since they are not understood very well and thus this experiment should help to understand some of their characteristics better. From our 245 mice. The mice will continue to be observed for a time . These three controls will help to reduce the chances that unexpected factors will alter the result. the second control will consist of 15 mice that will only be treated with MSCs cells and not infected with EAE. And second. Research materials and methods: Culture of Human Placenta MSCs: For our experiment. C3H. so 20 placentas will be collected after Caesarian sections are performed and the correct paperwork is filled out. which. we will be using mesenchymal stem cells from human placenta. we need our subjects to have a disease similar to the human version of multiple sclerosis. The mice should be maintained at normal room conditions and should have full access to food and water. weakness. The mice should be taken care of according to proper protocol according to the Institutional Animal Care and Use Committee. We will need about 20 healthy placentas. and therefore the mice are going to be induced into chronic experimental autoimmune encephalomyelitis (EAE). and even death. this experiment will be conducted for a longer time period. this experiment is conducted using placenta derived mesenchymal stem cells. although the placenta is a satisfactory source of these stem cells. paralysis. symptoms include tail tonicity. and along with the larger sample. of age 6-8 weeks old and approximate weight of 20 grams will be used. The third control will consist of another 15 mice that will only be infected with EAE. they are observed closely to see any physical signs that the disease is affecting them. as previously mentioned. the remaining 45 will be left as controls.7 Animals: We will be using mice as our test subject for this experiment. This will hopefully show what the long-term effects of MSC treatment are and give an accurate idea if this should be pursued to be tested in human trials in the future as a viable treatment for multiple sclerosis. only 200 of them will be used to test the MSCs. which simulates the disease in mice. Firstly. The process by which the mice will be treated to be placed into EAE is performed as was performed by Fisher-Shoval.SW or C57bl.method than others have tried. One control will consist of 15 mice that will be not be infected with EAE nor treated with MSCs. After about 10 days. the 100 mice should be injected intravenously with the prepared MSCs.

statistical test will be performed to see if there is any statistical significance to the results obtained. Zhu’s paper. Another concern would be the source of the placenta. EAE in mice. and bone marrow . Results: The expected result from the placenta MSC treatment is the regression of the experimental autoimmune encephalomyelitis (EAE) in our test subjects. And finally the MRI scans taken throughout the would show if there is any changes in the amount of white matter and grey matter in the brain along with if there is any more scar tissue. Another method to see the effectiveness of the placenta MSCs is to look at the brain tissue biopsies and see the amount of cell proliferation compared to the control. but how much resemblance can assure that MSC will be as effective in mice as it will be in human population? This question is very important if this experiment is to link research with actual medical practice. and can only be answered by further clinical trials.9 After all the data has been collected. One way to prove the effectiveness of the MSCs. which would indicate the deterioration of the disease. depends on where the inflammation is on the brain. The samples will then be processed according to the procedure of Dr. using immunohistochemistry.8 Cell proliferation will also looked at to see how the MSC affect the proliferation of different brain cells that can give information about how the MSC are helping treat MS. samples of them will be taken before the second dose of MSC are administered to the mice. The procedure for staining and collecting results. Potential problems and Their remedies: One of the major seeable problem in this experiment would be the fact that mice are very different from humans. placenta stem cell. Since MS often affects the spinal cord and cerebellum. like multiple sclerosis (MS) in humans.period of 1 year to see the long-term effects of treatment using placenta derived MSCs. Data Collection: The brain of the mice will all be looked at to see the effects of the EAE on the brain’s white matter and grey matter. which will be used in this experiment are described in Schönrock’s paper. would be to scale the physical condition of the rat to see if there is any deterioration of the EAE or if they get better. to five (the highest value) which would be complete paralysis or even death. Even though induced EAE in mice is similar to multiple sclerosis in human. Bone marrow stems cell are the least controversial out of embryonic stem cell. one (being the lowest value) would be minimal signs of EAE progression in the mice. therefore the mice will all have MRI scans performed before they are infected with EAE and then they will have routine scans to see if there are any visible lesions caused by the EAE. In order to rank the physical condition of the mice a scale would be used. If there is a regression of EAE in the mice there could be a possibility of it being a possible treatment for humans since EAE in mice is the equivalent of multiple sclerosis in humans. The mice should be injected biannually with another dosage of the MSC to ensure maximum efficiency of the treatment.

. Mesenchymal stem cells in health and diseases. Multiple sclerosis.. placenta stems cell have many advantages that are understood and some that are not yet known. or they will have different effects in the long run then what we have seen in the short term trials. Expected outcomes: If our data supports the idea that placenta derived MSCs help treat multiple sclerosis (MS).30-9. Dazzi F. References: University of California San Francisco Multiple Sclerosis Center.7 Our extension of the trial period will most likely support the results from previous research. 8-9.. the health of the mice dramatically improved. 726-736.. Seminars in immunopathology. A step that can be taken would be informing people of the potential advantages to saving the placenta. our findings could be used to expand on the idea. Nature Reviews Immunology. Pistoia V.. If we see a regression of the experimental autoimmune encephalomyelitis (EAE) in the mice. we can conclude that human trials could be the next step. Moretta L. The data we retrieve will most likely show that once administered the placenta-derived MSCs. Cell Biology International. There are many problems that can show up during the experiment. Compston A. and do everything possible to get accurate and reliable results from this experiment in order to show that placenta derived mesenchymal stem cells are a efficient treatment for multiple sclerosis. Chen L.stem cell itself. there is also the possibility that the placenta MSCs won’t work as efficiently as we have though they would. Zhang X. Coles A. 4 Marigo I. April 2011 5 Miao Z.The immunomodulatory properties of mesenchymal stem cells. Our most powerful tool to deal with these problems will be the mindset that we will try to solve all issues with the experiment as they show up. 3 National Multiple Sclerosis Society. But it is proven that bone marrow stem cells do not function as well as the other two.. Multiple Sclerosis... Future studies will need to focus on how to deal with the potential threat that MSCs pose because of their immunosuppressive effects on the subject. it can be at one’s disposal and could potentially help treat degenerative diseases like multiple sclerosis (MS).Huang W. If needed in the future. Multiple Sclerosis.. 1221-1231.. and to discover how placenta MSCs actually helps. Isolation of mesenchymal stem cells from human placenta: Comparison with human bone marrow mesenchymal stem cells. 359-9313. but there is no way to predict all of them. The Lancet... Jin J.. 6 Uccelli A. Qian H. 681-687...... and how willing are the individual new mothers to give up the placenta that was part of her and her baby a few hours ago? Besides ethnic questions and fact that multiple sclerosis is not EAE this experiment.5 But if MSC were to be used as a treatment then there will be a new market demand of human placenta. Zhao J. 1 2 .. This can be supported by the results of previous experiments such as the conclusion drawn by Yonit Fisher-Shova.. At least in terms of using stem cells to treat multiple sclerosis. Zhu J.

. January 2002. . Barhum. J Mol Neurosci..Y. Identification of glial cell proliferation in early multiple sclerosis lesions. 48..492-500. Li. Bruck.. 320-330.Y. J. Sadan. Ben-Zur... Du.. Kuhlmann... 10. T.24. Qiao. 176-184. Zhang. Bitsch. Experimental and Clinical Transplantation.. Adler. Y. Neuropathology and Applied Neurobiology.. Yust-Katz.. B. J. 8 Zhu. Q. Transplantation of Placenta-Derived Mesenchymal Stem Cells in the EAE Mouse Model of MS. 7 9 Schonrock. Transplanting of Mesenchymal Stem Cells May Affect Proliferation and Function of CD4+T Cells in Experimental Autoimmune Encephalomyelitis..Fisher-Shoval. O. S.

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