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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

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

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

In order to rank the physical condition of the mice a scale would be used. one (being the lowest value) would be minimal signs of EAE progression in the mice. which would indicate the deterioration of the disease. 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. 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. The samples will then be processed according to the procedure of Dr. EAE in mice. Another concern would be the source of the placenta. Bone marrow stems cell are the least controversial out of embryonic stem cell. 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. statistical test will be performed to see if there is any statistical significance to the results obtained. which will be used in this experiment are described in Schönrock’s paper. Even though induced EAE in mice is similar to multiple sclerosis in human. 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. Zhu’s paper. 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. 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.9 After all the data has been collected. placenta stem cell. 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. The mice should be injected biannually with another dosage of the MSC to ensure maximum efficiency of the treatment. and can only be answered by further clinical trials. The procedure for staining and collecting results. One way to prove the effectiveness of the MSCs. depends on where the inflammation is on the brain. 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. using immunohistochemistry. to five (the highest value) which would be complete paralysis or even death.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. Since MS often affects the spinal cord and cerebellum.period of 1 year to see the long-term effects of treatment using placenta derived MSCs. Results: The expected result from the placenta MSC treatment is the regression of the experimental autoimmune encephalomyelitis (EAE) in our test subjects. and bone marrow . samples of them will be taken before the second dose of MSC are administered to the mice. like multiple sclerosis (MS) in humans.

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

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