Yuanting Lu Akanksha Vaidya 4/12/2010 03-345 (M/F)

General mechanism for organ regeneration using stem cells Image credit: Evans, N, E. Gentleman, J. Polak. Scaffolds for stem cells. 2006. Materials today. 9: 26-33

Harvesting cells to use for organ regeneration Adult stem cells Bone-marrow derived stem cells most commonly used. Mesenchymal stem cells (MSCs) grow most easily in culture, but do not grow indefinitely. Used for forming heart tissue, bones, cartilage, and neurons. Do so by transdifferentiation. Embryonic stem cells Divide indefinitely and are able to form any many more cell types.

Directing the formation of a particular cell type Use media with growth factors for particular type. Eg: Media with vitamin C, b-glycerophosphate, dexamethasone used for producing osteoblasts. Inject stem cells directly into organs. This can cause tissue regeneration by: Image credit: Togel, F and C. Westenfelder. 2007. Adult Bone Marrow- Derived Stem Cells for Organ Regeneration and Repair. Developmental Dynamics. 236:3321-3331 Transdifferentiation: Environment of the organ causes differentiation and integration of stem cells. Cell fusion: Stem cells fuse with existing cells in organ. Stem cells release chemicals stimulating cells of organ to divide and repair organ.

Methods for selecting a particular cell type Use fluorescent tags that identify each cell type. Researchers at harvard used embryonic stem cells from mice to generate cardiac muscle cells. Fluorescent tags helped determine which cells would form specific parts of the heart. Image credit: http://singularityhub.com/2009/11/03/harvard- grows-heart-tissue-watches-it-beat/

Properties of good synthetic scaffolds Binds to the correct organs within the body. Glass ceramics bind bone and were used for bone transplants. Contain certain growth factors that direct the future fate of the stem cell. Adjusting hydroxyapatite to tri-calcium phosphate ratio in scaffold allows for cells to better form osteoblasts. Allows passage of nutrients within the body Porous materials are useful. Eg: Calcium phosphate, gels.

Disadvantages of synthetic scaffolds The body might generate an immune response against the scaffolds. Inorganic scaffolds might be too brittle or weak. The degradation products of the scaffolds might be toxic. The scaffold might not promote differentiation of stem cells.

Using natural scaffolds from old organs Researchers at the University of Minnesota removed the cells from a pigs heart and used the remaining skeleton of the heart as the scaffold. Stem cells were grown on this scaffold and resulted in a beating pigs heart. Last month, a british boy received a windpipe generated in a similar manner. Image credit: http://singularityhub.com/2009/06/23/stem-cells-used-to-grow-hearts-cool-new-pics-and-vid/

What are Human Embryonic Stem Cells? Human embryonic stem cells (hES) are grown from cells from the inner cell mass of mammalian blastocyst. Embryonic stem cells are pluripotent- capable of generating any and all cells under the right conditions. hES were isolated in 1998 Most widely studied are mouse embryonic stem cells. Source: http://www.sciencemag.org/cgi/content/full/282/5391/1145

Differentiated Human Embryonic Stem Cells and Insulin Expression Type I diabetes results from the destruction of pancreatic islet -cells. Homologous, undifferentiated hES cells are kept in mouse embryonic fibroblast and differentiated in suspension in bacterial-grade petri dishes. Immunohistochemistry shows cells being expressing insulin as early as 14 days of differentiation. Differentiated hES cells express -cell markers and insulin production. Normal Pancreatic Islets Insulin expressing cells after 19 days Source: Assady et al., (2001). Insuline production by human embryonic stem cells. Diabetes (50). 1691-1697.

Human Embryonic Stem Cells in Treating Multiple Sclerosis Multiple sclerosis (MS) is an immune mediated demyelinating disease of the CNS. Transplanted hES cells derived neural precursor cells into the animal model of MS. Found that this reduced the severity of clinical signs and tissue damage in CNS. Source: Aharonowiz et al.. (2008).

Human Embryonic Stem Cells in Parkinsons Disease Parkinsons disease is characterized by an extensive loss of dopamine neurons in the substantia nigra. Kim and Vellis suggested using neurons with dopamine phenotype generated from embryonic stem cells or neural stem cells. Transplanting neural stem cells in brain attenuates functional deficits associated with injury or disease in the CNS. Source: http://www.umm.edu/parkinsons/images/parkinson_disease.jpg

Parco M. Siu, Yan Wang, and Stephen E. Alway

Skeletal muscle mass is lost as a result of aging. Siu et. al. hypothesized that this loss in cells might be due to increased oxidative stress. Hence, the goals of the study were: To study whether oxidative stress caused due to Hydrogen peroxide would increase the number of proteins promoting apoptosis, and decrease those preventing apoptosis. To study the effect of various doses of hydrogen peroxide on the apoptosis of differentiated myotubes. Goals of the study

Hydrogen peroxide treatment causes morphological changes in the myotubes Image credit: Siu et. Al. 2009. Apoptotic signaling induced by H2O2 mediated oxidative stress. Life Science.

Hydrogen peroxide treatment causes fragmentation of DNA Image credit: Siu et. Al. 2009. Apoptotic signaling induced by H2O2 mediated oxidative stress. Life Science.

Image credit: http://www.biomedcentral.com/1471-2105/6/155/figure/F8

Proteins inhibiting apoptosis decrease after hydrogen peroxide treatment Image credit: Siu et. Al. 2009. Apoptotic signaling induced by H2O2 mediated oxidative stress. Life Science.

Image credit: http://www.biomedcentral.com/1471-2105/6/155/figure/F8

A. Cytochrome c present in the cytosol Proteins promoting apoptosis increase after hydrogen peroxide treatment Image credit: Siu et. Al. 2009. Apoptotic signaling induced by H2O2 mediated oxidative stress. Life Science.

Hypothesis for Oxidant/Antioxidant Treatment of Differentiated and Undifferentiated C2C12 Treatment of differentiated myotubes with hydrogen peroxide, an oxidant, should cause cell death and the myotubes to shrink in size. Hypothesize that treating undifferentiated C2C12 cells with hydrogen peroxide will prevent proper myotube formation and differentiation. L-ascorbic acid, an antioxidant, has been suggested to provide protection against the oxidative DNA damage caused by hydrogen peroxide. Source: http://www.cellsignal.com/products/images/2442_ific_ka_070322.jp g Source: http://coil.bio.ed.ac.uk/Images/Gallery/C2C12_undiff_tub_pcm_4_D3D.jpg

Undifferentiated C2C12 Cells Treatments L-ascorbic acid + H2O2 Day 1: Undiff -> Keep in 10% FBS Day 2: Add 11.16mM L-ascorbic acid to undifferentiated Day 3: Shift to 2% horse serum + add 4mM hydrogen peroxide for 48 hr Day 4: Stain with primary and secondary antibody. Hydrogen Peroxide Day 1: Undiff -> Keep in 10% FBS Day 2: Shift to 2% horse serum + add 4mM hydrogen peroxide Day 3: After 48 treatment, remove hydrogen peroxide Day 4: Stain with primary and secondary antibody

Differentiated C2C12 Treatments L-ascorbic acid + H2O2 Day 1: Undiff -> Shift to 2% horse serum Day 2: Add 11.16mM L- ascorbic acid to differentiated Day 3: Add 4mM hydrogen peroxide Day 4: Stain with primary and secondary antibody. Hydrogen Peroxide Day 1: Undiff -> Shift to 2% horse serum Day 2: Differentiated -> no change Day 3: Add 4mM hydrogen peroxide Day 4: Stain with primary and secondary antibody

Goals and Expectations L-ascorbic acid + H2O2 1)Undifferentiated: Expect L- ascorbic acid to protect myotubes and allow differentiation in the presence of hydrogen peroxide. Look same as differentiated control. 2)Differentiated: Expect myotubes to look the same as the differentiated control due to L- ascorbic acid protection from hydrogen peroxide damage. Hydrogen Peroxide 1) Undifferentiated: Expect hydrogen peroxide to prevent differentiation. 2) Differentiated: Expect myotubes to be damaged and look smaller than differentiated control. Source: http://upload.wikimedia.org/wikipedia/commons/archive/3/32/20071213041812!Hydrogen- peroxide-2D.png Source: http://www.vitamin-basics.com/uploads/pics/vitamin_C.jpg

Summary: The pluripotency of stem cells enables them to regenerate organs and also be used to treat diseases. This field has a bright future. For the first time, in January 2009 the FDA approved clinical testing of embryonic stem cells in humans for the biotech company Geron. In March 2010 a trachea generated using stem cells was transplanted into a 10 year old boy. Apoptosis of skeletal muscles in old age may be related to increased oxidative stress. Wild hypothesis: Vitamin C might help slow down aging!!

References 1)International Society of Stem Cell Research. (2008). requently Asked Questions. Available from: http://www.isscr.org/science/faq.htm. Accessed 2010 Apr 10 2)Itskovtiz-Eldor, J., Jones, J., Marshall, V., Shapiro, S., Swiegriel, J., Thomson, J. & Waknitz, M. (1998). Embryonic Stem Cell Lines Derived from Human Blastocysts. Science (282): 1145-1147. 3)Assady, S., Amit, M., Itskovitz-Eldor, J., Maor, G., Skorecki, K., & Tzukerman, M. (2001). Insulin Production by Human Embryonic Stem Cells. Diabetes (50): 1691-1697. 4)Aharonowiz, M., Ben-Hur, T., Einstein, O., Fainstein, N., Lassmann, H. & Reubinoff, B., (2008). Neuroprotective Effect of Transplanted Human Embryonic Stem Cell-Derived Neural Precursors in an Animal Model of Multiple Sclerosis. PLoS ONE 3(9): e3145. doi:10.1371/journal.pone.0003145. 5)Kim, S. & Vellis, J. (2009). Stem Cell-Based Cell Therapy in Neurological Diseases: A Review. Journal of Neuroscience Research (87): 21832200. 6)Siu P, Wang Y, Alway S. 2009. Apoptotic signaling induced by H 2 O 2 -mediated oxidative stress in differentiated C2C12 myotubes. Life Science. 84: 468-481. 7)Noroozi M, Angerson W, Lean M. 1998. Effects of flavonoids and vitamin C on oxidative DNA damage on human lymphocytes. American Journal of Clinical Nutrition. 67: 1210-8 8) Evans, N, E. Gentleman, J. Polak. Scaffolds for stem cells. 2006. Materials today. 9: 26-33 9)Togel, F and C. Westenfelder. 2007. Adult Bone Marrow-Derived Stem Cells for Organ Regeneration and Repair. Developmental Dynamics. 236:3321-3331 10)Sanez, A. 2009. Harvard grows tissue watches it beat. Singularity Hub. [Online] Available: http://singularityhub.com/2009/11/03/harvard-grows-heart-tissue-watches-it-beat/ http://singularityhub.com/2009/11/03/harvard-grows-heart-tissue-watches-it-beat/ 11)Sanez, A. 2009. Stem cells used to grow hearts. Singularity Hub. [Online] Available at: http://singularityhub.com/2009/06/23/stem-cells-used-to-grow-hearts-cool-new-pics-and-vid/ http://singularityhub.com/2009/06/23/stem-cells-used-to-grow-hearts-cool-new-pics-and-vid/ 12) Gadkar et al. BMC Bioinformatics 2005 6:155 doi:10.1186/1471-2105-6-155 13) Lister, S. 2010. Milestone moment as boy undergoes transplant to regenerate trachea. The Times. [Online]. Available from:http://www.timesonline.co.uk/tol/news/uk/health/article7068514.ece 14)Iwatani H, Imai E. 2010. Kidney reapir using stem cells: Myth or reality? Journal of Nephrology. 2: 143-6. 15)Wurmser, A and F. Gage. 2002. Stem cells: Cell fusion causes confusion. Nature. 416: 485-87.

Yuanting Lu Akanksha Vaidya 4/12/2010 03-345 (M/F)

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