Yuanting Lu Akanksha Vaidya 4/12/2010 03-345 (M/F)
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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
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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.
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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
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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.
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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
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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/
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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
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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.
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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.
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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/
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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
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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
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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.
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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).
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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
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Parco M. Siu, Yan Wang, and Stephen E. Alway
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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
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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.
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Hydrogen peroxide treatment causes fragmentation of DNA Image
credit: Siu et. Al. 2009. Apoptotic signaling induced by H2O2
mediated oxidative stress. Life Science.
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.
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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
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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
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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
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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
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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!!
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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
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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
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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
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