Enhancement of organogenesis with small molecule drugs: Difference between revisions
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== Research Goals == | == Research Goals == | ||
1. Produce and purify Pax3 protein from E. coli BL21 cells. | 1. Produce and purify Pax3 protein from E. coli BL21 cells. | ||
2. Obtain multi-layered mesenchymal aggregates which simulate tissue in embryonic stages by adding Pax3 protein to mesenchymal cell cultures. | 2. Obtain multi-layered mesenchymal aggregates which simulate tissue in embryonic stages by adding Pax3 protein to mesenchymal cell cultures. | ||
3. Hopefully obtain fluorescence data demonstrating angioblast differentiation into endothelium and/or the formation of blood vessels in the mesenchymal tissue with(and maybe without) the addition of statins to our cultures. | 3. Hopefully obtain fluorescence data demonstrating angioblast differentiation into endothelium and/or the formation of blood vessels in the mesenchymal tissue with(and maybe without) the addition of statins to our cultures. | ||
Revision as of 18:34, 13 May 2008
βββ -- using a bio-degradable scaffold. seed myocytes onto it. before scaffold breaks down, seed with angioblasts. use statins instead of VEGF to stimulate endothelial production. evaluate with fluoro microscopy.
Background
Vasculogenesis in vivo
- formation of new blood vessels through the movement and differentiation of angioblasts (endothelial precursor cells)[insert picture from book]
- induced by growth factors and extracellular matrix
- during embryonic development
- followed by angiogenesis (sprouting of new blood vessels from old existing ones) to form complete vascular tissue
Necessity of vasculogenesis in vitro
- current state of organogenesis
- possible to make thin patches of tissue
- e.g. NASA researchers able to make small patch of cardiac tissue ~0.1 mm thick (but need 5 mm or so at least)
- interior of patches have trouble obtaining enough oxygen.
- possible to make thin patches of tissue
- solution: induce blood vessel formation via vasculogenesis during organogenesis
Statins and Pax3 proteins (maybe just SMD's here)
- Small molecule drugs
- Statins (HMG-CoA reductase inhibitors) [insert picture of molecule]
- induce differentiation of angioblasts into endothelium (vasculogenesis at work!)
- same function as Vascular endothelial growth factor (VEGF)
- much smaller than VEGF (2 aromatic rings as opposed to entire 38 kDa protein)
- induce differentiation of angioblasts into endothelium (vasculogenesis at work!)
- Statins (HMG-CoA reductase inhibitors) [insert picture of molecule]
- Pax3 protein (how do we smoothly integrate this into our talk? maybe just mention this in our procedure?)
- induces mesenchymal stem cells to form multi-layered cell aggregates with epithelial characteristics
- simulates the dense, layered mesenchyme structure in embryo (refer to picture from book)
- induces mesenchymal stem cells to form multi-layered cell aggregates with epithelial characteristics
Research Goals
1. Produce and purify Pax3 protein from E. coli BL21 cells.
2. Obtain multi-layered mesenchymal aggregates which simulate tissue in embryonic stages by adding Pax3 protein to mesenchymal cell cultures.
3. Hopefully obtain fluorescence data demonstrating angioblast differentiation into endothelium and/or the formation of blood vessels in the mesenchymal tissue with(and maybe without) the addition of statins to our cultures.
Project Details and Methods
1. Grow mesenchyme cells in a Wharton's Jelly-like solution (significantly containing hyaluronan). Hyaluronan is a significant component of the extracellular matrix and has been shown to contribute to tumor growth. In this step, we need to grow a layer of mesenchyme cells roughly five cells-thick. Hopefully the Jelly will promote this growth pattern.
(Several day's growth...)
2. Transfect the cells with Invitrogen™ Qtracker® 505 dye.
3. Apply statins to layers of cells to induce differentiation.
(Several day's growth and differentiation...)
4. Transfect the cells with Invitrogen™ Qtracker® 655 dye.
5. Quickly freeze sections of the cell layers to enable preparation of thin cross-sectional slices of tissue for observation. Then view under fluorescent microscope.
Predicted Outcomes
Resources
- Researchers were able to grow rat ventricular cells in rotating bioreactors that quickly formed aggregates that rhythmically contracted in unison.
- R.E. Akins, R.A. Boyce, M.L. Madonna, N.A. Schroedl, S.R. Gonda, T.A. McLaughlin, C.R. Hartzell. Tissue Engineering. April 1, 1999, 5(2): 103-118. doi:10.1089/ten.1999.5.103. [Article]
- Asahara T, et al (1997). "Isolation of putative progenitor endothelial cells for angiogenesis." Science 275: 964-7. PMID 9020076. [Article]
- Asahara T, et al (1999). "Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization." Circulation Research 85 (6): 221-8. PMID 10436164. [Article]
- Hristov, et al (2003). "Endothelial progenitor cells: isolation and characterization." Trends in Cardiovascular Medicine 13 (5): 201-6. PMID 12837583. [Article]
- Shaw J, et al (2004). "Hematopoietic stem cells and endothelial cell precursors express Tie-2, CD31 and CD45.". Blood Cells, Molecules, and Diseases 32 (1): 168-75. PMID 14757432. [Article]
- Werner N, et al (2005). "Circulating Endothelial Progenitor Cells and Cardiovascular Outcomes.". New England Journal of Medicine 353: 999-1007. PMID 16148285. [Article]
- A specific study of HMG-CoA reductase inhibitors (statins). The researchers found that the statins controlled hematopoietic progenitor cell differentiation.
- Stefanie Dimmeler, Alexandra Aicher, Mariuca Vasa, Christiane Mildner-Rihm, Klaudia Adler, Michaela Tiemann, Hartmut Rütten, Stephan Fichtlscherer, Hans Martin and Andreas M. Zeiher. HMG-CoA reductase inhibitors (statins) increase endothelial progenitor cells via the PI 3-kinase/Akt pathway. J. Clin. Invest., 2001; 108(3): 391 - 397. [Article]
- The Pax3 protein induces dense multi-layer aggregation of mammalian mesenchymal cells and facilitates the mesenchymal to endothelial transition.
- Wiggan O'Neil, Mark Fadel, and Paul Hamel. Pax3 induces cell aggregation and regulates phenotypic mesenchymal-epithelial interconversion. Journal of Cell Science 115, 517-529 (2002) [Article]
- Pax3 protein production in E. coli.
- Stéphane C. Boutet, Marie-Hélène Disatnik, Lauren S. Chan, Kevin Iori and Thomas A. Rando. Regulation of Pax3 by Proteasomal Degradation of Monoubiquitinated Protein in Skeletal Muscle Progenitors. Cell 130, 342-362 (2007) [Article]
People
Steps (not for final page)
- http://science.nasa.gov/headlines/y2002/14feb_heart.htm
- http://www.nature.com/nbt/journal/v23/n10/full/nbt1005-1237.html
- http://www.nibib.nih.gov/HealthEdu/eAdvances/31May06
- http://www3.interscience.wiley.com/cgi-bin/fulltext/112395581/PDFSTART
- looking at cells in layers
- scaffolds
- statins and other signals for vasculogensis