Enhancement of organogenesis with small molecule drugs

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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.
  • solution: induce blood vessel formation via vasculogenesis during organogenesis

Hyaluronan, Statins, and Pax3 proteins (maybe just SMD's here)

  • Hyaluronan
    • a linear polysaccharide that is a major part of the extracellular matrix
    • all mechanisms not completely understood
    • leads to increased vascularization and aggregation of cells.
  • 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)
          • maybe integrates into cells easier due to being many magnitudes smaller
  • 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)

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. Mix mesenchymal stem cells in the following:

  • Hyaluronan, a significant component of the extracellular matrix that has been shown to contribute to tumor growth and vasculogenesis.
  • Pax3 (for appropriate samples) -- recombinant from E. coli, will get broken down as cells grow

2. Pour mixture in slabs approximately 1 mm thick. It may be necessary to spread a drop to achieve thinness.

3. Culture slabs at 37 °C, 5% CO2, with filtered, oxygenated Invitrogen™ StemPro® MSC Serum Free Medium circulated over the slabs at a rate of approximately 100 mL/min with a peristalic pump. Each slab will be grown with its own medium reservoir. -OR- culture slabs on top of osmotic membranes with a ~50 kDa cutoff. Observe daily, looking for cell aggregation.

4. Apply statins or VEGF to appropriate slabs to induce differentiation. (apply more Pax3? test for levels)

5. Let grow.

6. View cross-sections of slabs: quickly freeze, then slice with a microtome and view under visible light microscope.

Culture Pax3 VEGF A Statin
1, negative control
2, Pax3- control + +
3, Pax3 characterization +
4, VEGF characterization +
5, Statin characeterization +
6, VEGF comparison condition + +
7, Primary experiment condition + +
8, The Nebuchadnezzar + + +

Predicted Outcomes

1. Successful purification of Pax3 protein (seems easy enough if we have the right DNA sequences).

2. More densely-packed, layered cell aggregates will form in mesenchymal stem cell cultures with Pax3 protein as opposed to mesenchymal cells cultured without it. This can be viewed under a 40x light microscope?

3. Increased differentiation of angioblasts to endothelium with the addition of statins to mesenchymal cell layers and possibly more blood vessel formation as a result. (determined by fluorescence microscopy?)

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)

  • looking at cells in layers
  • scaffolds
  • statins and other signals for vasculogensis
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