Enhancement of organogenesis with small molecule drugs

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

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)
        • 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. Grow mesenchyme cells in a medium containing the following:

• Wharton's Jelly-substitute (significantly, hyaluronan). Hyaluronan is a significant component of the extracellular matrix and has been shown to contribute to tumor growth.
• Pax3 -- made separately in E. coli
• Invitrogen™ StemPro® MSC Serum Free Medium

3. Apply statins to layers of cells to induce differentiation.

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.

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

• Eddie Liu
• Zach Bjørnson

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