W/F Red

Overview
The mobilization of stem cells has been explored as a method of treatment for myocardial infarction, a disease affecting 1.1 million American's a year. This method of treatment arose when Quaini obtained a female heart who had a sex mismatched bone marrow transplant. Upon observation of the heart Y chromosome cells expressing cardiomyocyte, endothelial and smooth muscle markers were discovered. It was stipulated that the y chromosome bearing transplanted bone marrow stem cells had traveled to the heart and differentiated repairing the damaged tissue! Since then exploration of stem cell mobilisation for the treatment of heart disease has continued and has branched out to the repair of damaged tissue.

We wish to use this mobilization phenomenon to continue exploration of brain tissue repair in stroke victims. Our research aims to mobilize autologous mouse stem cells from bone marrow to a the damaged brain tissue behind the blood clot induced through middle artery occlusion(MCAo) in mice.

Stroke
Stroke is a loss in brain function due to disturbances in the blood supply. Known causes are ischemia, thrombosis, embolism and hemmorage; 80% of strokes are caused by blood clots

SDF-1α
SDF-1α is a chemokine naturally responsible for attracting stem cells to the heart. SDF-1α is secreted from epithelial cells in ischemic tissue and inactivated by CD26/DPP-IV.It is only present for a short time after damage to tissue after which time it is degraded.

CD26/DPP-IV
CD26/DPP-IV (dipeptidylpeptidase IV) cleaves SDF-1α at its position 2 proline. DPP-IV is a membrane bound extracellular peptidase. DPP-IV is inhibited by the antagonist, Diprotin-A.

CXCR-4
CXCR-4 is a homing receptor, expressed on many progenitor cells circulating in the blood. Active SDF-1α binds CXCR-4.

G-CSF
G-CSF (the granulocyte stimulation factor) stimulates neutrophil elastases which in turn cleave SDF-1 from endothelial or stromal cells leading to their mobalisation towards SDF-1.

Diprotin A
Diprotin A is a tripeptide (Ile-Pro-Ile) which acts as a competitive inhibitor for DPP-IV

Research Problem and Goals
This research seeks to improve the treatment of stroke victims and the repair of brain tissue. We plan to mobilize bone marrow derived mesenchymal stem cells(BMSCs) to ischemic tissue in mice with middle artery occlusion(MCAo). These stem cells should in turn differentiate and repair the damaged tissue. G-CSF will be used as a mobilizing factor and Diprotin A will be utilized at a inhibitor of the SDF-1 degradation process, hopefully increasing the SDF-1 signaling time and homing of stem cells towards the damaged tissue. FITC labeled antibodies will be used to track the BMSCs mobilization towards the ischemic tissue. The amount of neurological repair after treatment with or without Diaprotin A will be assessed through observation of mouse behavior.

Necessary Materials
Adult Male Sprague Dawley rats, and facilities for their care

Flow cytometer access

FITC-conjugated antibodies for EPC surface markers and GFP

Plasmid containing constitutionally expressed GFP and a neomycin resistance gene.

Geneticin

Access to a brain sectioner.

Access to a fluorescence detector

Resources
The Rockefeller University Press • 0022-1007/97/01/111/10
 * 1) Zaruba, M-M. et al. "Synergy between CD26/DPP–IV inhibition and G–CSF improves cardiac function after acute myocardial infarction." Cell Stem Cell 4 313–323 (2009).  http://www.ncbi.nlm.nih.gov/pubmed/19341621
 * 2) “Ischemic cardiomyopathy.” Medical Encyclopedia. 3 September 2008 http://www.nlm.nih.gov/medlineplus/ency/article/000160.htm
 * 3) "Performing RNAi Experiments in Animals." Applied Biosystems. http://www.ambion.com/techlib/tn/131/5.html
 * 4) Dong Yeon Lee. "Mobilization of endothelial progenitor cells in fracture healing and distraction osteogenesis" Bone. Volume 42, Issue 5, May 2008. 
 * 5) Tian, C., Rónai, A.Z., Bagosi, Z., Hiramatsu, H., Bird, A.P., et al. "Diprotin A." http://www.wikigenes.org/e/chem/e/94701.html
 * 6) Ye Wanga, Yubin Denga,, Guang-Qian Zhoub."SDF-1α/CXCR4-mediated migration of systemically transplanted bone marrow stromal cells towards ischemic brain lesion in a rat model." 2007 Elsevier B.V.
 * 7) Arman T Askari, Samuel Unzek, Zoran B Popovic, Corey K Goldman, Farhad Forudi, Matthew Kiedrowski, Aleksandr Rovner, Stephen G Ellis, James D Thomas, Paul E DiCorleto, Eric J Topol, Marc S Penn. "Effect of stromal-cell-derived factor 1 on stem-cell homing and tissue regeneration in ischaemic cardiomyopathy." THELANCET• Vol 362 • August 30, 2003 • www.thelancet.com.
 * 8) A. Aiuti, I.J. Webb, C. Bleul, T. Springer, and J.C. Gutierrez-Ramos."The Chemokine SDF-1 Is a Chemoattractant for Human CD34 Hematopoietic Progenitor Cells and Provides a New Mechanism to Explain the Mobilization of CD34 Progenitors to Peripheral Blood." J. Exp. Med.