3rd Annual BMRP Investigator Meeting - Abstract
Repair of Crohn’s Disease with Embryonic Stem Cells
Anand S. Srivastava1,a, Zhongling Feng1, Victor Arrunategui-Correa1, Hyun S Kim1 and Ewa Carrier1,2,b
1Department of Medicine and 2Rebecca and John Moores Cancer Center, University of California, San Diego (La Jolla, California, U.S.A.)
Objective: The primary objective of this work is to determine differentiation and repair potential of embryonic stem cells (ES) in murine model of Crohn’s disease (CD).
Hypothesis: It is hypothesized that the pluripotent nature of embryonic stem cells can be utilized to develop an effective therapy for CD. Furthermore, stem cell ® stem cell niche interaction is necessary for differentiation and stem cell niche is important to support incoming stem cells.
Rationale: Phase I studies demonstrated clinical improvement in patients with severe Crohn’s disease with autologous stem cell therapy, but biological mechanisms underlying this repair are not well understood. The intestinal environment, with its crypts and “niches,” will support incoming embryonic stem cells and allow them to engraft and differentiate. In the setting of elevated pro-inflammatory cytokines and other triggering factors produced locally in the intestine, stem cells migrate to the site of injury and differentiate into intestinal epithelial cells to replace the injured, ulcerated epithelial and submucosal layers and to repopulate intestine with immunologically-balanced, normally functioning lymphocytes. Newly formed lymphocytes cause Th1 ® Th2 shift, restoring immune balance in the intestine.
Methods: Colitis was induced in IL10-/- KO mice using piroxicam. The colitis in this model is patchy, progressive and leads to death unless rescue therapy is provided. Enhanced yellow fluorescent protein (EYFP) marked murine ES cells (R1/129) and ES-CCE (Stemcell Technologies, Inc., Vancouver, Canada) without marker fluorescence protein in vitro differentiation induced by TGF-b (2ng/ml), EGF (25ng/ml) and b-FGF (100ng/ml) for seven days. IL-10-/- KO mice were injected with pre-differentiated ES-YFP cells and sacrificed at two months post injection. Serial frozen sections of colon, small intestine, and thymus were made and EYFP staining was double detected using Bio-Rad MRC-1024 UV confocal microscope and determined by immunohistochemistry using anti-GFP antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, U.S.A.).
Results: Histopathological analysis demonstrated improvement in colon tissue. Fluorescent and confocal microscopy demonstrated presence of the ES-EYFP cells in the colon and small intestine. Anti-GFP staining was shown in these tissues using Leica DM microscope. The EYFP signal was not detected in sham and control IL10-/-KO mice. Immune studies by ELISA demonstrated Th1 ® Th2 shift indicated by significant decrease in the plasma and intestinal level of IL-12 cytokine in comparison to non-treated control IL 10-/- KO mouse suggesting immune recovery.
Conclusion: Our results suggest that the study of differentiation and repair potential of embryonic stem cells (ES) in a pathological Crohn’s disease model may lead to an alternative therapeutic potential for the treatment and prevention of inflammatory bowel disease (IBD).
aCo-investigator and Presenter; bPrincipal Investigator