Gastrointestinal Microbiome Manipulation and Mesenchymal Stem Cells to Combat Colitis

Nerissa Ignacio, Nabeetha Nagalingam, Jordan Mar, Susan Lynch University of California San Francisco, Department of Medicine

Division of Gastroenterology, Lynch Lab

I. Background Ulcerative Colitis(UC) is a chronic, idiopathic inflammatory bowel disease in the

colon and dysbiosis of microbial commensalism. It occurs in 35–100 people for every 100,000 in the United States and the numbers are only increasing 2. The commensal bacteria are crucial for human health since they provide defense against pathogen invasion, regulate host immune responses and metabolize complex carbohydrates. Therefore, the composition of commensal bacteria is significant to defend colonic health. Ulcerative Colitis is characterized by a loss of both microbiome diversity and gastrointestinal epithelial integrity. We hypothesized that mouse mesencyhmal cells(MSC) would repair epithelial damage and that supplementation with commensal bacteria will restore microbial diversity in the colon, thus promoting immune homeostasis and improving colitis outcomes. This study investigates the effectiveness of VSL#3 supplementation and stem cell therapy in isolation

and In combination in a dextran sodium sulfate(DSS) murine model of colitis.

II. Methods

Fig. A : Mice treated with DSS to induce colitis were treated with mesenchymal stem cells(MSC), VSL#3, or a

combination of both. Control included no DSS or no therapeutic intervention

•C57BL/6 mice were treated as illustrated in Fig.A. •Microbiome profiles were generated using 16s rRNA Phylochip 1. • Stem Cells were grown and harvested in Iscove’s Modified Dulbecco’s Medium at 37◦C • MSCs were tracked using fluorescently labeled stem cells and the IVIS imager.

Fig.3 Box plot of ileal bacterial diversity from control and treatment groups .VSL3-treated animals exhibit the greatest gain in bacterial diversity compared to all other groups. Fig.4 Box plot of colonic microbial diversity from control and treatment groups. Treatment did not significantly impact bacterial diversity in the colon of DSS-treated animals.

IV. Discussion All treatment groups improved colitis outcomes, though VSL3 treatment exhibited

the greatest efficacy in the DSS colitis mice. MSC treatment showed the least efficacy and a combination of MSC and VSL#3 exhibited an intermediate efficacy. This paralleled bacterial diversity in these treatment groups. Imaging labeled stem cells demonstrated that they trafficked to the gastrointestinal tract, and accumulated in the ileum, but that this was not associated with a significant increase in microbial diversity at this site. VSL3 significantly increased bacterial diversity and reduced disease presumably though down-regulation of pro-inflammatory responses in the gastrointestinal tract.

Caveats to the study include a prolonged study duration to determine whether the protective effect of these treatments persist past the duration of the study. Lastly, controlling for the stress of tail vein injection could be achieved by injection of controls with buffer to exclude the possibility that stress contributed to the observed results.

Fig. 1 Box plot of the disease score deriving from no treatment, MSC therapy, combination of MSC therapy and VSL #3, and VSL #3. Only the VSL#3 treatment group exhibit a significant decrease in disease score (p < 0.05).

Fig.2 Murine colonic histology. MSC result show poor epithelial recovery and only a slight reduction in inflammation. VSL#3 +MSC show an improved epithelial recovery and inflammation reduction than MSC only. VSL# 3 results show the best epithelial recovery and significantly reduced inflammation. Histological results mirror disease severity scores.

III. Results

1. Disease Score/ Histology

2. Microbial Diversity

3. Stem Cell Trafficking

V. References 1 Fujimura, Kei. "Microbes in House Dust from Dog-owning Homes Protect against a Common Viral Infection Associated with Increased Risk of Asthma Development." ASM 2012. UCSF, 19June 2012. Web. 26 July 2012. 2 NewsMedical. "What Is Ulcerative Colitis?" News Medical. Creative Commons Attribution-ShareAlike License, n.d. Web. 26 July 2012. <http://www.news-medical.net/health/What-is-Ulcerative-Colitis.aspx>.

VI. Acknowledgements I would like to give my sincerest appreciation to the Lynch Lab for having me this summer. I want express my utmost gratitude to Nabeetha Nagalingam and Jordan Mar for

mentoring me and Susan Lynch for giving me the opportunity to be part of this project. Lastly, I would like to thank Chris Lever and Lynn Huntsinger for establishing programs like this and giving students like me this great opportunity.

Fig.5 Fluorescent imaging of injected MSC-GFP. MSC has successfully entered the mice GI tract from the tail vein injection. MSCs present along the GI tract and not in other organs such as liver or spleen. Fig.6 Picture of MSC culture. MSC growth on Day 5

Control DSS MSC MSC and VSL#3 VSL#3

Fig.3 Fig.4

Healthy UC

A. MSC-gfp + no DSS B. MSC-gfp +DSS C. No MSC-gfp +DSS