UMBILICAL CORD TISSUE DERIVED MESENCHYMAL STEM CELLS: THERAPY FOR CYSTIC FIBROSIS

Introduction: The primary cause of morbidity and mortality in Cystic Fibrosis (CF) is infection and on-going inflammation. Human mesenchymal stem cells (hMSCs) can fight inflammation, infection, and tissue destruction. hMSCs have been derived from a variety of tissues including human cord blood and bone marrow. Bone marrow derived human mesenchymal stem cells have been shown to have anti-inflammatory and anti-microbial properties. The umbilical cord consists of both blood and cord tissue. hMSCs can be derived from umbilical cord tissue (HCT), which is usually discarded post-delivery. HCT hMSCs were evaluated for therapeutic potential in CF.

Methods: Cord Blood Registry® provided HCT hMSCs. hMSCs were cultured with or without lipopolysaccharide (LPS) or peptidoglycan (PEP) and cytokines: granulocyte macrophage colony- stimulating factor (GM-CSF), interleukin-1 beta (IL-Iβ), and interferon gamma (IFN-γ) for 2 or 24 hours. The hMSC supernatants and cell pellets were analyzed for secreted cytokines and gene expression. hMSC supernatants were analyzed for secreted cytokines using Luminex technology (mean±SEM pg/ml). Cord tissue cells were analyzed for gene expression by Real Time- Polymerase Chain Reaction (RT-PCR) (mean±SEM, change in cycle threshold, DCT). hMSCs functional activity was measured using Staphylococcus aureus (SA) and Pseudomonas aeruginosa (PA) for antimicrobial activity (colony forming units, CFUs) or ATP production by adenosine triphosphate (ATP) relative luminescence units (RFUs) using patient derived (A549) epithelial cells. Statistical analysis was done with Graph Pad Prism 6.

Results: Cord tissue hMSCs secreted cytokines MIP-1α, IL-6, TNFα , IL-1β, IL-8 and IL-17 and had increased hIL-6, hIL-8, CCL20 and CCL2 gene expression, all augmented by either pathogen agonists or cytokines compared to controls. Human Cord Tissue Secreted Products by Luminex Assay: HCT hMSCs secreted MIP-1α (45±23 pg/ml), IL-6 (95,339±81,389 pg/ml), TNFα (9.4±1.2 pg/ml), IL-1β (2616±1316.49 pg/ml), and IL-8 (93,147±13,684) (n=3, p≤0.05). LPS stimulation increased IL-6 and MIP-1α (121940±108,003 pg/ml and 50±26 pg/ml respectively, (n=3, p≤0.05).

Cord Tissue Gene Expression by RT-PCR: hMSC expressed IL-6 and IL-8 mRNA (-3.89±2.52 and - 2.15±3.42 respectively) which did not change with LPS. PEP stimulation increased both IL-6 and IL-8 mRNA synthesis (-6.76±1.5 and -5.93±2.71 respectively).

hMSCs supernatants had antimicrobial potency against Pseudomonas aeruginosa and Staphylococcus aureus. Human Cord Tissue Functionality Assays: Functionally, HCT hMSC supernatants were antimicrobial against both PA and SA decreasing from 37 to 28.5 (±0.104) CFUs and from 83.5 to 72.5 (±0.102) CFUs respectively. Further, the hMSCs supernatants showed cellular activation as evidenced by ATP production, which was enhanced by LPS (86,655±5725 RFUs to 115,770±1879 respectively, p≤0.05).

Conclusion: HCT hMSCs have potential as anti-microbial and anti-inflammatory therapeutics in cystic fibrosis.