Speaker
Description
"Introduction
Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, fatal form of diffuse interstitial lung disease which is associated with substantial mortality and a median survival of 3 years from the time of diagnosis. Acute exacerbations (AE) of IPF have been defined as acute, clinically significant respiratory deterioration of unidentifiable cause. Available data suggest that 46% of deaths in IPF are preceded by AE, and the median survival of patients with IPF who experience AE is approximately 3.5 months. Mesenchymal stem cells (MSC) derived from term amniotic fluid (TAF) are neonatal and can be propagated to extremely high amounts for use in cell therapy. TAF-MSC are derived from different tissues of the fetus. Therefore, sorting strategies using tissue-specific markers can prepare a sorted population of cells from each tissue-type. We hypothesize that lung-specific MSC can be used to reduce the overall severity of AE-IPF and lower the risk of mortality due to the pharmacological effects (anti-inflammatory, immunomodulatory, regenerative, proangiogenic, and antifibrotic) these cells may have.
Methodology
RNASeq data from TAF-MSC clones was used to identify tissue-specific markers present on these MSC. Several prospective lung markers were tested by flow cytometry of cultured TAF-MSC. One of these markers was used for cell-sorting using Tyto MACSQuant cell sorter and the positive cell population was expanded to passage 4 (TAF-lung-MSC). Rats were instilled intratracheally with bleomycin to induce fibrosis at day 0. At Day 4 TAF-lung-MSC or vehicle was administered by an IV injection. On day 28 lungs were prepared for histology, stained and scored.
Results
After collection, processing and expansion of TAF cells in a defined media system, different subtypes of MSC were identified. Of these, one type had unique RNA expression and cell surface phenotype expression profiles. This signature was partially similar to MSC collected from lung tissue. After identifying lung-specific clones, candidate marker genes were identified. The validity of the surface marker genes was established using flow cytometry with antibodies directed against the indicated lung surface markers. One of the prospective markers was used for lung-MSC sorting. The positive fraction was further propagated until passage 4 when it was used to treat bleomycin induced pulmonary fibrosis in a rat model. TAF-lung-MSC showed anti-fibrotic effect with significantly less fibrosis in the lungs of rats at 24 days after infusion of MSC than in the bleomycin-treated control group (p<0.05) as assessed both by histopathological evaluation (percent parenchyma affected) and fibrosis scoring using the Modified Ashcroft scale. Further, at the termination of the study on Day 28, TGF-β plasma levels were higher in bleomycin-treated control rats than in rats treated with TAF-lung-MSC. No adverse reactions from cell injections were reported. No remaining TAF-lung-MSC could be detected after Day 24, supporting that MSC were cleared from the rats after exerting their effect.
Conclusion
Several markers were found to be good lung-specific markers for sorting of TAF-MSC using the Tyto MACSQuant cell sorter instrument. Sorted cells were further propagated and TAF-lung-MSC were effective in reducing lung fibrosis in a rat bleomycin induced pulmonary fibrosis model."
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