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A5781 - Expression of Distinct Alpha6 Integrin Alternative Splicing Variants in Lung Resident Mesenchymal Stem Cells (LR-MSCs)
Author Block: Z. Zhou1, H. Chen1, J. Qu1, S. Liu2, D. Chanda1, V. J. Thannickal1, P. Chen3, Y. Zhou1; 1Division of Pulmonary, Allergy and Critical Care Medicine, University of Alabama at Birmingham, Birmingham, AL, United States, 2Division of Ophthalmology, University of Alabama at Birmingham, Birmingham, AL, United States, 3Department of Respiratory Medicine, the Second Xiangya Hospital, Central South University, Changsha, China.
RATIONALE: α6 integrin subunit is expressed by over 30 various populations of stem cells and is a “stemness” signature gene. In addition to acting as a biomarker for stem cells, there is evidence that α6 confers the functional role of the stem cell properties. Two major α6 variants have been identified owing to an alternative splicing of α6 transcripts in exon 25, which generates distinct α6 isoforms that differ in the cytoplasmic tails. Alternative splicing is a strategy for modifying the function of gene products. The current study aimed to investigate the potential role of distinct α6 isoforms in regulating lung resident mesenchymal stem cell (LR-MSC) propensity and niche interaction.
METHODS: LR-MSCs were obtained by isolation of CD45neg side population (SP) cells from adult mouse lung tissues using fluorescence-activated cell sorting (FACS). The self-renewal potential of isolated LR-MSCs was determined by a colony forming assay. The ability of LR-MSCs to differentiate into adipocytes, osteocytes and chondrocytes was determined by expression of specific cell markers. The mRNA level of a longer α6 variant (α6A) was determined by quantitative real-time PCR (qPCR) using specific PCR primers. The mRNA level of a shorter α6 variant (α6B) was determined by restriction enzyme (NsiI) digestion of α6 cDNA followed by qPCR.
RESULTS: The CD45neg lung SP cell population, which represents LR-MSCs, comprised 0.07-0.10% of total mouse lung cells. Large Giemsa-positive colonies were observed at 10-14 days. CD45neg SP cells were negative for hematopoietic marker CD45 and CD34, and were positive for Sca-1, CD90 and CD105. CD45neg SP cells were able to differentiate into adipocytes as demonstrated by Oil Red O stain, alkaline phosphatase-positive osteocytes and proteoglycan-expressing chondrocytes. Compared to differentiated lung mesenchymal cells/fibroblasts which predominantly expressed the longer α6 transcript (α6A), we observed a significant increase in expression of the shorter α6 transcript in passage 1 of LR-MSCs.
CONCLUSIONS: We have isolated mouse lung resident mesenchymal stem cells and confirmed the mesenchymal stem cell properties of LR-MSCs by established methods. Our preliminary studies show that LR-MSCs express α6 stemness signature gene. It appears that distinct α6 cytoplasmic variant expression is associated with LR-MSC fate determination.