Use of Single Cell RNA seq to Discern Molecular Pathways of Fibrosis and Predicting Emergence of CLAD

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<h3>Purpose</h3> CLAD occurs after airway epithelial cell damage in transplant. The mechanisms of fibrosis are poorly understood. Information of which genes are involved at a single-cell level is crucial. Challenges studying distal airway: 1) Microarray gene expression analysis is limited to pooling all cells in the sample, not single cells, adding uncertainty; and 2) Flow cytometry cannot identify specific epithelial cells. BAL has airway epithelial cells (AEC) and immune cells. The purposes of this pilot study are: 1) determine if AEC can be successfully analyzed with the single cell transcriptomic analysis on clinical BAL samples, 2) what cell populations are analyzed, 3) can rare cells be isolated? <h3>Methods</h3> 5 ml BAL samples were aliquoted from routine clinically scheduled graft monitoring procedures in 3 patients. Cells were washed in PBS, then plated onto array of picoliter wells. Beads bearing cell- and transcript-barcoding capture probes is added along single cell. Each mRNA within a cell is specifically labeled. cells lysed, mRNAs hybridize to beads, reverse transcribed, amplified, and sequenced. <h3>Results</h3> The digital gene expression profile for each cell is assigned to the cell of origin and quantified. Rare AECs were isolated, mphages abundant. Expression patterns from individual cells were quantified with DESeq to identify significant differences in molecular count data. DESeq groups data points that are similar (gene expression type and number of transcripts per gene) in the raw data, then presents them closely approximated to one another in 2D data space. Each cluster represents a cell type. Each point a single cell. Cells with similar HLA expression profiles are the same color. Figure 1 presents the results in a tSNE plot. The tables indicate gene expression data at single cell level. <h3>Conclusion</h3> In this pilot study, we discovered avenues of analysis applicable to clinical translational studies. Next-gen techniques are essential to gain the knowledge needed to counter CLAD and foster new therapeutics.