Comparison of Regulatory Type of Macrophages and PCMO Cells from perspective of RNAseq data.

Gene expression plasticity is central for macrophages? timely responses to cues from the microenvironment permitting phenotypic adaptation from pro-inflammatory (M1) to wound healing and tissue-regenerative (M2, with several subclasses). Regulatory macrophages (Mreg) are a distinct macrophage type, partially sharing some functionalities with both M1 and M2 cells. Mreg possess immunoregulatory, anti-inflammatory, and angiogenic properties, and are considered as a potential allogeneic cell therapy product to treat clinical conditions, e.g., non-healing diabetic foot ulcers. In this study we characterized clinically relevant regulatory macrophages, Mreg and Mreg_UKR, programmable cells of monocytic origin (PCMO) and comparator macrophages (M1, M2a and M0) using flow-cytometry, RT-qPCR, phagocytosis and secretome measurements, and finally RNA-Seq. We demonstrate that conventional phenotyping has a limited potential in deciphering all classes of produced cells. This limitation was ameliorated when global transcriptome characterization by RNA-Seq was employed. Using this approach we prove that macrophage manufacturing processes can result in a highly reproducible cell phenotype. At the same time, minor changes introduced in a protocol can consistently effect the phenotype of the end product as well. Additionally, we have identified a novel constellation of potential process specific biomarkers, which will not only support further clinical product development, but will lead to a better understanding of macrophage biology, differentiation and polarized activation. There are 7 immune cell types, comprised of 1 reference (CD14) and 6 differentiated types: M0, M1, M2a, Mreg, Mreg_UKR, and PCMO. There are 9 anonymized human donors, with a minimum of 3 donors per cell type. The donors act as biological replicates for the purpose of differential expression statistics.