CD13 is a bona-fide marker of bovine pre-adipocytes with potential in cultivated fat applications

Despite its significant potential, cultivated fat manufacture remains a relatively inefficient process, as it primarily uses source preparations of animal-derived mesenchymal stem/stromal cells (MSC) that are poorly defined and highly heterogenous in nature, containing only relatively minor fractions of bona-fide adipocyte progenitors. The aim of this study was to use RNA-sequencing of clonal MSC populations from cattle to identify cell surface marker(s) of bona-fide pre-adipocytes to be used for efficient enrichment of MSCs, with a view to future cultivated fat applications. Adipose-derived MSC populations (n = 5 animals) were grown clonally from single cells and subsequently tested for adipogenic capacity. Adipogenic (A) and non-adipogenic (N) clones (n = 10/group) thus identified were bulk RNA-sequenced. A total of 35 cluster of differentiation (CD) genes were identified among differentially expressed transcripts, of which CD13, CD141, CD36, CD55 and CD34 were selected for further testing using flow cytometry in bovine MSCs. All antigens except CD13 were detected at negligible levels. FACS was then used to sort MSCs (n = 4 animals) into CD13+ and CD13- fractions. Sorted CD13+ cells were larger and flatter, grew significantly slower, and expressed substantially higher levels of adipogenic regulators (PPARG and CEBPA) compared to CD13- cells. Moreover, on average, adipogenic efficiency was 10.3-fold higher in CD13+ than CD13- cells, as demonstrated by BODIPY staining and confirmed by differential expression of mature adipocyte markers (FABP4, ADIPOQ, LEP), while expression of alternative lineage markers (chondrogenic and osteogenic) and ability to differentiate into bone and cartilage were both similar for CD13+ and CD13- cells. In summary, we identified CD13 as a bona-fide marker of pre-adipocytes in bovine and demonstrated the potential of using CD13-based cell selection for enriching MSC populations for cultivated fat purposes.