Isolation and characterization of porcine monocyte-derived mesenchymal cells

Abstract

Monocytes are leukocytes in peripheral blood that differentiate into macrophages in the context of the inflammatory response. Leukocytes are easy to isolate from a blood sample by inexpensive standardized methods, such as the Ficoll-based density gradient. We have found that monocytes isolated from peripheral blood of pigs and grown using simple procedures produce large numbers of mesenchymal cells that exhibit differentiation into mesodermal lineages in vitro. Peripheral blood samples were obtained from 2, 4, and 6 months old male pigs. The cells were isolated by a Ficoll-based density gradient and cultured in 20% FBS in DMEM media, on uncoated tissue culture vessels. All isolates exhibited mesenchymal morphology and continued to expand at least to passage 7. The expansionary potential was greatest for the cells obtained from the 2 mo. old pigs. We isolated similar cells from porcine fetal livers (gestation day 60), at which time hematopoiesis is occurring in the liver. Therefore, these cells are present from at least mid-gestation through 6 months, the approximate age of puberty in pigs. In regards to immune-phenotype, the cells are strongly positive for the leukocyte maker CD 14 and SLA-DR-II. Approximately 50% of the cells are positive for CD 45, and they are negative for CD 105, CD 31, and CD 90. The monocyte-derived cells express mRNAs for TLR-3,4,5, 7, and 9. They also express the pluripotency-associated gene Nanog but only weakly express Sox-2 and Oct-4. In vitro the cells are capable of differentiation into adipogenic, osteogenic, and chondrogenic lineages. They also exhibit phagocytosis as measured by in vitro assay. We tested their ability to support the porcine reproductive and respiratory virus in vitro but they were not supportive using standard techniques. Initial attempts have also failed to support myogenic differentiation. The cells isolated in this study represent a novel subset of monocytes with characteristics overlapping those of mesenchymal stem cells. Swine are physiologically similar to humans and further work is needed to characterize these cells for regenerative medicine applications.