High-Throughput Screening of Surface Marker Expression on Undifferentiated and Differentiated Human Adipose-Derived Stromal Cells

Abstract

Adipose tissue contains an abundant source of multipotent mesenchymal cells termed ''adipose-derived stromal cells'' (ASCs) that hold potential for regenerative medicine. However, the heterogeneity inherent to ASCs harvested using standard methodologies remains largely undefined, particularly in regards to differences across donors. Identifying the subpopulations of ASCs predisposed toward differentiation along distinct lineages holds value for improving graft survival, predictability, and efficiency. Human ASCs (hASCs) from three different donors were independently isolated by density-based centrifugation from adipose tissue and maintained in culture or differentiated along either adipogenic or osteogenic lineages using differentiation media. Undifferentiated and differentiated hASCs were then analyzed for the presence of 242 human surface markers by flow cytometry analysis. By comprehensively characterizing the surface marker profile of undifferentiated hASCs using flow cytometry, we gained novel insights into the heterogeneity underlying protein expression on the surface of cultured undifferentiated hASCs across different donors. Comparison of the surface marker profile of undifferentiated hASCs with hASCs that have undergone osteogenic or adipogenic differentiation allowed for the identification of surface markers that were upregulated and downregulated by osteogenic or adipogenic differentiation. Osteogenic differentiation induced upregulation of CD164 and downregulation of CD49a, CD49b, CD49c, CD49d, CD55, CD58, CD105, and CD166 while adipogenic differentiation induced upregulation of CD36, CD40, CD146, CD164, and CD271 and downregulation of CD49b, CD49c, CD49d, CD71, CD105, and CD166. These results lend support to the notion that hASCs isolated using standard methodologies represent a heterogeneous population and serve as a foundation for future studies seeking to maximize their regenerative potential through fluorescence-activated cell sorting-based selection before therapy.

Ex Vivo Human Adipose Tissue Derived Mesenchymal Stromal Cells (ASC) Are a Heterogeneous Population That Demonstrate Rapid Culture-Induced Changes

“…It is possible that, in addition to culture-induced changes, differences in the cellular composition of these two populations may contribute to their different paracrine profiles and activity in differentiation assays. The latter option is feasible as heterogeneity in early passage cultured ASC has been reported previously (Baer et al, 2013;Walmsley et al, 2015;Barilani et al, 2018). Our analysis extends these previous results by showing that heterogeneity and donor-variability exists within uncultured ASC and aligns with recent reports of heterogeneity within this cell population (Merrick et al, 2019;Raajendiran et al, 2019).…”

Mesenchymal Stem/Stromal Cells in Regenerative Medicine: Can Preconditioning Strategies Improve Therapeutic Efficacy

“…Another study applied a broad antibody panel (>200 markers) to compare AT-MSC surface after osteogenic or adipogenic differentiation to undifferentiated AT-MSCs. Here, an increased expression of CD164 was associated with an osteogenic differentiation, whereas CD36, CD40, CD146, CD164, and CD271 were higher expressed after adipogenic differentiation [49]. …”

Design and characterization of calcium phosphate ceramic scaffolds for bone tissue engineering

“…The cost of in vivo animal studies and the loss of animal lives continue to motivate the development of in vitro screening assays. There are a number of different types of in vitro osteogenesis assays currently used that attempt to predict in vivo performance listed here in historical order (1) in vitro apatite forming ability measured by a simulated body fluid test 110 , (2) in vitro osteogenic differentiation assays involving seeding of human or rodent osteoprogenitor cells such as MSCs, calvarial bone progenitors, or cells lines derived from an osteosarcoma and evaluating their differentiation via bone protein expression and mineral content 111 , (3) high throughput assays involving flow cytometry screening of cells after exposure to osteogenic agents 112 . No method is fully predictive due to a variety of bone formation mechanisms that may be operational depending on the conditions of in vivo implantation, such as endochondral ossification in which cartilage formation precedes bone formation.…”