RESUMO
The stromal vascular fraction (SVF) of human adipose tissue is a heterogeneous population, with component cell types that may or may not contribute to its regenerative potential. Recent findings indicate that single-cell mechanical biomarkers are characteristic of cell type and can be used comparably to gene and protein expressions to identify cell populations. In this study, we characterized mechanical properties and differentiation potential of cell types present in the SVF. Fluorescence-activated cell sorting was used to isolate four distinct populations based on surface markers: endothelial cells (EC), adipose-derived stem cells (ASCs), pre-adipocytes, and smooth muscle cells (SMC). Atomic force microscopy was used to mechanically characterize sorted cell populations and unsorted SVF. Differentiation capabilities of sorted and unsorted populations were evaluated by quantifying lipid production and calcified matrix deposition. Cells populating the SVF exhibited a range of mechanical properties, with ECs, ASCs, pre-adipocytes, and unsorted SVF cells being significantly more compliant than SMCs. Lineage-specific metabolite production was most robust in SVF cells, followed by ASCs, with the other cell types showing little or no potential, suggesting the unsorted populations may benefit from a paracrine response that is lacking once the cells are sorted into more uniform cell populations.
RESUMO
INTRODUCTION: Mesenchymal stem cells have been increasingly used for cell-based therapies. Adipose-derived stem/stromal cells (ASCs) from the stromal vascular fraction (SVF) of fat tissue are a particularly attractive option for cell based therapy given their accessibility and relative abundance. However, their application in both clinical and basic science investigations is complicated by the isolation of differentiable cells within the SVF. Current enrichment strategies, such as monolayer passaging and surface marker-based sorting, can be time-consuming or overly stringent. Ideally, a population of cells with great regenerative capacity could be isolated with high yields so that extensive in vitro manipulation is not necessary. The objective of this study was to determine whether SVF cells sorted based on expression of alkaline phosphatase liver/bone/kidney (ALPL) resulted in populations with increased osteogenic differentiation potential. METHODS: SVF samples were obtained from four, human donors and processed to isolate initial, heterogeneous cell populations. These SVF cells underwent a four day osteogenic priming period, after which they were treated with a fluorescent, oligodeoxynucleotide molecular beacon probe specific for ALPL mRNA. Cells were separated into positive and negative groups using fluorescence-activated cell sorting (FACS) then differentiated down the osteogenic lineage. Differentiation was assessed by measuring calcified matrix production in each sample. RESULTS: Cells positive for ALPL expression (ALPL+) represented approximately 34% of the gated population, while cells negative for ALPL expression (ALPL-) represented approximately 18%. ALPL+ cells produced 3.7-fold and 2.1-fold more calcified matrix than ALPL- and unsorted SVF cells, respectively, indicating a significant improvement in osteogenic differentiation. Further, ALPL+ cells showed increases in metabolite production for both adipogenesis and chondrogenesis, suggesting that the enrichment process yields an enhanced multipotent phenotype. Osteogenic differentiation response and cell yields for ALPL+ cells were markedly improved over surface marker-sorted samples. CONCLUSION: This study demonstrates a novel method to enrich heterogeneous SVF cells for increased osteogenic potential. The procedure requires less time and results in higher yields of therapeutically useful cells than other existing approaches. Gene expression-based sorting of MSCs is a potentially paradigm-shifting approach that could benefit applications spanning from basic science to clinical therapy.