The adipose-derived lineage-negative cells are enriched mesenchymal stem cells and promote limb ischemia recovery in mice.

White adipose tissue (WAT) is a very attractive source of mesenchymal stem cells (MSCs) because of its availability and ease of harvest. However, the current method of isolating adipose tissue-derived MSCs often relies on the adhesiveness of the cultured stromal-vascular fraction (SVF). Unfortunately, the SVF is a heterogeneous cell population containing many cell types, including adipocyte precursor cells, endothelial cells, pericytes, multipotent MSCs, erythrocytes, and hematopoietic cells. Here we systematically characterized the adipose tissue-derived lineage-negative (Lin(-)) cell population using various surface markers and a set of cell proliferation and differentiation assays. We demonstrate clearly that the Lin(-) cell population represents enriched MSCs, which were identified by their high expression of MSC surface markers, and that these cells are a robust population with a vigorous growth capability and delayed aging. This cell population also demonstrated a much higher capacity for differentiation into osteogenic, chondrogenic and adipogenic cell lineages related to MSCs than did the SVF. These cells promoted recovery from limb ischemia, likely via production of vascular endothelial growth factor, an angiogenic factor. Our study demonstrates that Lin(-) cells are enriched in MSCs and provides a reliable method for isolating purer MSCs than SVF cells from the WAT, especially for obtaining fresh MSCs for clinical applications. In summary, this study identified a new, reliable method for enrichment of WAT MSCs with regenerative repairing features.