Phenotypic analysis of primary human neonatal ADSC. (A) Immunophenotypic analysis was performed using fluorescein-conjugated antibodies. For each sample, 10,000 events were read and the percent of positive cells expressing the respective surface marker is listed in the box. (B) Multilineage differentiation capacity of neonatal ADSC. Cells treated with osteogenic supplements showed an increased number of alkaline phosphatase-positive cells. Treatment of cells with adipogenic supplements resulted in the formation of adipocytic cells containing intracellular lipid droplets as detected by oil red staining. ADSC cultured in micromass showed a chondrogenic phenotype as detected by Alcian blue staining. (C) RT-PCR analysis of lineage-specific genes. Alkaline phosphatase (ALP) and osteocalcin (OCN) were used to indicate commitment to the osteogenic lineage. Lipoprotein lipase (LPL) and peroxisome proliferator-activated receptor gamma transcript variant 2 (PPARγ2) were used to indicate cells of an adipogenic lineage. SRY (sex determining region Y)-box 9 (SOX9), collagen type II (COLII), collagen type X (COLX), collagen type XI (COLXI), and aggrecan (AGN) were used to show commitment to the chondrogenic lineage. Top row shows the basal expression of these genes in control ADSC at 0 weeks. Bottom row shows expression of genes after three weeks of culturing in inductive media. (D) RT-PCR analysis for the expression of COL6A1, COL6A2 and COL6A3 genes in cultured ADSC using gene-specific primers. (E) Western blot analysis of individual (α1, α2, α3) collagen VI chains in ADSC using chain-specific antibodies. (F) Immunofluorescence analysis of individual (α1, α2, α3) chains of collagen VI was performed using chain-specific antibodies (red) and co-stained with human lamin A/C (green). ADSC, adipose-derived stem cells.