Newly recruited and pre-existing preadipocytes in cultures of porcine stromal-vascular cells: morphology, expression of extracellular matrix components, and lipid accretion.

Abstract

Expression of extracellular matrix (ECM) components during differentiation of pre-existing preadipocytes and preadipocytes recruited by dexamethasone (DEX) was examined with immunocytochemistry in primary cultures of adipose tissue stromal vascular (S-V) cells. Immunocytochemistry showed that a small proportion of preadipocytes (AD-3+) in 24-h cultures (d 0 to 1) contained lipid or expressed ECM. Two days of insulin treatment markedly increased preadipocyte ECM expression, and preadipocytes were "rounder" than those not treated with insulin. Dexamethasone with insulin increased preadipocyte recruitment two- to fivefold in completely serum-free cultures and in cultures serum-free after seeding and plating in serum for 1 to 3 d. Double staining demonstrated that ECM expression and lipid accretion were tightly coupled and lagged significantly behind preadipocyte recruitment (AD-3 expression). Double staining (lipid and AD-3) also demonstrated remarkable and unexpected cytological traits indicating a "reticuloendothelial" nature of newly recruited preadipocytes. Time-lapse phase contrast microscopy verified these observations and demonstrated that small adipocytes and preadipocytes migrated and formed cell-to-cell contacts while aggregating and clustering. Large clusters of lipid-free preadipocytes developed in DEX-treated cultures, but not in cultures treated with DEX + insulin. However, the influence of DEX on preadipocyte recruitment and ECM expression was independent of insulin. Preadipocytes on ECM substrata accumulated lipid but were "flat" and did not express ECM components, regardless of insulin or DEX treatment. These studies clearly indicate that preadipocytes express ECM components after recruitment, and the ECM may be critical for morphological development of adipocytes.