Generation of novel adipocyte monolayer cultures from embryonic stem cells.

Here we show a simplified and improved method to produce large quantities of evenly distributed monolayer cultures that display major characteristics of adipocytes. These cultures are applicable for quantitative analysis for biochemical and molecular events in adipogenesis during development and may provide a useful system for high-throughput drug screening assays of antiobesity drugs. In our method, we treated embryoid bodies (EBs) with all-trans retinoic acid (ATRA) for 3 days, 1 day after they attached to the gelatin-coated culture plates without further transfer. The cells were maintained in insulin and trioiodothyronine (T(3))-containing medium until day 12, when they were dispersed by enzymatic digestion and replated onto multiple culture plates. Two days later, adipocyte induction factors were added for 6 days and examined 6 days later. The amount of lipid droplet-laden adipocytes in the culture reached approximately 80%, with a nearly five-fold increase in GPDH activity. The cells expressed high levels of adipose-specific proteins (adipocyte markers), including PPARgamma2, ALBP, LPL, HSL, perilipin, and DGAT1. The adipocytes are functionally active, as evidenced by their response to lipolytic agents, such as forskolin, Bt2-cAMP, and isoproterenol, with more than 20-fold increases in glycerol release.

Inhibition of growth and differentiation of osteoprogenitors in mouse bone marrow stromal cell cultures by increased donor age and glucocorticoid treatment.

Primary cultures of bone marrow stromal cells (BMSC) from long bones of young (4-5 months) and old (22-25 months) C57BL/6 male mice were used to study how donor age affects growth and differentiation of osteoblasts and their sensitivity to dexamethasone (DEX). We assessed changes in the number and area of alkaline phosphatase-positive bone-forming osteolastic colonies (CFU-ALP) and in the total number of colonies (CFU-F) that include ALP negative colonies. Cell proliferation and apoptosis, specific activity of ALP, were also measured for growth and differentiation. We found that the number of nucleated cells harvested from old mice was significantly higher (approximately 20% more) than that from young mice. However, the number of colonies formed by old cells was fewer and the total area less than those formed by young cells plated at the same density. Young and old cells responded similarly to DEX showing a dose-dependent decrease in colony number and area with more inhibition for area than number. DEX affected CFU-ALP more than CFU-F indicating a greater inhibition for osteoprogenitor cells than other cell types. Inhibition of cell attachment at early culture was the major cause for the DEX reduction of colony number and the major cause of area reduction was inhibition of cell proliferation. This was demonstrated by a severe dose-dependent lowering of bromodeoxyuridine (BrdU) incorporation to less than 40% of the control. Although the number of apoptotic cells in the DEX-treated cultures was higher, apoptosis was not a major factor since the number of apoptotic cells was less than 5% even with DEX treatment. Despite these negative effects on colony number and size, DEX-enhanced osteoblastic differentiation activity by stimulating ALP activity of the colonies up to 25-fold in the young and 5-fold in the old. Our data suggest that increased age lowered the number of osteoprogenitor cells and their growth in BMSC cultures. DEX decreased the attachment and proliferation of BMSC in culture. These changes reflect age-related and glucocorticoid-induced osteopenia. Mouse BMSC cultures therefore may serve as a useful in vitro model to study the mechanisms of type II osteoporosis.