N. Hauser

Glucocorticoids induce a drastic inhibition of proliferation and stimulate differentiation of adult rat fat cell precursors.

The effects of physiological glucocorticoids such as cortisol and corticosterone, as well as dexamethasone, on proliferation and differentiation of rat fat cell precursors kept in primary culture were analyzed. In serum-containing medium (10%), glucocorticoids markedly decreased cell proliferation, either on subconfluent or on confluent cultures. This effect was independent of the presence of insulin. In contrast, acute amplification of adipose conversion was observed mainly when glucocorticoids and insulin were added simultaneously. Morphological quantification of lipid-containing cells confirmed acceleration of the maturation process, and an early and specific reorganization of the cytoskeleton was detected at the ultrastructural level. In the presence of insulin, glucocorticoids also enhanced the main marker enzymes, lipoprotein lipase, and glycerol phosphate dehydrogenase. Glucocorticoid effects on precursor proliferation and differentiation were clearly dose-dependent, dexamethasone being 10 times more potent than cortisol and corticosterone. Similar results were obtained in serum-free medium, as well as in preadipocyte cultures derived from different fat deposits. This study demonstrates that in addition to an acute inhibition of precursor growth, glucocorticoids exert a clear stimulation of adipose conversion, which depends mainly on the presence of insulin and the glucocorticoid concentration.

The stroma-vascular fraction of rat inguinal and epididymal adipose tissue and the adipoconversion of fat cell precursors in primary culture

Summary— The stroma‐vascular fraction (SVF) of inguinal and epididymal fat pads of 4 week‐old rats was studied by electron microscopy. Among the various cell types, endothelial cells and preadipocytes were found in both SVF, while mesothelial cells were only detected in the epididymal SVF. The resulting heterogeneity of primary culture and the adipoconversion of the fat cell precursors were studied in a serum‐supplemented medium enriched with insulin (14.5 nM) and exogeneous triglycerides. Despite the heterogeneity of the inoculum, the primary cultures were rather homogeneous, fat cell precursors being the main cell type. Distinctive contaminant fibroblast‐like cells were observed in both cultures, whereas epithelial‐like cells, which correspond most probably to mesothelial cells, were only found in epididymal cultures. Differentiation of fat cell precursors was assessed by the appearance of lipoprotein lipase (LPL) and glycerol‐3‐phosphate dehydrogenase (GPDH). LPL activity was found in the same level in cells of both deposits while GPDH activity was elevated in inguinal vs epididymal derived stroma‐vascular cells. The different adipose conversion pattern of both cultures was confirmed by morphological quantification: the maturation of epididymal fat cell precursors was faster but less extensive. These differences could be related mainly to regional localization rather than to different maturation of the two fat deposits.

Morphologic characterization of osteoblast-like cell cultures isolated from newborn rat calvaria.

SummaryTwo methods for harvesting osteoblast-like cell populations from newborn (10 days) rat calvaria were compared. The first one consisted in culturing the periosteum-free bones and then trypsinizing the cells on the bone surface. The second one involved the migration of the osteoblasts on glass fragments before trypsinization. Since the plating efficiency, the proportion of alkaline phosphatase-positive cells, the population doubling time, and the calcium deposition were more adequate, the second method was used to further characterize the behavior of the cultures. During the first week of culture, the cells featured shapes similar to those observedin vivo on the surface of periosteum-free calvaria. They formed multilayers and, in the presence of ascorbic acid, synthetized an organic matrix containing exclusively type I collagen. Later, small amounts of type III collagen appeared. The cells were embedded in the matrix and progressively acquired the morphologic phenotype of osteocyte-like cells. The matrix mineralized in the presence of β-glycerophosphate. The technique of dropinoculation (high concentration of cells in a small volume of medium) promoted the multilayer formation and the achievement of large mineralized plates (about 1 cm2) in 3 weeks of culture.

Ultrastructure and Cytochemical Detection of Alkaline Phosphatase in Long-Term Cultures of Osteoblast-like Cells from Rat Calvaria

Abstract. Two methods of collecting osteoblast-like cells from newborn rat calvaria were tested, either placing individual glass fragments or tipping dense glass beads onto the endocranial surface of periosteum-free bone. Inoculated at high density, cells collected by using these two methods form large mineralized plates after three weeks of culture. The main purpose of our investigation was to analyze the progressive formation of this mineralized structure and to localize alkaline phosphatase activity. At the beginning of the culture, flattened cells gathered into multilayers and synthesized collagen fibers. Cells in the upper layer became rapidly cuboidal in shape and continued to secrete collagen at their basal pole, whereas other cells became progressively embedded in the extracellular matrix. The upper cells featured ultrastructural characters of osteoblasts, whereas the embedded cells resembled osteocytes. After two weeks, the matrix began to mineralize: crystals appeared on collagen fibers, on matrix vesicles, and on cell debris. During the first days of the culture, the alkaline phosphatase activity was localized on the plasma membranes and on the collagen fibers. Thereafter, only the upper cells and collagen fibers that were juxtaposed to these cells showed alkaline phosphatase activity. In addition, the presence of mineralized matrix prevented the reaction product from being visualized on collagen fibers. The ultrastructural analysis reveals large mineralized plates with a structure resembling that of bone in vivo. This culture appears to be an appropriate model to study bone formation and regulation.

Effect of aging on the morphology of epididymal adipose tissue in the rat

Quantitative morphometrical parameters were compared in epididymal adipose tissue of adult (6 months old) and old (24 months old) Wistar rats, using light and electron microscopy of embedded material and freeze-etch replicas, and taking into account the functional unit of adipose tissue: the capillary-adipocyte. Despite an insignificant reduction of the adipocyte number and size in old rats when the whole population was sampled, the confounding factor of size of adipocytes could be excluded from morphometric computations in adult and old rats. Morphometric measures were performed on the whole transit from capillaries to adipocytes. They revealed that the plasma membranes, as seen in freeze etching, and the thicknesses of endothelial and adipocyte cytoplasms, as seen in ultra-thin sections, remained unaltered with aging. By contrast, the basement membranes were changed but differently around capillaries and adipocytes. The capillary-adipocyte distances were shorter and the vascularization density was higher in old rats.

Why Not Use Primary Cells

For the cell biologist, perhaps an ultimate goal is to explain how, from the unicellular zygote, the various cells of the organism proliferate, move, differentiate, and die. If the signals regulating these events are discovered, the complete developmental pattern would be explained. The program is currently under study in small animals, as Caenorhabditis and Drosophila (1,2). If one imagines a similar approach with more complex animals, man comprised, the key is found to take every desired cell at the good time and the good place, and then to govern its proliferation rate and differentiation. Needless to say that to have differentiated cells at work is a major biotechnological target, thanks to the variety of tests and products they can provide.