Determinations of adipose cell size and number in suspensions of isolated rat and human adipose cells

The osmic acid fixation-Coulter electronic counter method described for determining adipose cell size and number in intact adipose tissue fragments has been modified for use with suspensions of isolated rat and human adipose cells. Mean cell sizes in tissue fragments and isolated cell suspensions prepared from the same tissue are virtually identical in rats of various weights. No statistically significant difference in mean adipose cell size between tissue and isolated cell suspension was observed in human adipose tissue although the variability was much greater than in rat tissue. The distribution of cell sizes among replicate samples is more uniform in the isolated cell preparations, possibly reflecting the considerably larger quantities of tissue used in preparing isolated cells than in determining cell size and number directly from tissue fragments. An example of the utility of the modified method during routine metabolic studies with isolated rat epididymal adipose cells is described; isolated cells of increasing size can be obtained from rats of increasing body weight, or from the separated distal and proximal portions of the fat pads of rats of the same weight.     

Effects of insulin on glucose metabolism in isolated human fat cells

Isolated fat cells were used for the study of in vitro effects of insulin on glucose metabolism in human and rat adipose tissue. In human subcutaneous fat cells, effects of insulin could be detected at concentrations of glucose in the medium from 1 to 10 micro moles/ml. Cellular responsiveness was inversely proportional to the glucose level. At a constant concentration of 6 micro moles of glucose per ml, the effects of insulin at various concentrations up to 500 micro U/ml were investigated. At the highest concentration, which gave the maximal response, there was a 100% increase in the conversion of glucose-U-(14)C to glyceride-glycerol and a 40% increase in glucose oxidation. The dose-response curve was steepest between 2 and 20 micro U/ml. Rat epididymal fat cells were much more responsive to insulin. Glucose lipogenesis and pentose cycle activity could also be demonstrated in rat cells, whereas these activities could not be shown in fat cells from human omental and subcutaneous tissue. The findings for human cells are attributed to changes in cellular activity during preparation.     

Characteristics of human mesenchymal stem cells isolated from bone marrow and adipose tissue

Populations of human mesenchymal stem cells were derived from bone marrow and adipose tissue. Here analysis of six individuals is represented. Cells were isolated, expanded and evaluated by the expression of surface antigens using flow cytometry. These cells displayed similar characteristics for many markers. Cells isolated from bone marrow and adipose tissue were found to be homogeneously positive for CD13, CD44, CD90, CD105, and negative for CD45, CD34, CD31 and CD117. Besides, differences in surface antigene CD10 expression between narrow and adipose tissue-derived cells were detected. All these findings indicate that both bone marrow and adipose tissue are important sources of mesenchymal stem cells, which could be used in cell therapy protocols.     

Steroid metabolism by cells from human chorion laeve isolated on Percoll gradients

Collagenase-dispersed cells from human chorion laeve were examined on Percoll gradients. The 3 beta-hydroxysteroid dehydrogenase (a trophoblast marker) and steroid sulfatase activities of the cells were measured and a system was developed to isolate enriched preparations of the trophoblast cells. No cells were found to sediment at Percoll concentrations greater than 50%, and using continuous gradients of Percoll there appeared to be cells with different 3 beta-hydroxysteroid dehydrogenase (3 beta HSD): steroid sulfatase ratios sedimenting in different regions of the gradient. Cells with a high ratio were found in the denser region of the gradient. Continuous gradients provided inadequate separations of distinct populations of cells, thus to obtain a more reproducible system to isolate cells, discontinuous gradients of Percoll were studied. A discontinuous gradient composed of 5, 20, 40, and 60% Percoll was developed and three bands of cells were found sedimenting at the 20, 40 and 60% interfaces, respectively. The number and appearance of cells at the 20 and 60% interfaces varied from tissue to tissue. In contrast, the cells sedimenting at the 40% interface were less variable, a substantial number was found to be present in every tissue studied, they were similar in appearance to the trophoblast cells and had high 3 beta HSD:sulfatase ratios.     

Studies on the activity of 17 beta-hydroxysteroid dehydrogenase in human adipose tissue

The activity of 17 beta-hydroxysteroid dehydrogenase has been investigated in human subcutaneous adipose tissue. Using oestrone as substrate, oestradiol formation was linear with time and the concentration of protein in the tissue homogenate. The optimum pH was 8.0 and the Km for oestrone was 2.5 x 10(-6) M. With NADH, the production of oestradiol was about 30% of that with NADPH. Oestradiol was also a substrate for the enzyme although under the experimental conditions used reduction of oestrone appeared to be favoured in adipose tissue. In the presence of progesterone (31.8 x 10(-6) M) the Km for oestrone was increased fivefold.     

Studies of human achondroplasia: oxidative metabolism in tissue culture cells

Mitochondria prepared from the first growth of cells (fibroblasts) from skin biopsies from homozygous (but not heterozygous) achondroplastic human subjects were unable to carry out oxidative phosphorylation. However, successive crops of cells gained the ability to phosphorylate with normal P:O ratios with pyruvate-malate and succinate as substrates. Concentrations of cytochromes a + a3 were markedly and significantly lower in homogenates of homozygous achondroplastic tissue culture cells than in homogenates of normal cells. Levels of cytochromes a + a3 in the heterozygous achondroplastic cells were intermediate between the levels in normal cells and the homozygous achondroplastic cells. Activities of the mitochondrial oxidative systems (NADH, succinic and cytochrome oxidases) were not significantly lower in the achondroplastic cell preparations than in normal cell preparations under standard assay conditions (saturation levels of oxygen).     

The changes of multipotent mesenchymal stromal cells isolated from human adipose tissue during long-term cultivation

Multipotent mesenchymal stromal cells (MMSCs) isolated from human adipose tissue have been subcultured for over 50 population doublings. In three cultures that experienced more than 50 cellular doublings, there appeared cells lacking contact inhibition upon reaching the monolayer. Karyotype analysis (GTG banding) showed a normal diploid karyotype, and aneuploidy and restructurings were not registered. Flow-cytometric analysis of 20 surface antigens on MMSCs in early and late passages revealed changes in the share of cells positively stained with antibodies against CD10 (a zinc depended metalloproteinase); CD34 (sialomucin); CD49 a, d, f, (α1, α4, α6, integrins); and CD71 (a transferrin receptor). Long-term cultivation influenced cell adhesion to proteins of the extracellular matrix (ECM), such as fibronectin and laminin (ligands to α, integrins), as well as the functional abilities of MMSCs to form cells of adipose and bone tissues in vitro. These findings extend our knowledge of cell behavior in culture and allow us to get closer to a deeper understanding of the processes happening to precursor cells in vitro.