Isolation of fat cell precursors from adult rat adipose tissue

Summary The possible existence of adipocyte precursors in adult rat adipose tissue was investigated. Cells were isolated from the stromal fraction of adipose tissue and were grown in culture. Skin fibroblasts were used as controls. The stromal fraction cells were initially fusiform and proliferated; in culture, they accumulated lipid inclusions, became rounder and acquired an eccentric nucleus. In contrast, the skin fibroblasts from the same rat and grown under identical culture conditions, did not exhibit any appreciable lipid accumulation. The doubling time for both the stromal fraction cells and skin fibroblasts was 40–60 h. At confluency, the stromal fraction cells contained 5–7 times more glyceride-glycerol than skin fibroblasts.Thus, adipose tissue of adult rats contains cells with the potential to proliferate and acquire morphological characteristics similar to those of adipocytes.This work was supported by The Medical Research Council of Canada Grant MA-5827, The Ontario Heart Foundation, The Atkinson Charitable Foundation, The Banting Research Foundation and The J.P. Bickell Foundation     

Observatons on the growth and metabolic functions of cultured cells derived from human adipose tissue.

The growth and metabolic activity of cultured cells derived from human adipose tissue (CAT cells) were studied and compared to cultured skin fibroblasts. The morphological appearance of the CAT cells was distinctly different from that of fibroblasts. The growth rate of CAT cells as measured by 3H-thymidine incorporation was much slower than the fibroblast growth rate. Cultured CAT cells synthesized significantly 14C-glucose, while fibroblast cultures had a higher metabolic rate as measured by CO2 production. Insulin stimulated 3H-thymidine incorporation in both CAT and fibroblast cultures. The CAT cells did not show a consistent insulin response of lipid or CO2 production, but this may be a reflection of donor age or nutritional status. Even though the CAT cell may be a type of stromal cell peculiar to adipose tissue rather than a preadipocyte or adipocyte, it may prove useful in studies of human obesity.     

In vivo development of adipose tissue following implantation of lipid-depleted cultured adipocyte

The monolayer culture of isolated and disaggregated adipocytes from rat omental and perirenal sites, gave rise to a population of fibroblast-like cells, usually devoid of lipid inclusion. Similar fibroblast-like cells have been obtained in cultures of adipose tissue stromal cells and are thought to be undifferentiated adipocyte stem cells. Although the adipocyte-derived fibroblasts were morphologically indistinguishable from culture-derived fibroblasts of other origins, upon autoimplantation into the splenic bed they regained the lipid inclusion and developed again into adipose tissue. The findings suggest that the transformation of adipose cells into fibroblast-like cells is reversible modulation and not a dedifferentiation into the adipose tissue stem cell. This work also substantiates the increasingly recognized heterogeneity of fibroblasts.     

Interactions of adipocytes and their precursor cells with endothelial cells in culture

Factors involved in the growth of adipose tissue were examined by testing interactions under cell culture conditions between cellular components of this tissue and plasma from overfed rats. The cellular factors were capillary fragments, endothelial cells during growth and after confluence, fibroblasts, adipocytes and adipose precursor cells before determination (adipoblasts) and after determination (preadipocytes). Multiplying adipose precursor cells stimulated markedly the multiplication of endothelial cells, while their own multiplication was inhibited. The stimulatory effect was partially transferred into the culture medium but not remaining in culture dishes conditioned by preceding cultures of adipose precursor cells, removed by Tris-EDTA buffer or mechanically. The activity was apparently not dependent on feeding conditions. Plasma from overfed rats did not affect endothelial or adipose precursor cell multiplication, but caused more rapid lipid filling of the latter. Endothelial cells facilitated lipid accumulation of preadipocytes. These results indicate that when adipose tissue is expanding by adipocyte multiplication capillarization is stimulated secondarily, being then capable of facilitating triglyceride accumulation in adipocytes.     

Ultrastructural analysis of the in vitro differentiation of female rat preadipocytes

In an attempt to characterize the preadipocytes of the adipose tissue of female rat, we studied by electron microscopy the differentiation of the cells into mature adipocytes in in vitro cultures. The preadipocytes arose from the stroma-vascular fraction of perirenal and perigenital adipose tissue. Culture of the preadipocytes in an enriched medium consisting of Dulbecco's medium supplemented with 10% fetal calf serum, antibiotics, rat triglycerides (0.5%), insulin (290 nM) and Tween 80 (0.1 mg/ml) induced their adipose conversion. The morphology of preadipocytes changed progressively. They accumulated fat granules, droplets and finally globules, which fused together. The cell organelles featured qualitative and quantitative modifications. The nucleus migrated with most mitochondria and a part of the Golgi system towards the cell periphery; the rough endoplasmic reticulum, dilated at the initial stage of differentiation became less and less conspicuous; the perinuclear Golgi system was dispersed between lipid droplets during fat accumulation; thick bundles of microfilaments, localized beneath the plasma membrane disappeared; large lipid droplets were surrounded by a network of microfilaments; many microvesicles and some "rosettes" typical of mature adipocytes could be observed. Nevertheless, the ultrastructural criteria did not allow to clearly discriminate the undifferentiated cells: early preadipocytes (without lipid droplets), adipoblasts and fibroblasts, all of these being probably present in the culture system.     

Metabolism and fate of neutral lipids of fetal lung fibroblast origin

Fetal rat lung fibroblasts characteristically increase their triacylglycerol (TG) stores during development. Both fibroblasts and alveolar type II (TII) cells can synthesize TG de novo, but only fibroblasts can absorb TG from culture medium, and retain the TG in a stable state. When fibroblasts pre-labelled with [³H]triolein are recombined with TII cells in organotypic culture the radiolabel appears in TII cell disaturated phosphatidylcholine (disatPC). When fibroblasts are preloaded with increasing amounts of TG there is a commensurate increase in TII cell disatPC following organotypic culture. Comparison of [³H]triacylglycerol and [¹⁴C]glucose incorporation into type II cell phospholipids revealed preferential use of TG for the surface-active phospholipids disatPC (10-fold greater) and phosphatidylglycerol (23-fold greater). These in vitro data suggest that fibroblasts provide lipid substrate for TII cell surfactant phospholipid synthesis.     

Heterogeneity of fibroblast response in host-tumor cell-cell interactions in metastatic tumors

The spread and invasion of tumor cells into host tissues are associated with the release of elevated levels of collagenolytic activity of both host and tumor cell origins. However, the mechanisms of regulation of the enzyme activity is still unresolved. Histological examination of human and animal tumors revealed morphological changes in stromal fibroblasts and mast cells at the tumor periphery. Numerous mast cells appeared at microfoci along the tumor: host tissue junction and mast cell degranulation were associated with collagenolysis. In vitro studies, using rat mammary adenocarcinoma and human lung adenocarcinoma cells, showed that both tumor cells and host fibroblasts participate in matrix degradation. Tumor-associated stromal fibroblasts released higher levels of enzyme activity than normal fibroblasts and were more responsive to stimulation by tumor-conditioned media and soluble mast cell products. Host fibroblasts appear to be heterogeneous populations of responsive and nonresponsive subpopulations based on their response to tumor- or mast-cell-mediated stimulation of collagenase release. Fibroblast subpopulations were obtained by density fractionation of serum-deprived, synchronized confluent fibroblasts on discontinuous Percoll gradient. Density-fractionated fibroblast subpopulations differed in their response to stimulation by mast cell products and tumor-cell-conditioned media. The stimulatory activity of tumor-cell-conditioned media also varied as a function of the metastatic potential of the tumor cells. The data suggest that cellular interactions between tumor cells and select subpopulations of host fibroblasts at the tumor periphery play a key role in host tissue degradation. However, heterogeneity of stromal fibroblasts may determine the site and extent of the tissue damage at foci of tumor invasion.     

Complete differentiation of adipocyte precursors

Summary Evidence for the complete morphological maturation of precursor cells into adipocytes in vitro is presented. Cells were isolated from the stromal fraction of adipose tissue from adult humans and from rats and were grown in culture. Abdominal skin fibroblasts were used as controls. All cell strains were initially fusiform and replicated. On reaching monolayer confluency, they were transferred to an enriched growth medium in which the human and rat adipocyte precursors differentiated into a homogeneous population of cells, morphologically indistinguishable from mature adipocytes. In contrast, skin fibroblasts from the same person or animal, and grown under identical culture conditions, did not accumulate lipid and retained their fusiform contour. The same results were obtained in the first six subcultures that were studied. Thus, there is firm evidence that fat tissue of adult humans and rats contains adipocyte precursors that differentiate into mature fat cells. The culture system that has been described will facilitate the elucidation of the factors involved in replication and differentiation of adipocyte precursors.This work was supported by The Medical Research Council of Canada Grant MA-5827, The Ontario Heart Foundation, The Atkinson Charitable Foundation, The Banting Research Foundation, The J.P. Bickell Foundation, and the Physicians' Services Incorporated Foundation     

Studies on the fate of pulmonary surfactant in the lung.

1. Radioactively labelled pulmonary surfactant was prepared in an isolated perfused lung system provided with [14C]hexadecanoate. 2. After intratracheal administration of pulmonary surfactant radioactively labelled components were rapidly distributed into different lung fractions, including macrophages (free cells), but most of the radioactive label was accumulated by the lung tissue. 3. Alveolar macrophages, maintained in a variety of culture media in the presence and absence of mineral particles, incorporated a low percentage (11%) of radioactively labelled components when incubated with the surfactant, although evolution of labelled CO2 (6% of the original total activity) suggested that some breakdown of the components had taken place. 4. In similar cultures little intracellular accumulation or extracellular release of non-esterified fatty acids was demonstrated, indicating minimal catabolism of the high-molecular-weight lipid components of surfactant (particularly phosphatidylcholine). 5. However, experiments in vitro designed to simulate the lysosomal degradation of endocytosed surfactant indicated that the macrophage had enzymes capable of releasing non-esterified fatty acids, particularly hexadecanoate, from the lipoprotein complex. 6. It is argued that lung cells, other than alveolar macrophages, may also have a role in surfactant turnover.     

Chondrogenic potential of adipose tissue-derived stromal cells in vitro and in vivo.

Articular cartilage exhibits little intrinsic repair capacity, and new tissue engineering approaches are being developed to promote cartilage regeneration using cellular therapies. The goal of this study was to examine the chondrogenic potential of adipose tissue-derived stromal cells. Stromal cells were isolated from human subcutaneous adipose tissue obtained by liposuction and were expanded and grown in vitro with or without chondrogenic media in alginate culture. Adipose-derived stromal cells abundantly synthesized cartilage matrix molecules including collagen type II, VI, and chondroitin 4-sulfate. Alginate cell constructs grown in chondrogenic media for 2 weeks in vitro were then implanted subcutaneously in nude mice for 4 and 12 weeks. Immunohistochemical analysis of these samples showed significant production of cartilage matrix molecules. These findings document the ability of adipose tissue-derived stromal cells to produce characteristic cartilage matrix molecules in both in vitro and in vivo models, and suggest the potential of these cells in cartilage tissue engineering.     

Adipose conversion of mouse bone marrow fibroblasts in vitro: their alkaline phosphatase activity

By an in vitro colony assay and cytochemical staining, we investigated the capacity of mouse bone marrow fibroblasts to differentiate into adipocytes and to express alkaline phosphatase (ALP) activity. Glucocorticoids enhanced colony formation of the fibroblasts, and stimulated their adipose conversion (55-65% of the colonies became adipocyte-positive), but they did not affect ALP activity. The fibroblasts became heterogeneous in size and morphology after growing in vitro then differentiated into adipocytes. All the cell types had ALP activity, and more than 95% of the colonies contained ALP-positive cells. ALP staining was strongest in cells in the early stage of adipose conversion, gradually decreasing with maturation. Our results indicate that the majority of the mouse bone marrow fibroblasts that formed colonies under our culture conditions are preadipocytes. We conclude that these fibroblasts originate from adventitial reticular cells present in bone marrow stroma because reticular cells have been reported to possess high ALP activity and have been suggested to differentiate into adipocytes.     

Proteome of human subcutaneous adipose tissue stromal vascular fraction cells versus mature adipocytes based on DIGE

Adipose tissue contains a heterogeneous population of mature adipocytes, endothelial cells, immune cells, pericytes, and preadipocytic stromal/stem cells. To date, a majority of proteomic analyses have focused on intact adipose tissue or isolated adipose stromal/stem cells in vitro. In this study, human subcutaneous adipose tissue from multiple depots (arm and abdomen) obtained from female donors was separated into populations of stromal vascular fraction cells and mature adipocytes. Out of 960 features detected by 2-D gel electrophoresis, a total of 200 features displayed a 2-fold up- or down-regulation relative to each cell population. The protein identity of 136 features was determined. Immunoblot analyses comparing SVF relative to adipocytes confirmed that carbonic anhydrase II was up-regulated in both adipose depots while catalase was up-regulated in the arm only. Bioinformatic analyses of the data set determined that cytoskeletal, glycogenic, glycolytic, lipid metabolic, and oxidative stress related pathways were highly represented as differentially regulated between the mature adipocytes and stromal vascular fraction cells. These findings extend previous reports in the literature with respect to the adipose tissue proteome and the consequences of adipogenesis. The proteins identified may have value as biomarkers for monitoring the physiology and pathology of cell populations within subcutaneous adipose depots.     

Alkaline phosphatase-positive reticular cells of chicken bone marrow--in vivo and in vitro studies

We examined alkaline phosphatase (ALPase)-positive reticular cells from chicken bone marrow in vitro in relation to other varieties of adherent cells. ALPase activity was found in both reticular and adipose cells which formed epithelioid cell colonies and were negative for fibronectin. We observed transition between two cell types. ALPase-negative macrophages as small round cells in culture revealed positive fibronectin and transformed into ALPase-negative spindle cells in long-term culture. Thus, we suggest two cell lineages, each with distinct cell characteristics.     

Tunicamycin inhibits the differentiation of ST 13 fibroblasts to adipocytes with suppression of the insulin binding activity

ST 13 cells are a clonal line of murine fibroblasts that are capable of differentiating into adipocyte-like cells $\text{in}\text{vitro}$. When the cells were maintained as a confluent monolayer, they began to accumulate lipid droplets and to exhibit a rapid increase of insulin binding activity. Tunicamycin, a specific inhibitor of dolichol-mediated protein glycosylation, blocked this adipose conversion without affecting cell growth and total protein synthesis. The inhibitory effect of tunicamycin was dose-dependent and reversible. Enhancement of the incorporation of [¹⁴C]acetate into triglyceride fraction accompanying the adipose conversion was completely inhibited by tunicamycin, whereas the incorporation into phospholipid fraction was only partially affected. The insulin binding activity increased about 10-fold during differentiation, but was completely suppressed in tunicamycin-treated cells.     

Thiazolidinediones exhibit different effects on preadipocytes isolated from rat mesenteric fat tissue and cell line 3T3-L1 cells derived from mice

The effects of PPAR-gamma agonists, thiazolidinediones (TZDs), on preadipocytes isolated from rat mesenteric adipose tissue and murine cell line 3T3-L1 were compared using an in vitro cell culture system. After each cell formed a confluent monolayer under appropriate medial conditions, pioglitazone or troglitazone was applied at 10 microM to each medium for cell maturation. We observed morphological changes in each cell, especially the accumulation of lipid droplets in the cytoplasm, during the culture periods. At the end of culture, DNA content, triglyceride (TG) content and glycerol-3-phosphate dehydrogenase (GPDH) activity were determined. Adiponectin concentrations in each culture medium were also measured during appropriate experimental periods. Application of TZDs increased the DNA content, TG accumulation and GPDH activity in the 3T3-L1 cells but not in the mesenteric adipocytes. Although TG accumulation was unchanged, the number of lipid particles was decreased and the size of lipid particles in the mesenteric adipocytes was increased by TZD application. Although the TZDs increased adiponectin release from the 3T3-L1 cells, adiponectin release from mesenteric adipocytes was suppressed (P<0.05). Thus, the effects of TZDs differed between the primary culture of mesenteric adipose cells and the line cell culture of 3T3-L1 cells. The source of adipocytes is an important factor in determining the action of TZDs in vitro, and particular attention should be paid when evaluating the effect of PPAR-gamma agonists on adipose tissues.     

Studies on carcinogenesis by avian sarcoma viruses. VI. Differential multiplication of uninfected and of converted cells in response to insulin

Insulin can replace the factor(s) in calf serum whose amount is limiting for multiplication in cell culture of chicken embryo fibroblasts and of chicken embryo fibroblasts infected and converted by avian sarcoma virus. In serum-free, insulin-containing medium, converted cells multiply more than do uninfected cells. It appears, therefore, that the increased multiplication in cell culture of converted cells as compared with uninfected cells results from a decreased requirement by the converted cells for an insulin-like activity found in serum.     

Direct and indirect effects of leptin on preadipocyte proliferation and differentiation

Leptin has been shown to reduce body fat in vivo. Adipocytes express the leptin receptor; therefore, it is realistic to expect a direct effect of leptin on adipocyte growth and metabolism. In vitro studies examining the effect of leptin on adipocyte metabolism require supraphysiological doses of the protein to see a decrease in lipogenesis or stimulation of lipolysis, implying an indirect action of leptin. It also is possible that leptin reduces adipose mass by inhibiting preadipocyte proliferation (increase in cell number) and/or differentiation (lipid filling). Thus we determined direct and indirect effects of leptin on preadipocyte proliferation and differentiation in vitro. We tested the effect of leptin (0-500 ng/ml), serum from leptin-infused rats (0.25% by volume), and adipose tissue-conditioned medium from leptin-infused rats (0-30% by volume) on preadipocyte proliferation and differentiation in a primary culture of cells from male Sprague-Dawley rat adipose tissue. Leptin (50 ng/ml) stimulated proliferation of preadipocytes (P<0.05), but 250 and 500 ng leptin/ml inhibited proliferation of both preadipocyte and stromal vascular cell fractions (P<0.01), as measured by [3H]thymidine incorporation. Serum from leptin-infused rats inhibited proliferation of the adipose and stromal vascular fractions (P=0.01), but adipose tissue-conditioned medium had no effect on proliferation of either cell fraction. None of the treatments changed preadipocyte differentiation as measured by sn-glycerophosphate dehydrogenase activity. These results suggest that leptin could inhibit preadipocyte proliferation by modifying release of a factor from tissue other than adipose tissue.     

Cell-IQ visualization of motility,cell mass,and osteogenic differentiation of multipotent mesenchymal stromal cells cultured with relief calcium phosphate coating

The Cell-IQ continuous surveillance system allowed us to establish the following changes in a 14- day culture in vitro: a twofold suppression of the directional migration of multipotent mesenchymal stromal cells of human adipose tissue (MMSC-AT) towards the samples with a microarc calcium phosphate (CP) coating from synthetic hydroxyapatite; a tenfold decrease in the cell mass on the interphase with the samples, which was accompanied by a slight reduction in the expression of membrane determinants of stromal stem cells; and an enhancement of their osteogenic differentiation (osteocalcin secretion and mineralized matrix formation) on the 21st day of the study. Calcium phosphate particles, but not the calcium and phosphorus ions, may trigger the phenotypic transformation of the MMSC-AT behavior in vitro.     

Ultrastructural studies on white adipocyte differentiation in the mouse mammary gland following estrogen and relaxin

White adipocyte differentiation was studied ultrastructurally in the mouse mammary gland following stimulation with 17beta-estradiol and relaxin. These hormones have been previously demonstrated by us to induce hyperplasia and hypertrophy in the adipose cells of the mouse mammary gland. Following hormone treatment new fat cells are formed around the growing ducts. In these sites there is a close relationship between blood vessel growth and adipocyte development, and stromal areas featuring embryonic fat organs can frequently be found. In the sites of adipocyte differentiation the most numerous cell types are undifferentiated mesenchymal cells and preadipocytes, while fibroblasts and macrophages are much less common. No endothelial cells or pericytes were found detaching from the blood capillary walls. There were no fibroblasts or macrophages containing intracellular lipid deposits. Actively degranulating mast cells were frequent. The above findings strongly suggest the direct origin of adipocytes from perivascular mesenchymal cells, without the intermediate stages of well-differentiated fibroblasts, macrophages, endothelial cells or pericytes. The earliest morphologically recognizable stage in adipocyte differentiation is a 'pale preadipocyte', characterized by its irregular shape due to cytoplasmic processes, clear cytoplasmic matrix, well-developed organelles (especially ribosomes and Golgi apparatus), few and small lipid droplets, numerous pinocytotic vesicles and a very thin basal lamina. The next stage in adipocyte differentiation is a 'dark preadipocyte' showing a denser cytoplasmic matrix, reduced organelles and more abundant lipid accumulations. The pinocytotic vesicles are very numerous and the enveloping basal lamina is still thin. The subsequent maturation stages are the well-known globular multivacuolated adipocyte and finally the mature univacuolated, signet-ring, white adipocyte.     

Induction of cytokeratin expression in human mesenchymal cells

We studied the phenotypic features of some typical human mesenchymal cells, including decidual stromal cells and adult and fetal fibroblasts under different cell culture conditions by using antibodies to intermediate filament proteins and desmoplakins. In cell culture, the decidual stromal cells rapidly acquired typical fibroblastoid appearance with abundant arrays of vimentin filaments while the cytokeratin-positive epithelial cells, occasionally found in typical epithelioid colonies, lacked vimentin positivity and showed desmoplakin positivity. Within a few days, many of the stromal cells started to present cytokeratin positivity when cultured either in Condimed or in Chang medium. The cytokeratin positivity was first detected in small, scattered cytoplasmic dotted fibrils or in perinuclear dotlike aggregates with fibrillar projections. Later, denser cytokeratin-positive fibrillar arrays could also be seen in stromal cells, which lacked desmoplakin positivity as judged by two monoclonal antibodies. Decidual stromal cells were also cloned and in five out of ten clones some of the cells acquired a similar cytokeratin positivity when transferred into Chang or Condimed medium. Immunoblotting results indicated that cytokeratins 8, 18, and 19 can be found in these cultures. Similar cytokeratin positivity could also be seen in the same culture conditions in cultured fetal fibroblasts from skin, chorionic villi, and lung but not in young or adult skin fibroblast cultures. The present results suggest that decidual stromal cells as well as some embryonal mesenchymal cells can acquire epithelial differentiation in vitro as judged by the emergence of cytokeratin proteins. This ability appears to be lost in the corresponding adult cell. The results furthermore suggest that cytokeratin fibrils can be organized in the cytoplasm without an apparent organization center and that neither the appearance of desmoplakins nor the formation of cell-to-cell contacts are required for cytokeratin filament assembly.