Culture of proliferating and differentiating fat-storing cells in 3T3-conditioned medium

There is growing evidence suggesting that hepatic fat-storing cells (FSC) or Ito cells have an important function in vitamin A storage and metabolism and in the synthesis of connective tissue components in normal liver and during fibrogenesis. The purified FSC acquire a fibroblastic morphology and their vitamin A content decreases in culture. We cultivated cells under in vitro conditions that allowed the expression of FSC morphological and functional characteristics for 3–4 weeks of primary culture. Cells were isolated from rat liver by the collagenase-perfusion method without further purification and cultured with 3T3-conditioned medium, which seemed to stimulate the selective proliferation of the FSC. After 8–10 days, round and stellate cells grew actively from a few precursor cells in the primary culture and were not subcultivated; the stellate cells had the ability to become round and vice versa and were highly motile. The cells had intracytoplasmic lipid droplets, a well developed rough endoplasmic reticulum, Golgi complex, numerous vesicles filled with electron-dense material, and extracellular matrix (ECM) components on their surface. Both stellate and round cells showed the presence of desmin by immunofluorescence and vitamin A autofluorescence, but lacked peroxidase activity. The culture conditions we describe allowed the selective proliferation of cells with morphological and functional characteristics of the FSC in the normal liver, raising the possibility of studying FSC proliferation and differentiation.     

Changes in the lipid-accumulating cells of the liver during experimental hypervitaminosis A

Changes of the fat-storing cells (FSC) in the liver have been studied after oil solution of vitamin A have been injected to intact rats, when Kupffer's cells have been blocked with colloid silver and stimulated with prodigiosan. During these experiments (on the 4th, 7th, 14th days) the amount of FSC does not change, and their size increases. The blockade of Kupffer's cells does not influence the dynamics of the FSC changes, and their stimulation contributes to lipid accumulation in their cytoplasm and to earlier revealing of these elements (on the 2d day of the experiment). In parallel with increasing size of FSC, fatty infiltration of hepatocytes disappears and reticular fibers accumulate in the perisinusoid space. Participation of FSC in lipid metabolism and in synthesis of the III type collagen is proved, as well as existence of intercellular interactions of these elements with endotheliocytes, Kupffer's cells and hepatocytes.     

Effects of endogenous and exogenous cholesterol on the ultrastructure and steroid secretion of undifferentiated rat adrenocortical cells in primary culture

Summary The present study was undertaken to define the effects of lipoprotein-derived cholesterol and endogenous, de novo synthesized cholesterol on the ultrastructure and function of undifferentiated rat adrenocortical cells [lipoprotein (HDL₃ and LDL) receptor-negative, zona glomerulosa-like adrenocortical cells] in primary culture. For this purpose human plasma high density lipoprotein (HDL₃) or low density lipoprotein (LDL) was added to culture medium devoid of cholesterol. Steroid secretion remained at the low basal level even after addition of lipoproteins, and the amount of intracellular lipid droplets did not increase. When mevinolin (0.96 µg/ml), an inhibitor of cholesterol synthesis, was added to the culture medium, a low secretion of corticosterone was measured both in serum-free and serum-containing media. Ultrastructurally, lipid droplets disappeared after treatment with mevinolin in both media used. At this concentration of mevinolin cell proliferation was similar to that in the controls, but at higher concentrations (4.8 or 9.6 µg/ml) proliferation was inhibited to 42% and 26% in serum-free medium, and 20% and 12% in serum-supplemented medium, respectively. This study demonstrates that cell proliferation and synthesis of corticosterone by undifferentiated rat adrenocortical cells is identical in the absence or presence of exogenous lipoprotein cholesterol. Inhibition of de novo cholesterol synthesis by mevinolin over a period of 7 days does not inhibit corticosterone secretion or proliferation of cells but decreases the amount of intracellular lipid droplets, thus suggesting utilization of intracellular cholesterol esters. However, higher concentrations of mevinolin inhibit proliferation of cells both in serum-free and serum-containing media.     

Phenotypic characteristics of adipocytes generated from Meckel's chondrocytes in response to chick serum in vitro

When present in the culture medium, chick serum (CKS) modulated the phenotypic change from chondrocytes of Meckel's cartilage to adipocytes in vitro, as revealed by light and electron microscopy, the incorporation of BrdU, and immunocytochemistry. CKS inhibited DNA synthesis in chondrocytes and the proliferation of these cells, while it facilitated the differentiation to adipocytes. CKS contributed to phenotypic changes in undifferentiated chondrocytes, but did not affect the characteristics of differentiated chondrocytes. Electron microscopy revealed that the lipid droplets in adipocytes were enclosed by limiting membranes that fused to yield larger lipid droplets. Immunocytochemical staining of adipocytes with stage-specific antibodies revealed the presence of immunoreactive uncoupling protein (UCP-1) and peroxisome proliferator-activated receptor (PPARgamma) in immature adipocytes, and leptin and glucose transporter (Glut-4) in mature adipocytes. The adipocytes that were formed in the present study were multilocular adipocytes that contained many small lipid droplets, but in many ways they resembled white adipocytes. CKS contains a high level of estrogen, compared with fetal bovine serum, and it is possible that estrogen might have induced the differentiation to adipocytes.     

THREE-DIMENSIONAL STRUCTURE OF EXTRACELLULAR MATRIX REVERSIBLY REGULATES MORPHOLOGY,PROLIFERATION AND COLLAGEN METABOLISM OF PERISINUSOIDAL STELLATE CELLS (VITAMIN A-STORING CELLS)

The three-dimensional structure of the extracellular substratum was found to regulate reversibly the morphology, proliferation and collagen synthesis of perisinusoidal stellate cells (lipocytes, i.e. fat-storing ‘Ito’ cells). On non-coated polystyrene and type I collagen-coated culture dishes, the cells spread well and extended their cellular processes. On the surface of type I collagen gels, the cells gathered and formed a mesh-like structure. However, in type I collagen gel where the cells were surrounded by type I collagen three-dimensionally, the cells extended their fine cellular processes and resembled the star-shaped stellate cells seenin vivo. The cell proliferation was more prominent in culture dishes coated with type I collagen or in polystyrene culture dishes than on or in type I collagen gels. The collagen synthesis was affected in the same manner. These data indicate that the nature and the three-dimensional structure of the extracellular matrix (ECM) can regulate morphology, proliferation and functions of the perisinusoidal stellate cells. In order to examine the reversibility of these regulations, we liberated cultured cells with trypsin or with purified bacterial collagenase and re-seeded them onto or into each substratum. The cells changed their shape, rate of proliferation and collagen synthesis according to each new substratum. These results indicate that the three-dimensional structure of ECM reversibly regulates the morphology, proliferation rate and functions of the perisinusoidal stellate cells.     

INDUCTION OF CELLULAR PROCESSES CONTAINING COLLAGENASE AND RETINOID BY INTEGRIN-BINDING TO INTERSTITIAL COLLAGEN IN HEPATIC STELLATE CELL CULTURE

Cultered hepatic stellate cells were induced to elongate long, multipolar cellular processes by interstitial collagen gel used as a substratum, as compared to flattened or round cell shapes on polystyrene surface or on Matrigel containing the basement membrane components, respectively. The process induction was inhibited by several reagents as follows: (1) anti-integrin α2 antibody; (2) an oligopeptide, DGEA, an integrin-binding sequence in type I collagen molecule; (3) wortmannin, a phosphatidylinositol 3-kinase inhibitor. Protein tyrosine phosphorylation was enhanced throughout cells including cellular processes by culturing on type I collagen gel. Dual fluorescence staining showed that the core of the processes contained microtubules, whereas the periphery of the processes comprised fibrillar actin. Thus, the process extension was found to depend on integrin-binding to type I collagen fibres, followed by signal transduction and cytoskeleton assembly. The cellular processes included interstitial collagenase and vitamin A-containing lipid droplets. The lipid droplets and vitamin A-autofluorescence were increased by retinyl acetate addition to the culture medium, suggesting an important role of processes in hepatic stellate cell function.     

Constitutive myc expression impairs hypertrophy and calcification in cartilage.

The myc oncogene is expressed by proliferating quail embryo chondrocytes (QEC) grown as adherent cells and is repressed in QEC maintained in suspension culture. To investigate the interference of myc expression during chondrocyte differentiation, QEC were infected with a retrovirus carrying the v-myc oncogene (QEC-v-myc). Uninfected or helper virus-infected QEC were used as control. In adherent culture, QEC-v-myc displayed a chondrocytic phenotype and synthesized type II collagen and Ch21 protein, while control chondrocytes synthesized type I and type II collagen with no Ch21 protein detected as long as the attachment to the plastic was kept. In suspension culture, QEC-v-myc readily aggregated and within 1 week the cell aggregates released small single cells; still they secreted only type II collagen and Ch21 protein. In the same conditions control cell aggregates released hypertrophic chondrocytes producing type II and type X collagens and Ch21 protein. In the appropriate culture conditions, QEC-v-myc reconstituted a tissue defined as nonhypertrophic, noncalcifying cartilage by the high cellularity, the low levels of alkaline phosphatase enzymatic activity, and the absence of type X collagen synthesis and of calcium deposition. We conclude that the constitutive expression of the v-myc oncogene keeps chondrocytes in stage I (active proliferation and synthesis of type II collagen) and prevents these cells from reconstituting hypertrophic calcifying cartilage.     

Modulation of hepatic lipocyte proteoglycan synthesis and proliferation by Kupffer cell-derived transforming growth factors type beta 1 and type alpha.

Soluble mediators elaborated by activated Kupffer cells have been implicated in the activation of liver fat-storing cells. In the present study some of these factors were identified as TGF beta and TGF alpha affecting disparate reactions in the activation process. TGF beta is secreted in an inactive, latent form by Kupffer cells. It is activated after addition to primary FSC cultures and stimulates dose-dependently sulfated proteoglycan synthesis especially that of chondroitin sulfate, whereas the incorporation of [3H] thymidine is reduced significantly. These effects were neutralized completely by anti-TGF beta antibodies which ultimately converted the proliferation inhibitory effect of Kupffer cell medium in a proliferation stimulatory action. The latter is at least partially due to TGF alpha. Both cytokines are preferentially expressed in activated Kupffer cells. We conclude that Kupffer cells modulate the mitogenic activity of FSC in culture depending on the ratio of activated TGF beta and TGF alpha and affect chondroitin sulfate synthesis mainly by TGF beta. The results suggest a paracrine activation of FSC in injured liver by both transforming growth factors secreted by activated Kupffer cells.     

Distinct populations of hepatic stellate cells in the mouse liver have different capacities for retinoid and lipid storage

Hepatic stellate cell (HSC) lipid droplets are specialized organelles for the storage of retinoid, accounting for 50-60% of all retinoid present in the body. When HSCs activate, retinyl ester levels progressively decrease and the lipid droplets are lost. The objective of this study was to determine if the HSC population in a healthy, uninjured liver demonstrates heterogeneity in its capacity for retinoid and lipid storage in lipid droplets. To this end, we utilized two methods of HSC isolation, which leverage distinct properties of these cells, including their vitamin A content and collagen expression. HSCs were isolated either from wild type (WT) mice in the C57BL/6 genetic background by flotation in a Nycodenz density gradient, followed by fluorescence activated cell sorting (FACS) based on vitamin A autofluorescence, or from collagen-green fluorescent protein (GFP) mice by FACS based on GFP expression from a GFP transgene driven by the collagen I promoter. We show that GFP-HSCs have: (i) increased expression of typical markers of HSC activation; (ii) decreased retinyl ester levels, accompanied by reduced expression of the enzyme needed for hepatic retinyl ester synthesis (LRAT); (iii) decreased triglyceride levels; (iv) increased expression of genes associated with lipid catabolism; and (v) an increase in expression of the retinoid-catabolizing cytochrome, CYP2S1. CONCLUSION: Our observations suggest that the HSC population in a healthy, uninjured liver is heterogeneous. One subset of the total HSC population, which expresses early markers of HSC activation, may be "primed" and ready for rapid response to acute liver injury.     

Activation of collagen synthesis in primary culture of rat liver parenchymal cells (hepatocytes)

An increase in collagen synthesis by hepatic parenchymal cells (hepatocytes) was observed during 8 days in primary culture by the quantification of total [3H]hydroxyproline as a marker of total collagen synthesis and the ratio of [3H]hydroxyproline in the high-molecular-weight fraction to total [3H]hydroxyproline as a marker of collagen degradation after incubation of the cells with [3H]proline for 24 h. Type analysis of the collagen produced by the cells after 8 days in culture showed the presence of type I and type III collagens in addition to the components corresponding to type IV and type V (alpha A and alpha B) collagens. Only the latter two types were found in the collagens produced by the cells after 2 days in primary culture. The purity of the hepatocytes inoculated was 97%, and the majority of the contaminating small cells were erythrocytes. The rate of serum albumin synthesis, which is a typical function of the hepatocytes, was constant or increased during the culture period. Immuno-electron microscopic observation indicated the production of type I collagen by the hepatocytes after 8 days in primary culture. These results are explained only by the activation of collagen synthesis in the day-8 hepatocytes in primary culture.     

The stability of the collagen phenotype during stimulated collagen,glycosaminoglycan, and DNA synthesis by articular cartilage organ cultures

Rabbit articular cartilage slices were grown in organ culture for 9 weeks. Eightfold increases in the synthesis of both glycosaminoglycan and collagen were observed at 1 and 3 weeks, respectively. These levels of synthesis gradually declined in parallel to fourfold at 9 weeks. DNA synthesis was stimulated more than 30-fold at 3 weeks and then declined to sevenfold at 9 weeks. In contrast, the content of glycosaminoglycans and collagen per milligram of original wet slices did not vary significantly, while the number of cells increased 1.7-fold by the end of the study. The collagen phenotype of these cultures was determined by sodium dodecyl sulfate electrophoresis of recently synthesized, [³H]proline-labeled intact collagen chains and CNBr peptides. Throughout the study the major collagen synthesized was type II, ranging from 95 to 68% of the collagen synthesized at 0 and 5 weeks, respectively. Increases in the proportions of X₂Y and type III collagen were first observed at 3 weeks in culture. The synthesis of type I collagen was detected only after 5 weeks in culture and never represented more than 11% of the total collagen synthesized. The synthesis of type I trimer could not be verified at any time. This study demonstrates that in vitro organ culture of articular cartilage slices allows chondrocytes to maintain the normal chondrocyte collagen phenotype of predominantly type II synthesis while stimulating their proliferation and matrix synthesis.     

Influences of construct properties on the proliferation and matrix synthesis of dedifferentiated chondrocytes cultured in alginate gel

To investigate whether the chondrocytes-alginate construct properties, such as cell seeding density and alginate concentration might affect the redifferentiation, dedifferentiated rat articular chondrocytes were encapsulated at low density (LD: 3 x 10(6) cells/ml) or high density (HD: 10 x 10(6) cells/ml) in two different concentrations of alginate gel (1.2% or 2%, w/v) to induce redifferentiation. Cell viability and cell proliferation of LD culture was higher than those of HD culture. The increase in alginate gel concentration did not make an obvious difference in cell viability, but reduced cell proliferation rate accompanied with the decrease of cell population in S phase and G2/M phase. Scan electron microscopy observation revealed that chondrocytes maintained round in shape and several direct cell-cell contacts were noted in HD culture. In addition, more extracellular matrix was observed in the pericellular region of chondrocytes in 2% alginate culture than those in 1.2% alginate culture. The same tendency was found for the synthesis of collagen type II. No noticeable expression of collagen type I was detected in all constructs at the end of 28-day cultures. These results suggested that construct properties play an important role in the process of chondrocytes' redifferentiation and should be considered for creating of an appropriate engineered articular cartilage.     

The different effects of type I and IV collagens on the survival and proliferation of cultured BSC-1 cells

The effects of type I and IV collagens on the survival and proliferation of cells were investigated to clarify a possible involvement of the substratum in the regulation of cell function. BSC-1 cells attached, spread and sustained their viability in the absence of calf serum on culture dishes coated with type IV collagen, but were unable to spread and survive on untreated culture dishes. The effects of adding type IV collagen in solution were similar to those of type IV coating. The fraction of the solution of type IV collagen with molecular mass of more than 100 kDa enhanced spreading and survival of cells, but the fraction of less than 100 kDa did not. Type I collagen did not support cell viability in the absence of calf serum. Moreover, coating of culture dishes with type I collagen, but not with type IV collagen, inhibited DNA synthesis and cell proliferation in the presence of calf serum. The cells grown on type I collagen were long, thin and spindle-shaped, and their stress fibers were not well developed, but the cells grown on type IV collagen, as well as those grown on untreated culture dishes, were polygonal in shape with well-developed stress fibers. These results indicate that the interactions of BSC-1 cells with the substratum, when it is derived from type I and IV collagens, differentially modulate the survival and proliferation of BSC-1 cells.     

Ascorbic acid phosphate stimulates type IV collagen synthesis and accelerates adipose conversion of 3T3-L1 cells

We showed that the synthesis and secretion of type IV collagen, entactin, and laminin were enhanced when adipose conversion of 3T3-L1 cells at confluence was stimulated by hormones (Y. Aratani and Y. Kitagawa (1988) J. Biol. Chem. 263, 16163-16169). Ascorbic acid phosphate (Asc-P) stimulated the synthesis and secretion of type IV collagen and other collagens from both 3T3-L1 preadipocytes and adipocytes. The synthesis and secretion of laminin and entactin were not affected by Asc-P. The continuous addition of Asc-P stimulated cell growth and increased cell density at confluence 1.3-fold. Concomitantly, Asc-P remarkably accelerated the emergence of lipoprotein lipase, glycerophosphate dehydrogenase, and Oil Red O-stainable lipid droplets. These findings suggest an important role for type IV collagen in adipocyte differentiation.     

Cell hypertrophy and type X collagen synthesis in cultured articular chondrocytes

Articular cartilage is a permanent tissue whose cells do not normally take part in the endochondral ossification process. To determine whether articular chondrocytes possess the potential to express traits associated with this process such as cell hypertrophy and type X collagen, chondrocytes were isolated from adult chicken tibial articular cartilage and maintained in long-term suspension cultures. As a positive control in these experiments, we used parallel cultures of chondrocytes from the caudal portion of chick embryo sternum. Both articular and sternal chondrocytes readily proliferated and progressively increased in size with time in culture. Many had undergone hypertrophy by 4-5 weeks. Analysis of medium-released collagenous proteins revealed that both articular and sternal chondrocytes initiated type X collagen synthesis between 3 and 4 weeks of culture; synthesis of this macromolecule increased with further growth. Immunofluorescence analysis of 5-week-old cultures showed that about 15% of articular chondrocytes and 30% of sternal chondrocytes produced type X collagen; strikingly, there appeared to be no obvious relationship between type X collagen production and cell size. The results of this study show that articular chondrocytes from adult chicken tibia possess the ability to express traits associated with endochondral ossification when exposed to a permissive environment. They suggest also that the process of cell hypertrophy and initiation of type X collagen synthesis are independently regulated both in articular and sternal chondrocytes.     

TIP47 associates with lipid droplets

Most mammalian cells package neutral lipids into droplets that are surrounded by a monolayer of phospholipids and a specific set of proteins including the adipose differentiation-related protein (ADRP; also called adipophilin), which is found in a wide array of cell types, and the perilipins, which are restricted to adipocytes and steroidogenic cells. TIP47 was initially identified in a yeast two-hybrid screen for proteins that interact with the cytoplasmic tail of the mannose 6-phosphate receptor, yet its sequence is highly similar to the lipid droplet protein, ADRP, and more distantly related to perilipins. Hence, we hypothesized that TIP47 might be associated with lipid droplets. In HeLa cells grown in standard low lipid-containing culture media, immunofluorescence microscopy revealed that the cells had few lipid droplets; however, TIP47 and ADRP were found on the surfaces of the small lipid droplets present. When the cells were grown in media supplemented with physiological levels of fatty acids, the amount of neutral lipid stored in lipid droplets increased dramatically, as did the staining of TIP47 and ADRP surrounding these droplets. TIP47 was found primarily in the cytosolic fractions of HeLa cells and murine MA10 Leydig cells grown in low lipid-containing culture medium, while ADRP was undetectable in these fractionated cell homogenates. When HeLa and MA10 Leydig cells were lipid-loaded, significant levels of ADRP were found in the floating lipid droplet fractions and TIP47 levels remained constant, but the distribution of a significant portion of TIP47 shifted from the cytosolic fractions to the lipid droplet fractions. Thus, we conclude that TIP47 associates with nascent lipid droplets and can be classified as a lipid droplet-associated protein.     

Effects of plasma exposure on cultured hepatocytes: Implications for bioartificial liver support

In order to examine their potential for use in a bioartificial liver, hepatocytes maintained in a collagen sandwich configuration were cultured for 9 days in heparinized rat plasma. The cells exhibited a progressive accumulation of cytoplasmic lipid droplets which proved to be mainly triglyceride (TG). The rate of TG accumulation correlated with the free fatty acid (FFA) content of the plasma. Removal of FFA and TG from plasma by ether extraction significantly reduced the rate and extent of TG accumulation. A smaller reduction in the rate and extent of TG accumulation was observed when cells were maintained in an oxygen enriched environment. The lipid accumulation suppressed urea synthesis, but clearance of the drug diazepam, although constitutively depressed in plasma, appeared unaffected by the accumulation. The functional and morphological effects of plasma exposure could be fully reversed after at least 6 days of plasma exposure by returning the cells to culture medium.The results indicate that elevated FFA in plasma induces lipid accumulation, which inhibits urea synthesis in cultured hepatocytes. This suggests that estimates of the cell number needed for effective liver support should not be based upon function measurements conducted in culture media. Furthermore, optimization of bioartificial liver support device use may have to be governed by the need to limit the plasma exposure of cultured hepatocytes. However, the highly responsive nature of these cultures and the reversibility of the plasma effects suggest that the collagen sandwich culture system is a promising foundation for the development of an effective bioartificial liver support system. (c) 1996 John Wiley & Sons, Inc.     

Effect of phase of growth and hyperlipidemic serum on the synthesis of collagen in rabbit aortic smooth muscle cells in culture

The aim of this study was to elucidate wheter long-term cultivation in the presence of hyperlipidemic serum is able to induce changes in the rate of synthesis of collagen and other proteins by arterial smooth muscle cells. Rabbit aortic medial cells were grown in 10% sera and their collagen and total protein synthesis were studied by incubation of the cells with radioactive proline. When the cells were grown in fetal calf serum, their collagen synthesis was low after trypsinization but reached a constant level in one week, whereafter it remained within 4--5% of total protein synthesis for up to 30 days. Cultivation in hyperlipidemic rabbit serum for up to 14 days caused an accumulation of lipid droplets in the cells, but there were no detectable changes in the rate of collagen of total protein synthesis when compared with cells grown in normal rabbit serum.     

Intracellular lipid droplet targeting by apolipoprotein A-V requires the carboxyl-terminal segment

The expression of apolipoprotein A-V (apoA-V) in hepatoma cells results in homing of this protein to intracellular lipid droplets. When hepatoma cells transfected with a full-length apoA-V-green fluorescent protein fusion protein were cultured in medium that was not supplemented with oleic acid (OA), intracellular lipid droplet size and number were reduced compared with those of cells supplemented with OA. Confocal microscopy studies revealed that apoA-V associates with lipid droplets under both conditions. To define the structural requirements for apoA-V lipid droplet association, hepatoma cells were transfected with a series of C-terminal truncated apoA-V variants. Confocal microscopy analysis revealed that, in a manner similar to mature full-length apoA-V (343 amino acids), truncation variants apoA-V(1-292), apoA-V(1-237), and apoA-V(1-191) associated with lipid droplets, while apoA-V(1-146) did not. Western blot analysis of the relative abundance of apoA-V in cell lysates versus conditioned medium indicated that apoA-V variants associated with lipid droplets were poorly secreted while apoA-V(1-146) was efficiently secreted. Ultracentrifugation of conditioned medium revealed that, unlike full-length apoA-V, which associates with lipoproteins, apoA-V(1-146) was present solely in the lipoprotein-deficient fraction. Deletion of the N-terminal signal peptide from apoA-V resulted in an inability of the protein to be secreted into the medium, although it associated with lipid droplets. Taken together, these data suggest that the C terminus of apoA-V is essential for lipid droplet association in transfected hepatoma cells and lipoprotein association in conditioned medium while the signal peptide is required for extracellular trafficking of this protein.