Scanning electron microscopy and the study of Fc and C3b receptors of cultured fat-storing cells from rat liver

Non-parenchymal fat-storing cells (FSCs) were isolated from rat liver by Metrizamide density centrifugation after liver perfusion with media containing pronase and/or collagenase. These cells were cultured in Williams' E medium with 20% fetal calf serum under the usual conditions. FSCs, identified by vitamin A-specific fluorescence (VAF), were viewed under conventional light microscopy 24, 48 and 72 h after seeding. At 24 h, some FSCs remained hemispheric; at 48 h all FSCs were well spread out, and at 72 h the number of FSCs had increased and the VAF intensity had become weaker. Scanning electron microscopy revealed that the features of the FSCs clearly differed from those of the other non-parenchymal cells. The in vitro FSC features closely resembled the in vivo features. FSCs cultured for 48 h had flattened, triangular or oval shapes with fairly smooth surfaces that showed scattered microvilli 0.1 micron long. Two types of processes, long slender (130 microns maximum length) and short broad ones, protruded from the cell body. These processes had smaller projections 0.2 micron wide that looked like fern leaves. Many lipid droplets could be seen under the cell surface. IgG-coated sheep erythrocytes (EAs) and IgM-C3-coated ox erythrocytes (EACs) were used to investigate whether there are Fc and C3b receptors on the FSCs. No rosette-formation characteristic of EAs or EACs was present on the FSCs; proof that FSCs have neither receptor.     

Peculiar Distribution of Fodrin in Fat-Storing Cells

Fat-storing cells (FSCs) show unique morphology containing many lipid droplets in the cytoplasm. In this study, we found that a membrane skeletal protein, fodrin, shows peculiar distribution in FSCs of rat liver. By immunofluorescence microscopy of FSCs in culture, intense labeling for fodrin was seen as coarse filaments in the cytoplasm. Especially in FSCs isolated from vitamin A-treated rats, the labeling was often seen as many small rings in the cytoplasm. In contrast, labeling for fodrin in human fibroblasts or rat adipocytes in culture was seen diffusely in the cell cortex. Distribution of actin, tubulin, vimentin, and desmin in FSCs was also examined, but none of them appeared correlated with fodrin. By immunoelectron microscopy using nanogold labeling with silver enhancement, positive labeling for fodrin was seen around some lipid droplets in FSCsin vivo.We assume that the peculiar distribution of fodrin may be related to the morphological characteristics of FSCs.     

The Different Immune Responses by Age Are due to the Ability of the Fetal Immune System to Secrete Primal Immunoglobulins Responding to Unexperienced Antigens

Among numerous studies on coronavirus 2019 (COVID-19), we noted that the infection and mortality rates of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) increased with age and that fetuses known to be particularly susceptible to infection were better protected despite various mutations. Hence, we established the hypothesis that a new immune system exists that forms before birth and decreases with aging.Methods: To prove this hypothesis, we established new ex-vivo culture conditions simulating the critical environmental factors of fetal stem cells (FSCs) in early pregnancy. Then, we analyzed the components from FSCs cultivated newly developed ex-vivo culture conditions and compared them from FSCs cultured in a normal condition.Results: We demonstrated that immunoglobulin M (IgM), a natural antibody (NAb) produced only in early B-1 cells, immunoglobulins (Igs) including IgG3, which has a wide range of antigen-binding capacity and affinity, complement proteins, and antiviral proteins are induced in FSCs only cultured in newly developed ex-vivo culture conditions. Particularly we confirmed that their extracellular vesicles (EVs) contained NAbs, Igs, various complement proteins, and antiviral proteins, as well as human leukocyte antigen G (HLA-G), responsible for immune tolerance.Conclusion: Our results suggest that FSCs in early pregnancy can form an independent immune system responding to unlearned antigens as a self-defense mechanism before establishing mature immune systems. Moreover, we propose the possibility of new solutions to cope with various infectious diseases based on the factors in NAbs-containing EVs, especially not causing unnecessary immune reaction due to HLA-G.     

Acetylcholine increases intracellular Ca2+ in the rat pituitary folliculostellate cells in primary culture

Pituitary folliculostellate cells (FSCs) are thought to partially inhibit pituitary hormone secretion through a paracrine mechanism. In this process, one of the important questions is what factors regulate the function of FSCs. Because ACh is synthesized in and possibly released from the corticotrophs and lactotrophs, we examined whether FSCs respond to ACh by the method of Ca2+ imaging in primary cultured FSCs from male Wistar rats. ACh (30 nM-3 microM) increased intracellular calcium concentration ([Ca2+](i)) of FSCs in a concentration-dependent manner, with an initial rapid rise followed by a relatively sustained increase. The complete block of the response by atropine and pirenzepine suggests involvement of muscarinic receptors. Depletion of the stored Ca2+ by thapsigargin blocked the response completely. Blockers of phospholipase C, U-73122 and neomycin, suppressed significantly the rise of [Ca2+](i). These results suggest that ACh increases [Ca2+](i) in FSCs by activating phospholipase C, presumably through activation of M(1) receptors. The rise in [Ca2+](i) could trigger a variety of Ca2+-dependent cellular processes, including the synthesis and release of bioactive substances, which in turn act on endocrine cells.     

Biosynthesis of major plasma proteins in the primary culture of fat body cells from the silkworm, Bombyx mori

Plasma proteins termed "SP1" and "30K proteins" are synthesized by the fat body cells of the silkworm, Bombyx mori, in a sex- and stage-specific manner during larval development. We successfully established a primary culture of the fat body cells in order to investigate the regulatory mechanisms of plasma protein gene expression. The primary cultures of fat body cells contained at least two cell types: small oval cells, and large spherical cells. The cells adhered to and migrated on the cultured dish after plating. By the 7th day of cultivation, the cells clustered to form fat body-like structures, which were maintained for at least 3 months. Plasma proteins were actively synthesized in the primary cultures of the fat body cells isolated from the final instar larvae only when the cells tightly adhered to and clustered on the cultured dish. Immunocytochemical analysis revealed that only 10-15% of the clustered cells synthesized plasma proteins in our culture system, indicating that the primary culture comprises heterogeneous cells that are morphologically and functionally distinct. The patterns of SP1 syntheses in primary cultures faithfully reproduced their sex-dependency in vivo.     

Fibromatosis stem cells rather than bone-marrow mesenchymal stem cells recapitulate a murine model of fibromatosis

Palmar fibromatosis is a benign fibroproliferative tumor of unknown etiology, with a high rate of recurrence after excision. The offending cells of palmar fibromatosis are myofibroblasts and the cellular origin of other myofibroblasts has previously been reported to be the bone marrow. However, further clarification of the relationship between bone marrow precursors and palmar fibromatosis is required. Stem cells (SCs) are known to exist in various tissues, but whether SCs can be isolated from fibromatosis tissue is still unclear. The purpose of this study was to isolate and identify stem cells from human palmar fibromatosis, and to evaluate the differences in the differentiation and fibrogenic capacities of bone marrow stem cells (BMSCs) and fibromatosis-derived stem cells (FSCs). We found that FSCs had better fibrogenic differentiation potential than BMSCs, whereas BMSCs had better adipogenic and chondrogenic differentiation capacities. Treatment with transforming growth factor-β1 increased the expression of α-smooth muscle actin, and types III and I collagen significantly more in FSCs than in BMSCs. An in vivo study further confirmed the results of fibrogenesis and suggested that FSCs can recapitulate the fibromatosis nodule. In summary, their myofibroblastic differentiation both in vivo and in vitro makes FSCs a potential cell source for future applications in murine models of fibromatosis or fibrogenesis.     

Ultrastructure of bovine embryos developed from in vitro–matured and –fertilized oocytes: Comparative morphological evaluation of embryos cultured either in serum‐free medium or in serum‐supplemented medium

The ultrastructure of bovine embryos developed from in vitro‐matured and ‐fertilized oocytes, cocultured with bovine cumulus/granulosa cells either in a serum‐free medium (IVMD101) or in a serum‐containing medium (TCM199+CS) was compared. Embryos up to the eight‐cell stage had many cellular organelles and cytoplasmic components that were randomly distributed in the cytoplasm. Mitochondria were spherical or ovoid and had only a few peripheral cristae. There were no obvious differences in the ultrastructure between embryos developed in IVMD101 and TCM199+CS up to the eight‐cell stage. However, conspicuous differences in the ultrastructural features between the embryos cultured in IVMD101 and TCM199+CS were observed at the morula and blastocyst stages. At the morula stage, embryos cultured in IVMD101 had cells containing elongated mitochondria, well‐developed Golgi apparatus, lipid droplets, and large vesicles resembling lysosomes. The lysosome‐like vesicles were partially filled with electron‐dense materials and were frequently fused with lipid droplets. The blastomeres of morulae cultured in TCM199+CS contained numerous large lipid droplets and fewer lysosome‐like vesicles than those cultured in IVMD101. In blastocysts cultured in IVMD101, lysosome‐like vesicles were frequently observed in the trophoblast cells and lipid droplets were present in the cytoplasm of trophoblast and inner cell mass (ICM)‐cells, but they were not abundant. On the other hand, the blastocysts developed in TCM199+CS contained fewer lysosome‐like vesicles and large numbers of lipid droplets. This accumulation of lipid droplets was higher in the trophoblast cells than in the ICM‐cells. This study showed major differences in the ultrastructural features between the morulae and blastocysts from serum‐free and serum‐supplemented cultures, suggesting that the ultrastructural differences may reflect physiological characteristics of embryos. Mol. Reprod. Dev. 53:325–335, 1999. © 1999 Wiley‐Liss, Inc.     

Up-regulation of ADRP in fatty liver in human and liver steatosis in mice fed with high fat diet

The present study was performed to examine a role of adipose differentiation-related protein (ADRP) in the process of liver steatosis. Immunohistochemical findings indicated that ADRP expression is increased in the hepatocytes in patients with fatty liver when compared with normal liver. ADRP expression is localized in the surface of lipid droplets in the hepatocytes. Increased expression of ADRP mRNA and protein was similarly observed in fatty liver in ob/ob mice and the liver steatosis induced by high fat diet in mice. The up-regulation of ADRP mRNA and protein in the liver by high fat diet was identified in the surface of lipid droplets in a time-dependent manner. Recent studies demonstrated that up-regulation of PPARgamma in the hepatocytes is deeply involved in liver steatosis. To clarify whether ADRP expression is increased by PPARgamma activation in hepatocytes, we examined the effect of a PPARgamma ligand, troglitazone, on ADRP mRNA expression in HepG2 cells. ADRP mRNA expression was increased by troglitazone in dose- and time-dependent manners. All these results suggest that ADRP is up-regulated in liver steatosis in human and mice, and that high fat diet increases expression of ADRP through PPARgamma activation, followed by induction of liver steatosis.     

Flow cytometric analysis of mature adipocytes

Flow cytometry is an excellent method for studying the physiological function in adipocytes because their response to hormones, especially insulin, varies with cell maturity and therefore size. Adipocytes present a unique technical challenge. A freshly prepared adipocyte suspension contains cells and fat droplets ranging from 10 to greater than 120 microns in diameter. Stored fat occupies 90-98% of the cell volume, making it difficult to distinguish cells from fat droplets. Other difficulties include buoyancy, large size, fragility, and tendency to aggregate and clog the sample tube and nozzle. These obstacles were overcome by 1) maintaining the sample, sample line, sheath fluid, reservoir, and nozzle assembly at 37 degrees C; 2) using a 200 microns diameter orifice; 3) using a short, 300 microns inside diameter Teflon sample delivery line; 4) injecting the sample at constant flow rate into the sheath fluid at low pressure; and 5) using the pH-sensitive vital stain, biscarboxyethylcarboxyfluorescein (BCECF) to distinguish cells from fat droplets. Stained cells are brightly fluorescent when excited at 488 nm. Because fat droplets do not fluoresce, they can be distinguished from fat cells by gating on the BCECF emission. The cytosolic pH of intact, viable, mature adipocytes was derived from the ratio of the fluorescent emission intensities at 520 and 620 nm and was estimated to be 7.2. Unlike BCECF, several other useful fluorescent probes of cell function, e.g., the intracellular calcium indicator, indo-1, label both fat cells and fat droplets.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunocytochemistry of the Microtubules of fat-laden cells. Brown fat cells and adrenocortical cells in primary monolayer culture

Cytoplasmic microtubules of fat-laden cells containing fine lipid droplets, such as brown fat cells and adrenocortical cells, were studied in relation to the metabolism of intracellular lipid. In these cells, the amount and distribution of lipid droplets reflect the state of inherent cellular function. Materials used were primary monolayer culture of fetal rat brown fat cells and that of bovine adrenocortical cells. The method was the immunocytochemistry with anti-tubulin antibody. When brown fat cells were being lipolyzed or the steroidogenesis of adrenocortical cells were being stimulated, the cytoplasmic microtubules in the cells were organized in a radial pattern in response to the behavior of the lipid droplets. It is assumed that the microtubules were in the regulation of cellular function in terms of the metabolism of lipid droplets in these cells. We have devised, in the course of the current study, a double fluorescence technique as an observational method whereby microtubules were observed immunocytochemically and lipid droplets by a secondary fluorescence with phosphine E staining.     

The ultrastructure of mammary explants from virgin ovariectomized goats during hormone-induced lactogenesis

The effect of insulin (I), cortisol (F) and prolactin (P) on the ultrastructural morphology of epithelial cells of cultured mammary explants from virgin ovariectomized (OV-X) goats were studied. The epithelial cells showed little structural organization and were devoid of fat droplets and secretory protein granules at zero time of culture. The cytoplasm contained few profiles of smooth and rough endoplasmic reticulum and the Golgi apparatus was rudimentary. After being cultured in Waymouth's medium without added hormones the epithelial cells were indistinguishable from epithelial cells of uncultured explants. The addition of I induced changes mainly in the appearance of nucleoli. The nucleoli were enlarged and fibrillogranular areas with light spaces were observed. The most obvious cytological changes of epithelial cells of explants cultured in the presence of I and F are translocation of the nucleus into the basal cytoplasm, increase of rough endoplasmic reticulum, an increase in the size of the Golgi apparatus, presence of one or two lipid droplets and in some cells vacuoles with protein granules were present. Mitochondria were more abundant. The epithelial cells of explants cultured in the presence of I, F and P were characterized by the polarization of organelles within the cytoplasm and by the formation and release of protein granules and small and large fat droplets. The cell nucleus was in the basal cytoplasm, the Golgi apparatus was supranuclear. The rough endoplasmic reticulum was extensively developed and formed large sacs. Golgi vacuoles contained protein granules.(ABSTRACT TRUNCATED AT 250 WORDS)     

Association of SND1 protein to low density lipid droplets in liver steatosis

Although the human homologue of SND p102, p100 coactivator, was initially described as a nuclear protein, the p100 coactivator protein family members have non-nuclear localization in mammalian cells with active lipid handling, storage, and secretion. However, their role in lipid homeostasis remains unresolved. Here, we investigate the distribution of the rat homologue SND p102 (also called SND1) and its association with newly formed lipid droplets in the liver parenchyma and cultured hepatocytes. Sucrose gradient fractionation showed that SND p102 cofractionated with endoplasmic reticulum and Golgi markers. Such cofractionation was not altered in regenerating steatotic rat liver. However, SND p102 was also detected in lipid droplets from regenerating liver, showing a specific directionalization to the least dense ones. Confocal microscopy of cultured hepatocytes confirmed the findings of gradient fractionation. In addition, p100 coactivator was consistently encountered in microsomes and lipid droplets in control and oleate-treated HepG2 cells. The total amount of SND p102 in hepatocytes was similar in both conditions, suggesting a specific translocation of the protein. Our findings indicate that SND p102 and the human p100 coactivator have a ubiquitous cytoplasmic distribution in hepatocytes and that steatogenic conditions promote the targeting of SND p102 from other cell compartments to specific low density lipid droplets.     

Nuclear lipid droplets and nuclear damage in Caenorhabditis elegans

Fat stored in the form of lipid droplets has long been considered a defining characteristic of cytoplasm. However, recent studies have shown that nuclear lipid droplets occur in multiple cells and tissues, including in human patients with fatty liver disease. The function(s) of stored fat in the nucleus has not been determined, and it is possible that nuclear fat is beneficial in some situations. Conversely, nuclear lipid droplets might instead be deleterious by disrupting nuclear organization or triggering aggregation of hydrophobic proteins. We show here that nuclear lipid droplets occur normally in C. elegans intestinal cells and germ cells, but appear to be associated with damage only in the intestine. Lipid droplets in intestinal nuclei can be associated with novel bundles of microfilaments (nuclear actin) and membrane tubules that might have roles in damage repair. To increase the normal, low frequency of nuclear lipid droplets in wild-type animals, we used a forward genetic screen to isolate mutants with abnormally large or abundant nuclear lipid droplets. Genetic analysis and cloning of three such mutants showed that the genes encode the lipid regulator SEIP-1/seipin, the inner nuclear membrane protein NEMP-1/Nemp1/TMEM194A, and a component of COPI vesicles called COPA-1/α-COP. We present several lines of evidence that the nuclear lipid droplet phenotype of copa-1 mutants results from a defect in retrieving mislocalized membrane proteins that normally reside in the endoplasmic reticulum. The seip-1 mutant causes most germ cells to have nuclear lipid droplets, the largest of which occupy more than a third of the nuclear volume. Nevertheless, the nuclear lipid droplets do not trigger apoptosis, and the germ cells differentiate into gametes that produce viable, healthy progeny. Thus, our results suggest that nuclear lipid droplets are detrimental to intestinal nuclei, but have no obvious deleterious effect on germ nuclei.     

Structural changes produced in Kupffer cells in the rat liver by injection of lipopolysaccharide

Summary The fine structure of Kupffer cells has been studied at various times after an intravenous injection of lipopolysaccharide of Salmonella abortus equii. The most prominent effects were: an increase in the number and dimensions of phagocytic vacuoles (often containing ingested LPS and neutrophilic granulocytes); mitochondrial damage, including disintegration of the matrix and cristae; an increase in the amount of dilated, lucent rough endoplasmic reticulum; presence of fat droplets in the cytoplasm. Five days after injection of lipopolysaccharide, the Kupffer cells had resumed their normal ultrastructure.Several minutes after injection of lipopolysaccharide, platelets adhered to the Kupffer and endothelial cells. Between one and six hours, neutrophilic granulocytes accumulated in the liver sinusoids. The resulting obstruction of the hepatic microcirculation most probably affected cellular ultrastructure by ischaemia. At three days, the number of Kupffer cells was doubled, and increased further at later time intervals.     

REVERSAL BY ADENINE OF THE ETHIONINE-INDUCED LIPID ACCUMULATION IN THE ENDOPLASMIC RETICULUM OF THE RAT LIVER : A Preliminary Report

Within 3.5 to 4 hours after thionine administration, numerous small osmiophilic bodies, liposomes, appear in the endoplasmic reticulum of the liver cells. By fusion, the liposomes lead to the formation of larger collections of fat, giant liposomes. Adenine administration to ethionine-treated rats removes the liposomes from the hepatocytes and causes the transitory appearance of osmiophilic droplets in the sinusoidal space of Disse. The characteristic disaggregation of hepatic polysomes seen in the liver after ethionine administration is corrected by the injection of adenine.     

Integrins control the positioning and proliferation of follicle stem cells in the Drosophila ovary

Adult stem cells are maintained in specialized microenvironments called niches, which promote self-renewal and prevent differentiation. In this study, we show that follicle stem cells (FSCs) in the Drosophila melanogaster ovary rely on cues that are distinct from those of other ovarian stem cells to establish and maintain their unique niche. We demonstrate that integrins anchor FSCs to the basal lamina, enabling FSCs to maintain their characteristic morphology and position. Integrin-mediated FSC anchoring is also essential for proper development of differentiating prefollicle cells that arise from asymmetrical FSC divisions. Our results support a model in which FSCs contribute to the formation and maintenance of their own niche by producing the integrin ligand, laminin A (LanA). Together, LanA and integrins control FSC proliferation rates, a role that is separable from their function in FSC anchoring. Importantly, LanA-integrin function is not required to maintain other ovarian stem cell populations, demonstrating that distinct pathways regulate niche-stem cell communication within the same organ.     

Recapitulation of fibromatosis nodule by multipotential stem cells in immunodeficient mice

Musculoskeletal fibromatosis remains a disease of unknown etiology. Surgical excision is the standard of care, but the recurrence rate remains high. Superficial fibromatosis typically presents as subcutaneous nodules caused by rapid myofibroblast proliferation followed by slow involution to dense acellular fibrosis. In this study, we demonstrate that fibromatosis stem cells (FSCs) can be isolated from palmar nodules but not from cord or normal palm tissues. We found that FSCs express surface markers such as CD29, CD44, CD73, CD90, CD105, and CD166 but do not express CD34, CD45, or CD133. We also found that FSCs are capable of expanding up to 20 passages, that these cells include myofibroblasts, osteoblasts, adipocytes, chondrocytes, hepatocytes, and neural cells, and that these cells possess multipotentiality to develop into the three germ layer cells. When implanted beneath the dorsal skin of nude mice, FSCs recapitulated human fibromatosis nodules. Two weeks after implantation, the cells expressed immunodiagnostic markers for myofibroblasts such as α-smooth muscle actin and type III collagen. Two months after implantation, there were fewer myofibroblasts and type I collagen became evident. Treatment with the antifibrogenic compound Trichostatin A (TSA) inhibited the proliferation and differentiation of FSCs in vitro. Treatment with TSA before or after implantation blocked formation of fibromatosis nodules. These results suggest that FSCs are the cellular origin of fibromatosis and that these cells may provide a promising model for developing new therapeutic interventions.     

油红O染色原代未活化胰腺星状细胞脂肪滴的实验观察

目的观察油红O染色原代培养的未活化胰腺星状细胞脂肪滴,并予鉴定。方法选取接种于6孔板中培养4天的原代未活化胰腺星状细胞,予100g/L甲醛液固定,磷酸盐缓冲液漂洗后,加入5g/L油红O饱和液染色,镜下动态观察。胞浆内的脂肪滴一旦着色,即可洗去油红,苏木素衬染胞核,漂洗分化脱色后镜下成像。结果未活化的胰腺星状细胞脂肪滴被油红0染成鲜艳的红色,大小不一的串珠样"油珠子"分散于胞质中,簇集成"环状",呈戒环样包绕在细胞核周围。结论油红染色原代未活化胰星状细胞脂肪滴是鉴定该细胞的重要方法之一。