Morphological features of brown adipose cell maturation in vivo and in vitro

The maturation of brown adipose cells, derived at various time intervals from interscapular brown fat pads of neonatal rats, has been investigated electron microscopically. The major changes in in vivo brown adipose cells are related to lipid accumulation. In older animals, fat droplets are separated by a membrane complex, and membranous fragments are seen within large lipid inclusions. The ultrastructural characteristics of monolayer cultures of brown fat cells, isolated from neonatal brown fat pads by tryptic dissociation, and maintained for one to six days on a reconstituted collagen matrix, have also been investigated. Adipose cells in vitro are morphologically similar to control brown fat cells, and lipid accumulation closely parallels that seen in vivo. In vitro lipid droplet coalescence, impossible to visualize in vivo, has been documented by time-lapse cinematography, and membrane complexes similar to those observed in vivo are seen. Major changes in cultivated fat cells, compared to in vivo cells, involve extensive glycogen deposition and breakdown of cytoplasmic ribosomal rosettes. Older cultures contain adipose cells exhibiting characteristics of white fat cells. Effects of tissue culture environment upon the brown fat cells, and the possible conversion of the brown fat cell into a white fat cell are discussed.     

An Electron Microscopic Demonstration of Induction of Chondrogenesis in Neonatal RAT Muscle Outgrowth Cells in Monolayer Cultures

Second passage fibroblast-like cells grown from explants of neonatal rat muscle continue to demonstrate fibroblast-like properties for many days when cultured on plastic surfaces. Such cells can be induced to change to a chondrocyte-like mode of expression by the addition of effector materials prepared from bovine cortical bone decalcified with 0.6 NHCl. Other studies show that similar demineralized bone particles and extracts from them have, in vivo, osteoinductive properties. Optimum conditions for this differentiation in monolayer culture were found in the use of 2% fetal calf serum with Dulbecco's modified Eagles medium. At 10% fetal calf serum the chondrogenic changes could not be detected. Light microscopy showed a sequence of morphological changes, after 36 h in culture, which resembled those seen at the beginning of osteogenesis in vivo Induced cultures showed abundant extracellular proteoglycan production. Isotope incorporation studies showed stimulation of glycosaminoglycan synthesis in response to effector materials in soluble form. Type II collagen could be detected after three days. Electron microscopic analysis of induced and control cultures showed unequivocal evidence for marked production of an extensive extracellular matrix in the region of effector particles. The cells themselves change shape and develop an abundant system of lysosome-like vesicles and a very active, highly engorged endoplasmic reticulum and Golgi apparatus. After nine days in culture, evidence for the formation of a ruthenium red stained structure on the surface of the cells in contact with inductive particles, was observed. The simple monolayer culture system described provides a direct means by which the presence of active chondrogenic fractions may be assessed, and in which the mechanism of action of the effectors can be studied.     

Temperature-dependent malignant invasionin vitro by frog renal carcinoma-derived PNKT-4B cells

The northern leopard frog,Rana pipiens, may be afflicted with a herpesvirustransmitted renal carcinoma which has the interesting property that its metastatic behavior is temperature-related. PNKT-4B is a cell line derived from a pronephric carcinoma arising in a tadpole. We sought to ascertain if invasion of normal tissue by PNKT-4B cells in three-dimensional confrontation culturein vitro is similarly temperature-dependent. Normal fragments of tadpole and frog organs are invaded by PNKT-4B cells at 28°C but not at 20°C or 21°C. PNKT-4B cells failed to invade tadpole tissues at 7°C. A temperature critical for invasion was sought. Temperatures of 21°C and cooler are invasion-restrictive and 23°C and warmer are invasion-permissive under the conditions of this study. Identification of a critical permissive temperature allows for the characterization of biochemical events which may be activated at the same temperature. The biochemical changes, which are selectively activated and subsequently repressed as tumor cells are cycled through invasion-permissive and invasion-restrictive temperatures, become compelling candidates as reactions involved in. or causal for, malignant invasion.     

Development of T cell precursor activity in the murine fetal liver

The generation of T cell precursors in the liver of murine embryos was studied. The total number of T cell precursors in the liver was measured in thymic organ cultures by a limiting dilution assay. Sixty T cell precursors were detected in the liver at day 11 of gestation. By day 12 the number of precursors showed a 20-fold increase, half of which could be explained by in situ proliferation as ascertained by a fetal liver organ culture assay. By day 13 a further 2 – 3-fold increase was observed. Whereas the number of total liver cells continued to increase, that of T cell precursors declined in the following days, suggesting a massive exit of these cells after day 13. The capacity to generate a TCRB repertoire in the cells was evaluated by a PCR assay. T cell precursors in day 11 fetal liver developed a TCRB repertoire at day 8 of culture. The cells from days 12 – 15 developed an identically diverse repertoire by day 6, suggesting that day 11 precursors are more immature than those of later days. A mechanism for yielding a single wave of T cell precursors in the fetal liver is discussed with a proposed model.     

Expression of cytoskeletal proteins in hepatic stellate cells isolated from normal and cirrhotic rat liver

Hepatic stellate cells (HSC) are located in Disse spaces of normal rat liver. In their quiescent state they serve as a storage site for vitamin A. In fibrotic liver they become activated, proliferate and they undergo transdifferentiation into myofibroblast-like cells. Changes in the cell phenotype are accompanied by changes in the cellular cytoskeleton. We have studied the expression of alpha-smooth muscle actin and intermediate filament proteins vimentin, desmin and glial fibrillary acidic protein (GFAP) by immunocytochemistry in HSC cultured for 2 or 7 days after isolation. Normal or cirrhotic rat liver was perfused with solutions of pronase and collagenase and HSC were isolated by density gradient centrifugation of the resulting cell suspension. Liver cirrhosis was produced in rats by repeated carbon tetrachloride administration. Vimentin was detected in all cells from normal and cirrhotic liver. The concentration of desmin in the cells from cirrhotic liver was slightly higher than that in normal cells and it increased with time in culture. GFAP could be detected only in normal cells 2 days after their isolation. In contrast, alpha smooth muscle actin (alpha-SMA) was absent from normal cells at this time but its expression was pronouced later. In most cells from cirrhotic liver this antigen was already present on the second day of culture and its expression further increased.     

Osteological development of a small and fast metamorphic frog,Microhyla fissipes (Anura,Neobatrachia, Microhylidae)

Describing osteological development is of great importance for understanding vertebrate phenotypic variations, form-functional transitions and ecological adaptations. Anurans exhibit dramatic changes in their morphology, habitat preferences, diet and behaviour between the tadpole and frog stages. However, the anatomical details of their cranial and postcranial development have not been extensively studied, especially in Microhylidae. In this work, we studied the microhylid Microhyla fissipes, commonly known as the ornamented pygmy frog, a small-sized frog with fast metamorphosis. Its osteological development was comprehensively described based on 120 cleared and stained specimens, including six tadpoles for each stage between 28 and 45, six juveniles and six adults. Additionally, 22 osteological traits of these specimens involved in food acquisition, respiration, audition and locomotion were selected and measured to reflect the changes in tadpole ecological functions during metamorphosis. Our study provides the first detailed qualitative and quantitative developmental information about these structures. Our results have confirmed that skeletal elements (viz., neopalatines, omosternum, clavicles and procoracoids) absent in adults are not detected during development. Our data reveal that morphologically, radical transformations of the cranial structures related to feeding and breathing are completed within stages 42–45 (72 h), but the relative length and width of these skeletons have changed in earlier stages. The postcranial skeletons correlated with locomotion are well developed before stage 42 and approach the adult morphology at stage 45. Indeed, the relative length of the pectoral girdle and forelimb reaches the adult level at stage 42 and stage 45, respectively, whereas that of the vertebral column, pelvic girdle and hind limbs increases from their appearance until reaching adulthood. Based on published accounts of 19 species from Neobatrachia, Mesobatrachia and Archaeobatrachia, cranial elements are among the first ossified skeletons in most studied species, whereas sphenethmoids, neopalatines, quadratojugals, mentomeckelians, carpals and tarsals tend to ossify after metamorphosis. These results will help us to better understand the ecomorphological transformations of anurans from aquatic to terrestrial life. Meanwhile, detailed morphological and quantitative accounts of the osteological development of Microhyla fissipes will provide a foundation for further study.     

Effects of oncostatin M on secretion of vascular endothelial growth factor and reconstruction of liver-like structure by fetal liver cells in monolayer and three-dimensional cultures

Vascular endothelial growth factor (VEGF) is crucial for the development and regeneration of the liver. However, there have been no reports about VEGF secretion by cultured fetal liver cells (FLCs). In the present study, the effects of oncostatin M (OSM), which strongly stimulates the growth and albumin secretion of FLCs, on VEGF secretion and morphological changes of long-term cultured FLCs were investigated under three-dimensional (3-D) and monolayer conditions. The cultured FLCs proliferated well and showed stable secretion of VEGF for up to 1 month under both monolayer and 3-D culture conditions. The addition of OSM to cultured cells strongly enhanced VEGF secretion. Compared with 3-D cultures, VEGF secretion per cell was higher in monolayer cultures. After 1 month in culture, the FLCs in 3-D cultures formed large aggregates like liver tissue, and FLCs also formed colonies and duct-like structures after several months of culture even under monolayer conditions. In conclusion, OSM stimulated the secretion of VEGF by cultured FLCs, which seemed to contribute to the development of a liver-like structure.     

Structure and development of the secretory cells of the hen's ovary

Summary The granulosa and the luteal cells have been studied in the ovary of the adult laying hen by histochemistry, autoradiography, polarized light and electron microscopy. It is concluded that the granulosa cells are specialized for the synthesis of proteins, whereas the luteal cells mainly secrete steroids. These conclusions have been tested by examining the cells under three naturally-occurring physiological conditions. (a) In the discharged follicles, the granulosa cells exhibit degenerative changes, and the luteal cells show reduced reactions in the tests for steroids. (b) In the ovaries of old, off-lay hens, the granulosa cells resemble those of the laying hen; the luteal cells however, whilst still showing positive reactions for steroids, have undergone morphological changes, some of which suggest that the cells are atrophic. (c) Luteal are recognisable in embryonic ovaries as early as nine days of incubation. By fifteen days, these cells show strongly positive reactions in tests for steroids.     

肝损伤大鼠血清诱导培养脐带间充质干细胞向肝样细胞的分化

背景：脐带间充质干细胞来源于新生个体脐带组织,来源广泛,无社会伦理学因素制约,有望代替骨髓间充质干细胞成为细胞移植和再生医学的理想种子细胞。 目的：探讨肝衰竭大鼠血清对人脐带间充质干细胞肝向诱导分化作用,为临床上利用脐带间充质干细胞治疗终末期肝病提供实验依据。 方法：腹腔注射10%四氯化碳溶液建立急性肝损伤大鼠模型,对照组腹腔注射等剂量的大豆油,48 h后腹主动脉取血离心分离血清。取第3代人脐带间充质干细胞,分别用体积分数为20%肝损伤大鼠血清和体积分数为20%胎牛血清诱导培养,观察诱导前后人脐带间充质干细胞形态学改变,检测培养液上清甲胎蛋白、白蛋白水平。 结果与结论：肝损伤大鼠血清培养第1天细胞形态变化不大,呈梭形,仍呈编织状或漩涡状生长,第2天细胞呈短梭形,第3天细胞呈类圆形,第4天呈圆形,并有极少量细胞漂浮。肝损伤大鼠血清培养人脐带间充质干细胞4 d,其上清液白蛋白水平较诱导前和对照组均有升高趋势(P < 0.001),甲胎蛋白水平无明显变化。结果表明肝损伤大鼠血清可使脐带间充质干细胞向肝样细胞分化。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

重型肝病患者血清诱导脐血间充质干细胞表达白蛋白和细胞角蛋白18

背景：脐血富含间充质干细胞,刺激后进入细胞周期的速度以及自分泌生长因子的能力均强于骨髓及外周血干细胞。 目的：观察肝衰竭患者血清对人脐血间充质干细胞的诱导分化作用。 方法：密度梯度离心和贴壁培养法相结合分离纯化脐血间充质干细胞并鉴定,以20%肝衰竭患者血清诱导培养,培养7,14,21 d采用免疫组织化学方法检测白蛋白和细胞角蛋白18的表达。 结果与结论：实验分离获得了高纯度贴壁生长的间充质干细胞,人脐血间充质干细胞高表达CD44和CD29,不表达CD34,体外可以分化为脂肪细胞;人脐血间充质干细胞在肝病患者血清的低糖DMEM培养基中形态发生明显改变,镜下观察细胞变得稍大而扁平,呈类上皮样细胞,SP染色显示实验组培养7 d时仅少数细胞表达白蛋白和细胞角蛋白18,培养14,21 d,白蛋白和细胞角蛋白18表达阳性率逐渐升高,与对照组相比差异有显著性意义(P < 0.05)。提示密度梯度离心和贴壁培养法相结合可以获得纯度较高的脐血间充质干细胞,肝衰竭患者血清可诱导间充质干细胞表达白蛋白和细胞角蛋白18。     

Morphologic and functional studies of mouse hepatocytes in primary culture

Mouse liver cells in primary culture were evaluated by high-resolution light microscopy (HRLM) and transmission electron microscopy (TEM). Cells after 2 hours of culture in L-15 medium supplemented with 10% fetal bovine serum were spherical in shape, and were either individual or in small clusters of up to ten cells. Following 1 day in culture, hepatocytes were flattened and usually found in groups. Bile canaliculus-like structures were apparent between hepatocytes. Tight junctions and desmosomes were also present along adjacent plasma membranes. Autophagic vacuoles were seen within the cytoplasm. After 2 days in culture, hepatocytes appeared more elongated and flattened than in earlier sampling periods. Both autophagic and clear vacuoles were seen in the cytoplasm. Mitochondria were present in a variety of shapes and sizes. Small bundles of microfilaments were frequently seen in the basal region of cross-sectioned cells. From the fourth until the eighth day in culture, hepatocytes displayed further progression of the morphologic changes seen after 2 days. Nuclear elongation and the projection of cytoplasmic pseudoinclusions into the nucleus were also evident after 4 days. Cytoplasmic and nuclear changes were eventually observed in all hepatocytes by the eighth day of culture. DNA synthesis in the cells during culture was investigated by autoradiography. The percentage of S-phase labeled cells was 0.1% after 1 day of culture. The labeling index increased to 1.02%, 3.14%, and 5.88% after 2, 4, and 6 days of culture, respectively. Synthesis of albumin by the liver cells was also detectable during the first 8 days of primary culture. A gradual drop in albumin synthesis was noted with increased time in culture. The percentage of hepatocytes that histochemically stained for gamma glutamyl transpeptidase (GGT) progressively increased from 0.01% of the cells after 2 hours culture to 3.14% of the cells after 8 days of culture.     

Long-term culture of fetal liver cells using a three-dimensional porous polymer substrate

To develop a bioartificial liver, long-term culture of fetal liver cells over a month's time was performed under three different culture conditions, i.e., stationary cultures and shaken-flask cultures, both by using a substratum made of porous polyvinyl formal (PVF) resin and conventional monolayer dish cultures as controls. Time course changes in cell numbers and albumin secretion were evaluated in cultures using Williams' E medium (WE) or minimum essential medium alpha (aMEM) supplemented with serum and hormones. In the WE medium, the numbers of fetal liver cells in all culture conditions gradually decreased with time, and albumin secretion rates rapidly decreased. In the stationary cultures using PVF, however, a significant increase in albumin secretion was observed after two weeks of culture. When cells were cultured in aMEM, the fetal liver cells exhibited sufficient proliferation in stationary and monolayer cultures, although albumin secretion rates per single cell were lower than those in WE. On the basis of these results, another series of culture experiments were performed, in which aMEM was used for the first 10 days to encourage cell proliferation, and the medium was changed to WE afterward. In these cultures, albumin secretion rates in the stationary cultures dramatically increased after the medium exchanges and were maintained at these high levels throughout the remaining culture period.     

In vitro modulation of the metastatic phenotype. I. Analysis of differentiation forms of the B16 melanoma expressing Met-72 determinants and metastatic activity

In vitro cultures of a highly metastatic B16 melanoma clone (BL6-10) were found to undergo dramatic changes in morphology and differentiation upon transfer to another culture medium. Specifically, BL6-10 melanoma cells which had been originally selected and adapted for growth in Eagles' Hanks' amino acid supplemented media with 10 per cent newborn calf serum were amelanotic and epitheliod in shape. When these cells were shifted into Dulbecco's modified Eagles medium with 10 per cent fetal calf serum, they became highly melanotic and of spindle/dendritic morphology within 4 days of culture. These morphological changes as well as other parameters were all characteristic of established criteria of melanoma differentiation. Alterations in the differentiation state of our highly metastatic variant, BL6-10, did not result in any change in tumorigenicity but did have profound effects on metastatic potential. All of the morphological and functional characteristics of the differentiated melanoma were found to be reversible by re-plating the cells in their original growth medium and 4 days of in vitro growth.These studies have allowed us to follow and more firmly establish Met-72 antigen expression as a surface marker for metastatic cells of the B16 melanoma, and have provided direct experimental evidence that the less differentiated, Met-72 positive melanoma form is the dominant cell type capable of metastatic potential.     

A quantitative assessment of the structural changes the rat's liver following obstruction of the common bile duct.

A study has been made of sequential changes in the rat's liver from 1 to 40 days after obstruction of the common bile duct. The qualitative changes have been described and illustrated. The volume proportions of hepatocytes, bile duct epithelium and biliary stroma have been quantified by histological analysis using a point counting technique. The proliferation of hepatocytes and bile duct cells have been measured by labelling with tritiated thymidine. The absolute quantity of hepatocytes in each liver has been estimated and expressed as a percentage of body weight. Over 40 days there is a relative fall in the volume proportion of hepatocytes and an increase in bile duct cells and biliary stroma. These changes in volume proportions are related directly to the period of jaundice. Biliary stroma increases in support of new bile duct tissue and there is no excessive fibrosis. Hepatocytes proliferate at a greater rate than normal after obstruction of the common bile duct and the degree of proliferation reaches a maximum of 24 times that of normal 4 days after obstruction. Similarly, the proliferation of bile duct epithelium is increased in obstructive jaundice but in this instance it reaches a maximum of 50 times that of normal 24 h after ligation of the common bile duct. The absolute quantity of hepatocytes in the liver probably falls during the period of jaundice. However, the fall is less than anticipated from the volume proportion of hepatocytes because of the overall increase in liver size.     

Formation of short‐lived multinucleated giant cells (MGCS) from cultured gilthead seabream macrophages

Monocytes/macrophages obtained from the head kidney and peritoneal cavity of gilthead seabream (Sparus aurata) were cultured using plates from three different manufacturers, and were maintained under different conditions. The effects on the morphology and fusion of monocytes/macrophages of initial cell loading, removal of non‐adherent cells at different times after plating, and addition of serum and antibiotics were evaluated by light microscopy, and transmission (TEM) and scanning (SEM) electron microscopy. Despite variations in adherence, the behaviour and the morphological changes in kidney monocytes/macrophages were similar in all three types of plates. When foetal calf serum (FCS) was added to the incubation medium, most of the cells resembling monocytes/macrophages were connected by cytoplasmic extensions that formed bridges after 24 hr in culture. After 30 hr, the monocytes/macrophages started to fuse, forming multinucleated giant cells (MGCs) which gradually increased in size until the culture was 4–5 days old. After 5 days the MGCs started to die, and after a week most had disappeared from the cultures. Cells incubated with medium without serum showed changes similar to those fed with FCS, but some cells survived for 3 weeks. The addition of fish serum to the medium appeared to accelerate all processes: the monocytes/macrophages and MGCs died after 3 days in culture. Antibiotics had no apparent effect on the cultures. Removal of non‐adherent cells at different times after plating did not appear to affect cell fusion. Coating the wells with extracellular matrix proteins reduced adherence but did not inhibit cell fusion. Curiously, not all macrophages fused with MGCs, and, unlike MGCs, these macrophages phagocytosed sheep red blood cells (SRBCs). Peritoneal macrophages also fused and formed MGCs in culture, similarly to kidney cells. Anat Rec 267:204–212, 2002. © 2002 Wiley‐Liss, Inc.     

Retention of liver-specific antigen by hepatocytes in filter-well culture

The precipitation band produced by hepatocytes from freshly-dispersed adult rat liver against liver-specific antiserum in agar gel immunodiffusion plates is still exhibited after the cells have been maintained in organized culture on Millipore membranes for 10 days.     

Pathological Changes Following the Inoculation of Chick Embryos with Adult Cells: I. Spleen Cells

An account has been given of the sequence of changes in the spleen and liver following the inoculation of fifteen-day-old chick embryos with adult cock spleen cells. These changes have been divided into two phases:

(i) Extensive proliferation of reticulum cell foci (which consisted of primitive reticulum cells, activated reticulum cells and blast cells, with a scattering of granulocytes), and a blast cell and granulocytopoietic response. These changes resulted in a rapid enlargement of the spleen and to a lesser extent the liver. This phase, which has been termed the splenomegaly syndrome, lasted 7 days and ended at the time when the only deaths were encountered.

(ii) Lymphoid transformation of the reticulum cell foci and a gradual return to normal structure in both liver and spleen. This phase followed the first and lasted at least until 23 days after treatment.

Pathological changes were not observed in the livers and spleens of chick embryos treated with adult rabbit spleen cells, bovine serum albumin, or Hanks's saline.

These findings have been discussed in relation to the observations of Biggs and Payne (1959). They found that mitotic figures examined in spleens enlarged five-to twenty-fold were of both host and donor origin, and were present approximately in the proportion of 1: 1. It has been suggested that the reticulum cell foci together with some of the blast cells are of donor origin and that the majority of blast cells and developing granulocytes are of host origin.

     

Morphology of adipose cells in lambs at birth and during subsequent transition of brown to white adipose tissue in cold and in warm conditions

Light and electron microscopy were used to study the morphology of adipose cells in newborn Merino lambs (Ovis aries). Postnatal changes in morphology were also examined in naturally fed Merino lambs held at 26°C or at 3°C for up to 32 days from the time of birth, and in lambs fasted for up to three days at 26°C or at 3°C since birth. All adipose cells examined in the newborn lambs showed the morphological characteristics of brown adipose cells; no white adipose cells could be found. The brown adipose tissue in normally fed lambs was replaced progressively by white adipose tissue during the first two or three weeks of life, and this replacement was retarded in the lambs held at 3°C. During replacement a continuous spectrum of cells with morphological characteristics between those of brown and white adipose cells were seen. No degenerating brown adipose cells were observed; and, apart from brown adipose cells, no cells were identified that could have been precursors of white adipose cells. This evidence suggests that in various body regions of lambs white adipose cells are derived from brown adipose cells. Lambs fasted at 3°C or under conditions that approached thermoneutrality (26°C) showed rapid depletion of lipid from brown adipose cells; hence brown adipose tissue of the lamb differs from that in the newborn rabbit in which this tissue is not readily depleted of fat during starvation under thermoneutral conditions.