Biological characteristics of cultured cells derived from various types of human brain tumors

We placed in culture brain tumors from 45 cases (7 cases of astrocytoma, 2 from oligodendrogliomas, 2 glioblastomas, 2 ependymomas, 13 meningiomas, 6 pituitary adenomas, 5 neurinomas, a malignant lymphoma, a choroid plexus papilloma, and 6 metastatic tumors) and succeeded in making a primary culture from 33, and maintained 17 in vitro over a considerable period of time (greater than three months). In the early period of the primary cultures, the astrocytoma cells had cytoplasmic processes which contacted each other, the oligodendroglioma cells were small and spindle-shaped, the glioblastoma cells were neoplastic with pleopmorphic features and possessed cytoplasmic processes, the ependymoma cells formed a rosette-like cell arrangement, the meningioma cells were spindle- or round-shaped cells and characterized as forming psammoma bodies, the pituitary adenoma cells were round- or oval-shaped cells and produced growth hormone (GH), adenocorticoid tropic hormone (ACTH), prolactin, or other hypophyseal hormones, the choroid plexus papilloma cells were round-or polygonal and showed a papillary cell arrangement, the neurinoma cells were spindle- or fibrous-shaped cells, and the malignant lymphoma cells were round and formed cell aggregates floating in the culture medium.     

Characterization of the population of phagocytic cells in thymic cell suspensions

Rat thymic phagocytic cells were characterized in vitro using various light- and electron-microscopical techniques. Thymic cell suspensions were mechanically prepared and enriched for non-lymphoid cells, which were predominantly phagocytic and of three types. Type I showed acid phosphatase (APh) activity in small granules dispersed throughout the cytoplasm and were mostly Ia antigen-positive, although the Ia membrane label varied in intensity and distribution among individual cells. Only a few cells had endogenous peroxidase activity. The type-I cells could not be clearly distinguished morphologically from type-II or -III cells, and most likely comprise precursors of both these cell types. Type-II were large pale cells with many slender cell processes. These cells had APh activity centrally positioned, were strongly positive for Ia on the cell membrane and were negative for endogenous peroxidase. The cytoplasm frequently contained Birbeck granules, which unequivocally classifies these cells as the in vitro equivalent of the interdigitating cells present in the medullary area of the thymus in situ. Type-III cells were rounded with a smooth or ruffled cell membrane and contained vacuoles and many phagolysosomes. They were strongly positive for APh which was present throughout the cytoplasm. About 50% of these cells were positive for endogenous peroxidase in a pattern resembling resident macrophages. The cells were negative for Ia antigens. Type-III cells mostly likely represent the macrophages found in the cortical area of the thymus.     

Differentiation of B lineage cells from liver of neonatal mice: generation of immunoglobulin isotype diversity in vitro

The development and differentiation of B cells expressing different immunoglobulin (Ig) isotypes was studied in cultures of murine neonatal liver cells. Before culture, 5 to 15% of the liver cells were mu + pre-B cells; 1 to 3% had surface IgM and less than 0.1% had slgG. During 4 days in culture the number of pre-B cells declined, whereas the number of IgM B cells increased greater than 20-fold; IgG B cells also increased in number. Of the four subclasses, IgG3+ and IgG2b+ cells predominated, each representing 3 to 10% of the total B cells at day 4. IgG1+ and IgG2a+ cells were present in lower numbers, representing 1 to 5% and 0.3 to 2.5% of B cells, respectively. Most IgG+ cells also expressed sIgM. Only a minority (less than 10%) of the sIgM+ cells were sIgD+, and most sIgG+ cells were sIgD-. Few T cells were present in these cultures (less than 0.5% in newborn liver), and sIgG+ cells were generated in normal frequencies in cultures of cells from nude mice. The numbers of B cells expressing each IgG subclass were similar in cultures from athymic nu/nu mice, nu/+ heterozygous littermates, and normal BALB/c mice. Plasmablasts and plasma cells appeared over a 14-day culture interval, and these expressed cytoplasmic IgM, IgG3, IgG1, IgG2b, IgG2a, and IgA. Measurable amounts of the first four isotypes were detected in the culture supernatants by radioimmunoassay. These results indicate that neonatal B cells can undergo isotype switching in the absence of T cell help, and that the expression of sIgD may not be a prerequisite for cells to switch Ig isotypes.     

An immunohistochemical study of endocrine cells in the pancreas of the Red-bellied frog (Bombina orientalis)

The regional distribution and frequency of pancreatic endocrine cells in the red-bellied frog, Bombina orientalis, were studied by the immunohistochemical peroxidase anti-peroxidase (PAP) method using five types of specific mammalian antisera to insulin, glucagon, somatostatin, bovine pancreatic polypeptide (PP) and secretin. The frequency was calculated as the mean number of each endocrine cell type/1,000 total cells (including exocrine and endocrine cells) using an automated image analysis process. The percentage of each immunoreactive (IR) cell species to the total IR cell population was also calculated. In the pancreas of the red-bellied frog, all five endocrine cell types were demonstrated. Insulin IR cells were located in the pancreas as single cells or islet-like clusters. The latter were localized in central regions. The insulin-IR cells showed a frequency of 65.40 plus/minus 14.56/1,000 cells. Glucagon IR cells were also detected as single cells or as clusters but in the case of clusters, two distributional patterns were detected - a central core type and a marginally distributed type. They showed an abundance of 32.70 plus/minus 7.32/1,000 cells. Somatostatin-IR cells were dispersed throughout the pancreatic parenchyma as single cells, three to four cells, or clusters. The clusters were located in the marginal regions. The somatostatin-IR cell frequency was 19.40 plus/minus 6.52/1000 cells. PP-IR cells were randomly distributed throughout the pancreatic parenchyma as single cells with a frequency of 14.70 plus/minus 4.92/1,000 cells. Secretin-IR cells were demonstrated as clusters or as single cells, and as clusters they occupied the central regions. They showed a frequency of 39.60 plus/minus 10.36/1,000 cells. This is the first report of the presence of secretin-IR cells in amphibian pancreatic endocrine cells. Overall, there were 37.20 plus/minus 6.84% insulin-, 21.90 plus/minus 5.55% glucagon-, 11.60 plus/minus 4.33% somatostatin-, 8.60 plus/minus 2.72% PP- and 23.40 plus/minus 4.45% secretin-IR cells.     

Possible involvement of bone cells in a new cementum formation

Cells from the gingival lamina propria, bone-derived granular tissues and periodontal ligament (PDL) were isolated after periodontal surgery and subsequently cultured in vitro. The resulting cells were defined as gingival cells, bone cells and PDL cells, respectively. Under a phase contrast microscope, the cultured cells exhibited a spindle and/or a polyhedral shape. On the basis of their appearance under an electron microscope, spindle-shaped cells and polyhedral-shaped cells were identified as fibroblasts and osteoblasts, respectively. Bone cells, a homogeneous population of osteoblasts, had a more rapid growth ability than PDL cells, which were a heterogeneous population of fibroblasts and osteoblasts. Of particular interest was that only bone cells produced bone matrix in the multilayers in vitro. These results support the hypothesis that the phenotype expressed by cells from the alveolar bone establishes a new concept for progenitor cells in the formation of cementum.     

Freeze-fracture study of the lateral-line canal organ of the Japanese sea eel,Lincozymba nystromi

Summary Specializations of apical surfaces of hair cells, supporting cells and marginal cells in the lateral-line canal organ of Japanese sea eel, Lincozymba nystromi, were examined with a freeze-fracture technique. Apical surfaces of hair cells have a lower density of intramembrane particles (IMP) than those of the surrounding supporting cells. Density of IMP on the streocilia is almost the same as that on the apical surface of hair cells. Junctions between hair and supporting cells were tighter than those between two supporting cells; those between supporting and marginal cells were tighter than those between hair and supporting cells, and those between two marginal cells were the tightest in the lateral-line canal organ.     

Morphology and behaviour of neural crest cells of chick embryo in vitro

Summary Neural primordia of chick embryos were cultured for three days and the behaviour of migrating neural crest cells studied. Somite cells were used as a comparison. Crest cells were actively multipolar with narrow projections which extended and retracted rapidly, contrasting to the gradual extension of somite-cell lamellae. On losing cell contact, somite cells were also more directionally persistent. The rate of displacement of isolated crest cells was particularly low when calculated over a long time base. Both crest and somite cells were monolayered; contact paralysis occurred in somite cell collisions but was not ascertained for crest cells. However, crest cells in a population were far more directionally persistent than isolated cells. Contact duration between crest cells increased with time and they formed an open network. Eventually, retraction clumping occurred, initially and chiefly at the periphery of the crest outgrowth. Crest cells did not invade cultured embryonic mesenchymal or epithelial populations but endoderm underlapped them. No effects were observed on crest cells prior to direct contact. Substrate previously occupied by endoderm or ectoderm caused crest cells to flatten while substrate previously occupied by the neural tube caused them to round up and clump prematurely.     

Flow cytometry and sorting of meiotic prophase cells of female rabbits

We present a new, flow cytometric method by which cells in various stages of the meiotic prophase can be quantitated and sorted in partly enriched fractions. Ovarian cells of 3-16-day-old rabbits were mechanically dispersed and fixed in ethanol and aldehydes. The cell suspension was stained with the DNA fluorochrome mithramycin and analysed and sorted in a FACS IV cell sorter according to the fluorescence and forward light scatter distribution. Cells sorted onto slides were stained with haematoxylin and eosin and differentially counted in the microscope. In the diploid fraction, preleptotene cells were more fluorescent than somatic cells. Leptotene cells were found throughout the S fraction and the tetraploid fraction. Zygotene and pachytene cells caused a major peak in the tetraploid region with 10-25% more fluorescence than somatic cells. Cells in diplotene had 5-15% more fluorescence than somatic cells. Mitotic cells were 20-40% more fluorescent than somatic cells and scattered the light more intensely than did meiotic cells with the same fluorescence.     

Induction of mammotroph development by a combination of epidermal growth factor,insulin, and estradiol-17beta in rat pituitary tumor GH3 cells

Several reports have indicated that prolactin-secreting cells (PRL cells) are generated from growth hormone-secreting cells (GH cells). We have shown that treatment with a combination of epidermal growth factor (EGF), insulin, and estradiol-17beta (E (2)) induces the appearance of PRL cells in pituitary tumor GH3 cells. The aim of the present study was to clarify the involvement of mitosis in the cytogenesis of PRL cells in rat pituitary and GH3 cells. The effects of the treatment with EGF, insulin and E(2) on DNA-replication were studied by detecting the uptake of bromodeoxyuridine (BrdU) into the nucleus. In cultured rat pituitary cells, BrdU-labeled PRL cells were observed irrespective of the hormone treatment. In GH3 cells, BrdU-labeled GH cells and mammosomatotrophs (MS cells) were detected; BrdU-labeled PRL cells were not detected, however, when GH3 cells were treated with BrdU for 3 hr and then immediately examined for BrdU-labeling. BrdU-labeled PRL cells were found only when GH3 cells treated with BrdU were allowed to grow for another 3 days. This finding suggests that during the additional 3-day culture, BrdU-labeled PRL cells were generated from BrdU-labeled cells other than PRL cells. These results indicate that PRL cells are transdifferentiated from GH cells or MS cells in GH3 cells by a combined treatment with EGF, insulin and E(2), while PRL cells in rat pituitaries are able to proliferate in response to the hormone treatment. Thus, there may be two pathways for cytogenesis of PRL cells: the transdifferentiation of GH cells or MS cells, and a self-duplication of PRL cells.     

Differentiation of Langerhans Cells from Interdigitating Cells Using CD1a and S-100 Protein Antibodies

The present study shows that Langerhans cells can be differentiated from Interdigitating cells at the light microscopic level. Superficial lymph nodes and skin taken from necropsies and the lymph nodes of dermatopathic lymphadenopathy (DPL) were used for this experiment. Sections of lymph node and skin were embedded using the acetone, methyl benzoate and xylene (AMeX) method and dendritic cells were immunostained with anti S-100 protein antibody (S-100, and OKT-6 (CD1a) using the restaining method. Langerhans cells in the skin were positive for both CD1a and S-100. Dendritic cells positive for both CD1a and S-100, and dendritic cells positive for S-100, but not for CD1a were observed in superficial lymph nodes. In normal superficial lymph nodes, there were more interdigitating cells than Langerhans cells. The majority of the dendritic cells in the DPL were Langerhans cells. We conclude that the S-100 and CD1a positive cells are Langerhans cells, and the S-100 positive-CD1a negative cells are interdigitating cells.     

Identification of subsets of human T cells capable of enhanced transendothelial migration.

A critical step in immunologically mediated inflammation is the migration of T cells between endothelial cells of postcapillary venules and into the tissues. To determine whether specific cells are capable of transendothelial migration, T cells that had migrated through endothelial monolayers were retrieved and analyzed. To accomplish this, human umbilical vein endothelial cells (EC) were cultured to confluence on collagen gels and incubated with human T cells. T cells that were nonadherent to the EC, those that bound to the endothelium, and cells that had migrated through the endothelial monolayer and into the collagen were individually harvested and characterized. After a 4-h incubation with EC, T cells distributed themselves such that 77 +/- 2% were nonadherent, 13 +/- 2% were bound to EC, and 10 +/- 1% had migrated into the collagen. The CD4+ T cells that had migrated into the collagen were predominantly CD29bright/CD45RObright and CD45RA-. CD8+ T cells demonstrated a greater transendothelial migratory capacity than the CD4+ T cells. The migrated CD8+ T cells were mainly CD29bright but CD45RA+. Additional phenotypic analysis of the migrating cells indicated that they contained fewer cells that expressed L-selectin. Moreover the surface expression of CD7 was less dense in the T cells that had migrated than in the nonadherent T cells. Finally the T cells that migrated were not enriched for CD45RBdim T cells. Prolonging the incubation with EC to 36 h increased the number of T cells that migrated but did not alter the predominance of CD29bright T cells in the migrated population. Stimulation of EC with IL-1 or IFN-gamma also increased the number of adherent and migrating T cells, respectively, but did not alter the phenotype of the migrating cells. These results indicate that the capacity for transendothelial migration is an intrinsic ability of certain subpopulations of T cells and is related to their stage of differentiation as identified by their surface phenotype.     

Immunocytochemical localization of pepsinogen in rat stomach

The localization of pepsinogen in rat stomachs was investigated by a postembedding immunoferritin method. When the preparations embedded in Epon were used, the secretory granules of chief cells were stained heavily and the granules of mucous neck cells were stained moderately. The secretory granules of cells intermediate between mucous neck cells and chief cells showed a bizonal staining; the electron dense parts were stained heavily and the electron lucent parts were stained moderately. The secretory granules of pyloric gland cells, on the other hand, were labeled faintly. However, the secretory granules of surface mucous cells, foveolar mucous cells, endocrine cells, cardiac mucous cells and cardiac serous cells were not stained by the method. The protein A-gold method showed a similar staining pattern of pepsinogen to that of the immunoferritin method. When the samples embedded in Lowicryl K4M were used to enhance the stainability of pepsinogen, essentially the same staining pattern as that of the samples embedded in Epon was obtained. In addition, the Golgi apparatus and the rough surfaced endoplasmic reticulum were more easily stained.     

Embryologic development of rat adrenal medulla in transplants to the anterior chamber of the eye

The morphological development and plasticity of embryonic and postnatal rat adrenal medullary cells were studied in homologous adrenal grafts to the anterior chamber of the eye. The eyes of recipient rats were adrenergically denervated 10 days prior to grafting by extirpation of the superior cervical ganglion in order to increase levels of NGF and NGF-like activities in the iris. Grafts taken at the 15th day of embryonic development (E15), i.e., at the beginning of immigration of medullary progenitor cells into the adrenal cortical anlagen, contained no cortical or mature medullary cells after 2 weeks in oculo. Numerous sympathoblastic cells, however, were located at the anterior surface of the iris. E 16 and E 17 transplants showed abundant mature cortical tissue after 2 weeks. Small groups of medullary cells with the ultrastructural characteristics of mature pheochromoblasts or young chromaffin cells were interspersed among cortical cells without forming a discrete medulla. Neuronal cells were exclusively found outside the cortical cell mass. Sympathoblasts grew at the surface of the iris, while young sympathetic nerve cells, which were invested by Schwann cells and received synaptic axon terminals, were embedded into the stroma of the iris. Grafting of E 21 adrenals yielded very similar results except that, in a few instances, young chromaffin cells were located outside the cortex and sympathetic nerve cells were seen to be in close contact with cortical cells. In transplants of adult medullary cells typical mature adrenaline and noradrenaline cells were clearly distinguishable after 8 weeks even in the absence of cortical cells. The only indication of phenotypical changes in these cells was the formation by some of them, of neuritic processes which could be visualized in glyoxylic acid-treated whole mounts of irises. These results are compatible with the idea that embryonic adrenal medullary cells have the environmentally controlled potential to develop along the neuronal or endocrine line, but could also be interpreted in terms of a selection of a specific subpopulation with predetermined potentialities by a specific microenvironment. Moreover, these results suggest that increasing differentiation of medullary cells is accompanied by progressive restrictions in their genetic program, which eventually prevent full transdifferentiation of mature chromaffin into neuronal cells.     

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.     

Effects of proliferation on the decay of thermotolerance in Chinese hamster cells

Development and decay of thermotolerance were observed in Chinese hamster HA-1 cells. The thermotolerance kinetics of exponentially growing and fed plateau-phase cells were compared. Following a 10-min heat exposure at 45 degrees C, cells in both growth states had similar rates of development of tolerance to a subsequent 45-min exposure at 45 degrees C. This thermotolerant state started to decay between 12 and 24 hr after the initial heat exposure. The decay appeared to initiate slightly sooner in the exponentially growing cells when compared to the fed plateau-phase cells. During the decay phase, the rate of thermotolerance decay was similar in the two growth conditions. In other experiments, cells were induced to divide at a slower rate by chronic growth (3 months) in a low concentration of fetal calf serum. Under these low serum conditions cells became more sensitive to heat and the rate of decay of thermotolerance remained the same for exponentially growing cells. Plateau-phase cells were also more sensitive, but thermotolerance decayed more rapidly in these cells. Although dramatic cell cycle perturbations were seen in the exponentially growing cells, these changes appeared not to be related to thermotolerance kinetics.     

Expression of Schwann cell markers by mammalian neural crest cells in vitro

During embryonic development, neural crest cells differentiate into a wide variety of cell types including Schwann cells of the peripheral nervous system. In order to establish when neural crest cells first start to express a Schwann cell phenotype immunocytochemical techniques were used to examine rat premigratory neural crest cell cultures for the presence of Schwann cell markers. Cultures were fixed for immunocytochemistry after culture periods ranging from 1 to 24 days. Neural crest cells were identified by their morphology and any neural tube cells remaining in the cultures were identified by their epithelial morphology and immunocytochemically. As early as 1 to 2 days in culture, approximately one third of the neural crest cells stained with m217c, a monoclonal antibody that appears to recognize the same antigen as rat neural antigen-1 (RAN-1). A similar proportion of cells were immunoreactive in cultures stained with 192-IgG, a monoclonal antibody that recognizes the rat nerve growth factor receptor. The number of immunoreactive cells increased with time in culture. After 16 days in culture, nests of cells, many of which had a bipolar morphology, were present in the area previously occupied by neural crest cells. The cells in the nests were often associated with neurons and were immunoreactive for m217c, 192-IgG and antibody to S-100 protein and laminin, indicating that the cells were Schwann cells. At all culture periods examined, neural crest cells did not express glial fibrillary acidic protein. These results demonstrate that cultured premigratory neural crest cells express early Schwann cell markers and that some of these cells differentiate into Schwann cells. These observations suggest that some neural crest cells in vivo may be committed to forming Schwann cells and will do so provided that they then proceed to encounter the correct environmental cues during embryonic development.     

Requirements for stimulation of T cell responses against virus-infected cells: nature of ectromelia virus-infected cells capable of stimulating cytotoxic T cells in a secondary response in vitro.

The nature of infected stimulator cells in the in vitro secondary cytotoxic T cell response to ectromelia infection was investigated. It was found that macrophages were better stimulator cells than spleen cells. B cells (Ig-positive cells) were superior to T cells (Ig-negative cells) both on a relative proportion and on a cell-to-cell basis. Concanavalin A and lipopolysaccharide-stimulated lymphocytes were also effective stimulator cells but appeared to be slightly inferior to spleen cells. Spleen cells depleted of Ia-positive cells were markedly inferior to normal spleen cells as stimulators. It was also found that primary and secondary cytotoxic T cells were largely Ia-negative. These findings are discussed in relation to the likely events during T cell responses to infection in vivo.     

Morphological and cytochemical studies of circulating Hodgkin's and Reed-Sternberg cells

Morphological and cytochemical studies of circulating neoplastic cells were carried out in a patient who presented a preterminal leukaemic phase of Hodgkin's disease (HD). Three types of abnormal cells were found in the peripheral blood: abnormal mononuclear cells, Hodgkin's cells and Reed-Sternberg cells. All neoplastic cells were cytochemically negative to Sudan black B, peroxidase and alkaline phosphatase. Some neoplastic cells were positive to PAS and all were positive to acid phosphatase, alpha-naphthylacetate esterase and beta-glucuronidase. The origin of the neoplastic population in HD is discussed.     

Effect of tryptophan on growth and morphology of Hansenula schneggii cells

Sundhagul, Malee (Illinois Institute of Technology, Chicago), and L. R. Hedrick. Effect of tryptophan on growth and morphology of Hansenula schneggii cells. J. Bacteriol. 92:241-249. 1966.-When Hansenula schneggii cells were cultured aerobically in a tryptophan-glucose medium, 70 to 90% of the cells were elongated; no growth occurred under anaerobic conditions. The size of the elongated cells was 15 to 25 mu by 2 to 4 mu, as compared with 2.5 to 5 mu for ellipsoidal cells. Formation of elongated cells occurred essentially during the logarithmic growth period; the highest percentage of elongated cells was found soon after the end of this growth phase. In the later stationary phase, some of the cells formed spherical buds which became spherical cells. The rate of cell division during this period was greatly reduced, but the spherical cells formed decreased the percentage of elongated cells in the population. Cells cultured in a membrane-filter filtrate of a tryptophan-glucose medium (with limiting tryptophan), in which elongated cells had been grown, were ellipsoidal until nitrogenous nutrients were exhausted; thereafter the cells were elongated if tryptophan was added. Of compounds related to tryptophan, kynurenine was the only one which induced a high percentage of the cells to elongate. Some amino acids, such as cystine, histidine, phenylalanine, tyrosine, and threonine, induced elongation in about 15% of the cells. Growth of cells with other amino acids, or the addition of most of the other amino acids to tryptophan-glucose medium, resulted in a population of spherical cells. Several consecutive sequential transfers of cells into tryptophan medium induced elongation in 90% of the cells, but one transfer from a culture with elongated cells into a medium with ammonium sulfate, or a mixture of amino acids, gave a culture with ellipsoidal cells. Growth in media at pH 4 or 5 favored formation of elongated cells; as the pH was increased, the percentage of elongated cells decreased. Carbon sources other than glucose did not affect the percentage of elongated cells, except for the alcohols mannitol and erythitol, which gave comparable growth but reduced the percentage of elongated cells from 70 to 50%. Cell wall analyses of the two types of cells indicated that elongated cells have 2.5 times as much mannan as cell walls of ellipsoidal cells. This suggests that tryptophan, kynurenine, and, to a limited extent, some of the other amino acids cause a diversion of polysaccharide biosynthesis to mannan in the elongated cells rather than to glucan as in ellipsoidal cells.