Cytological and cytochemical effects of a liver extract from an adult rat on chick embryo hepatocytes and fibroblasts cultivated in vitro]

The effects of an extract from adult liver on chick embryo hepatocytes and fibroblasts cultivated in vitro are studied with cytological and quantitative cytochemical methods. This substance inhibits the multiplication of hepatocytes adn prevents these cells from entering into the S phase of the cycle. No effect is observed on the chick embryo fibroblasts. Not any morphological alterations are observed in the two cell types.     

Comparative characteristic of Mytilus muscle cells developed in vitro and in vivo

The mussel cells from premyogenic larval stages are capable of differentiation into smooth muscle cells in vitro. However, the behavior and protein composition of these cells are not completely identical to those of smooth muscle cells of adult mussels. In this study we compared some properties of mussel muscle cells forming from cells of trochophore (premyogenic larval stage) in vitro with those of muscle cells of veliger and adult mussel. We found a substantial difference between the contractile apparatus protein composition of veliger muscle and cultivated cells. Myorod, one of the molecular markers of the phenotype of mollusc smooth muscle cells (Shelud'ko et al., 1999, Comp Biochem Physiol 122:277-285), is not a constituent of the contractile apparatus of veliger muscle. At the same time the protein composition of contractile apparatus in cultivated cells was similar to that of adult Mytilus muscles. There were only few quantitative differences between them. The contractile activity of cultivated cells was changing in time. The kinetic parameters of first spontaneous contractions were similar to those of phasic contractions, while their period was close to that of tonic contractions. After 50-55 hrs cultivation the cells produced both phasic and tonic contractions, but the character of contractile activity of cultivated cells was regulated after six days of cultivation only. However, there were no muscle cells in vitro, whose contractile activity was similar to that of veliger muscle cells. So, we concluded that properties of muscle cells forming from premyogenic larval mussel cells in culture are similar to those of muscle cells of the adult mussel, but not of veliger.     

Cultivation and growth of dissociated neurons from chick embryo cerebral cortex in the presence of different substrates

Summary Dissociated cells from 7-day old chick embryo cerebral hemispheres were cultivated for one month in Rose chambers. Four different culture conditions were employed in the composition of the matrix on which the cells were cultivated: collagen alone, collagen plus embryonic extract, collagen plus plasma and collagen plus plasma and embryonic extract.Within the first 48 hours of cultivation the cells formed processes under all four culture conditions. In the presence of plasma the dissociated cells remained well isolated; in the other culture conditions many cells reassociated into clumps.After 2–3 weeks in cultures on collagen or collagen plus embryonic extract many polygonal cells developed and formed a layer upon which typical neurons and oligodendrocyte-like cells were observed. After 3 weeks the polygonal cells began to transform into astrocyte-like cells. In the presence of plasma the cell bodies of the neuroblasts remained small and round. The processes developed generally consisted of one long and many short thick fibres; all processes had a bulbous appearance. In 3–4-weeks old cultures the cells which remained viable, were morphologically unchanged.The differences in the morphological aspects of the cells cultivated on plasma and those cultivated on collagen alone or with embryonic extract are discussed.This work was supported in part by the Délégation Générale à la Recherche Scientifique et Technique and the Mind Science Foundation. Thanks to Prof. Dr. Z. Lodin, Czechoslovak Academy of Sciences, for his continuing help. We are grateful to Mrs. M. F. Knoetgen for technical assistance.     

谷氨酸对大鼠脂肪干细胞诱导分化神经细胞的影响及机制

目的:研究谷氨酸(Glu)对大鼠脂肪干细胞Adipose-derived stem cells(ADCSs)诱导分化神经细胞的作用及机制。方法:取成年大鼠腹股沟脂肪组织进行体外细胞培养,采用免疫组化方法检测证实为ADSCs。对照组为正常培养的ADSCs并诱导分化神经细胞,谷氨酸(Glu)处理组加入不同浓度的Glu,MTT比色法观察脂肪干细胞的存活率。结果:从ADSCs诱导分化的细胞包括神经元及神经胶质细胞,免疫组化结果显示神经元特异性烯醇化酶(NSE)染色阳性和胶质纤维酸性蛋白(GFAP)染色阳性。Glu处理组给药24h后,与对照组比较ADSCs存活率明显降低,50μmol·L-1Glu组细胞存活率为83.98%,与对照组比较差异无显著性(P>0.05);100及1000μmol·L-1Glu组干细胞存活率(分别为66.82%和17.08%)低于对照组(P<0.05,P<0.01)。结论:Glu对ADSCs有损伤作用,随着Glu剂量的增加,ADSCs的存活率逐渐降低,二者呈剂量依赖关系。     

Effects of different Sertoli cell types on the maintenance of adult spermatogonial stem cells in vitro

Spermatongonial stem cells (SSCs) are unique testis cells that are able to proliferate, differentiate, and transmit genetic information to the next generation. However, the effect of different Sertoli cell types on the expression of specific SSC genes is not yet well understood. In this study, we compare the in vitro effect of adult Sertoli cells, embryonic Sertoli cells, and TM4 (a Sertoli cell line) as feeder layers on the expression of SSC genes. SSCs were isolated from the testis of adult male mice and purified by differential plating. Following enrichment, SSCs were cultivated for 1 and 2 wk in the presence of various feeders. The expression of SSC-specific genes (Mvh, ZBTB, and c-kit) was evaluated by real-time polymerase chain reaction. Our results revealed that expression of the specific SSC genes was significantly higher in the embryonic Sertoli cells after 1 and 2 wk compared to the adult Sertoli cells and the TM4 group. Our finding suggest that co-culturing of SSCs with embryonic Sertoli cells is helpful for in vitro cultivation of SSCs and might improve the self-renewal of these stem cells.     

In vivo and in vitro activity of Streptococcus faecium extract on Herpes simplex virus

The effects of substance or substance extracted from Str. faecium SF 68 on HSV-1 are evaluated. The "in vivo" assay show that bacterial extract introduced i.p. in mice simultaneously with HSV-1 brought about 100% of survival, but bacterial extract after virus challenge brought about complete mortality of mice. "In vitro" assays show that bacterial extract reduce significantly PFU number. It seemed that Str. faecium extract affected the virus at the stage of adsorption on the host cells.     

Application of <Emphasis Type="Italic">Chlorella sorokiniana</Emphasis> (Chlorophyceae) as supplement and/or an alternative medium for the in vitro cultivation of <Emphasis Type="Italic">Schomburgkia crispa</Emphasis> (Orchidaceae)

Schomburgkia crispa Lindley (Orchidaceae) is an epiphytic species found in gallery forests and dry vegetation in the Brazilian Cerrado. It is typically unable to germinate or exhibits low germination because of dependency on mycorrhizal associations. In vitro cultivation techniques have helped circumvent difficulties involved in propagation from seeds. Alternative media and organic biostimulant substances that reduce costs and promote satisfactory in vitro growth are constantly sought. This study evaluated in vitro multiplication and rooting of S. crispa in a modified culture medium containing extract of the microalga Chlorella sorokiniana. We analyzed supplementation of WPM (Woody Plant Medium) with microalgae suspended in NPK medium, or as the supernatant resulting from the centrifugation of a culture in NPK medium. The extracts were added to WPM instead of distilled water. The compounds 6-benzylaminopurine (BAP) and indolebutyric acid (IBA) were used as reference in the in vitro multiplication and rooting of S. crispa, respectively. Both growth regulators were tested at 0, 2.5, and 5.0 mg L^?1. During in vitro multiplication of S. crispa, WPM supplemented with 5.0 mg L^?1 BAP favored the formation of more sprouts, whereas WPM containing 2.5 mg L^?1 IBA supplemented with microalgae extract stimulated in vitro rooting. Schomburgkia crispa explants cultivated in medium supplemented with microalgae suspension or the supernatant of C. sorokiniana showed growth similar to explants cultivated in WPM alone. Therefore, it is possible to use the microalga C. sorokiniana as a supplement and/or alternative to WPM for the in vitro cultivation of S. crispa.     

Development of self-tolerance in normal mice. Appearance of suppressor cells that maintain adult self-tolerance follows the neonatal autoantibody response

T cell populations from BALB/c mice at different ages were analyzed to determine when in development Ts cells specific for the anti-mouse RBC (MRBC) autoantibody response become activated. Previous studies have shown that adult CD8+ T cells actively suppress this autoimmune response and adult spleen cells depleted of CD8+ cells can generate an anti-MRBC response in culture with MRBC. The present results demonstrate that T cells from mice less than 1 wk of age do not suppress the in vitro anti-MRBC response of adult spleen cell populations depleted of CD8+ Ts cells. By 2 wk of age Ts cells are detectable in this anti-self response and reach adult levels by 3 wk of age. Non-specific "natural suppressor" cells normally present in neonatal spleen cell populations are unable to suppress this autoantibody response, although they are active in suppressing anti-SRBC responses in the same cultures. Before the appearance of Ts cells active in the anti-MRBC response, neonatal spleen cell populations can generate anti-MRBC antibody-forming cells, both spontaneously in vivo and in vitro. The in vitro anti-MRBC response of neonatal spleen cells was shown to be Ag driven and Ag specific. The ability of unfractionated spleen cells to generate this response in vitro declines with age and is relatively low by 3 wk. This decline in responsiveness occurs simultaneously with the appearance of suppression specific for the anti-MRBC response, suggesting that the two events may be causally related.     

The retinal pigment epithelial cells of the adult newt and rat under conditions of in vitro organotypic culture

To understand why the retinal pigment epithelium (RPE) has different potentials for neural differentiation in lower and higher vertebrates, the RPEs of adult newts and rats were compared under similar in vitro cultivation conditions. The RPEs of both animal species were organotypically cultivated within the posterior eye wall under constant rotation in the serum medium free of growth factors. Comparison of the cell morphology, proliferation, and expression of pan-neural markers demonstrated that the RPE cells of adult newts and rats under similar in vitro conditions displayed both similarities and differemces. They were able to synthesize DNA but rarely divided mitotically. In addition, part of the RPE cells of both the newt and the rat were dislodged from the layer, migrated, and acquired a macrophage phenotype. However, the majority of the cells retained the initial morphology and remained within the layer. In several cases, these cells displayed the initial characteristics of neural differentiation, namely, expression of pan-neural proteins. The difference between the newt and rat RPE cells was in the ability of the former to generate in vitro an additional row of dedifferentiated NF-200-positive cells, characteristic of in vivo newt retinal regeneration. These data demonstrate that the RPE cells of the adult newt and rat retain the potential of manifesting neural cell traits; however, more advanced changes towards differentiation are characteristic of only the newt RPE.     

Action of "myelauxins", substances capable of accelerating granulocytic hematopoiesis, in vivo in rabbits]

Extracts from homologous blood serum had been shown by the author to stimulate the granulocytopoiesis of cells from chickens' embryonic spleen cultivated in vitro. The results presented here show that extracts obtained with the same method from rabbits' serum can induce the same activity in vivo in the adult mammals. Data relating to the nature of those active substances will be published later.     

Enzyme activities of monoamine oxidase,cathechol-o-methyltransferase and γ-aminoubutyric acid transaminase in primary astroglial cultures and adult rat brain from different brain regions

The activities of monoamine oxidase (MAO), cathechol-O-methyltransferase (COMT) and -aminobutyric acid transaminase (GABA-T) were measured in primary cultures from newborn rat cultivated from 6 different brain regions. These primary cultures contained mostly astroglial cells, evaluated by the presence of the glial fibrillary acidic protein (GFAp, -albumin) and the S-100 protein. The enzyme activities in the corresponding brain areas from adult rat were also quantified. MAO activities were on the same level in 14-day old cultures and in adult rat brain homogenates, with significantly lower values in brain stem as compared to the other brain regions examined. COMT activities were on a higher level in the cultures than in adult rat brain homogenates. Astroglial cells from hippocampus were found to have the highest and those from brain stem the lowest COMT-activities. GABA-T activities were lower in the cultures than in adult rat homogenates. No significant differences were seen in the various astroglial cultures. Accumulation of [³H]dopamine and [³H]-aminobutyric acid (GABA) visualized by autoradiography showed only a slight uptake of dopamine in comparison with the uptake of GABA. It is concluded that astroglial cells in culture have enzymatic properties similar to those of astroglial cells in different brain regions of adult rat brain. Studies are in progress to evaluate if the regional heterogeneity observed among cultivated astroglial cells is affected by in vivo differentiation until cultivation and/or time in culture.     

Age-related and carcinogen-induced alterations of the extracellular growth factor requirements for cell proliferation in vitro

Cells from thigh muscles of fetal rats proliferate readily in vitro in medium containing homologous adult rat "plasma". As the donor animals mature, these cells become unable to make DNA and proliferate in "plasma" medium, but retain an ability to proliferate in medium containing fetal bovine serum (FBS). This age-related loss of the ability to proliferate in "plasma"-medium is due to an increasing need by the cells for an exogenous prereplicative promoter which is found in FBS and a crude preparation of bovine luteinizing hormone. Adult cells (and possibly fetal cells) also require a "cycle-completion" factor which is found in FBS and adult rat "plasma". The requirements for such external proliferative promoters is completely eliminated by neoplastic transformation in vivo, and neoplastic adult cells isolated from a nickel sulfide-induced mixed rhabdomyo-fibro-sarcoma can make DNA and proliferate in vitro in the complete absence of exogenous growth factors.     

Effect of Solcoseryl on nerve tissue under in vitro conditions]

Explants from trigeminal ganglia and skin of chick embryos and hippocampus from fetal rats were cultivated in Maximow assembly in the presence of Solcoseryl (Solco AG, Basel), a blood extract of calf. Solcoseryl in vitro did not influence the regeneration of nerve fibers from CNS explants. A stimulatory effect of Solcoseryl in vitro by 1% concentration on the outgrowth of new processes in explants of PNS was demonstrated. It is discussed: under optimal concentration Solcoseryl may be important for the influence of the composition of the medium in which explants of the nerve system and skin are cultivated.     

Cultivation,characterization and modulation of rat thymic nonlymphoid cells in vitro

Thymic fragments of young adult rats were cultivated in vitro using an explant technique. Two weeks later, non-lymphoid cells were characterized by enzyme-cytochemistry and immuno-cytochemistry. Based on cytomorphological criteria and cell-specific markers approximately 95% of cells were classified into 3 main types: epithelial cells, macrophages and fibroblasts. The effect of dexamethasone and amphotericin B, known modulators of cell growth in culture was also studied. Quantitative analysis showed that amphotericin B inhibited proliferation of macrophages and epithelial cells, while dexamethasone suppressed proliferation of fibroblasts and promoted growth of epithelial cells.Abbreviations TNLC Thymic Non-Lymphoid Cells - NSE Non-Specific Esterase     

Cell cycle commitment of rat muscle satellite cells

Satellite cells of adult muscle are quiescent myogenic stem cells that can be induced to enter the cell cycle by an extract of crushed muscle (Bischoff, R. 1986. Dev. Biol. 115:140-147). Here, evidence is presented that the extract acts transiently to commit cells to enter the cell cycle. Satellite cells associated with both live and killed rat myofibers in culture were briefly exposed to muscle extract and the increase in cell number was determined at 48 h in vitro, before the onset of fusion. An 8-12-h exposure to extract with killed, but not live, myofibers was sufficient to produce maximum proliferation of satellite cells. Continuous exposure for over 40 h was needed to sustain proliferation of satellite cells on live myofibers. The role of serum factors was also studied. Neither serum nor muscle extract alone was able to induce proliferation of satellite cells. In the presence of muscle extract, however, satellite cell proliferation was directly proportional to the concentration of serum in the medium. These results suggest that mitogens released from crushed muscle produce long-lasting effects that commit quiescent satellite cells to divide, whereas serum factors are needed to maintain progression through the cell cycle. Contact with a viable myofiber modulates the response of satellite cells to growth factors.     

The effects of solcoseryl on the growth and multiplication of chick embryo fibroblasts cultivated "in vitro"

The action of Solcoseryl, a free protein extract of calf blood, was studied on chick embryo fibroblasts cultivated in vitro. Solcoseryl stimulates the permitotic DNA synthesis and increases the number of mitoses.,     

Remote Consequences of Irradiation for the Progeny of Mammalian Cells

It is shown that -irradiation has remote consequences for mammalian cells cultivated in vitro. Many generations in the progeny of cells surviving acute and chronic irradiation at high and low doses are characterized by a number of abnormalities, including delayed cell death, the formation of micronuclei and giant cells, an increased frequency of sister chromatid exchanges, a reduced potential for repair, the loss of adaptive response, and increased radiosensitivity. These phenomena are regarded as manifestations of genomic instability induced by ionizing radiation.     

Effect of andrographolide and Kan Jang--fixed combination of extract SHA-10 and extract SHE-3--on proliferation of human lymphocytes, production of cytokines and immune activation markers in the whole blood cells culture.

The immunomodulatory properties of a diterpene lactone andrographolide and Kan Jang--a standardized fixed combination of Andrographis paniculata extract SHA-10 and Eleutherococcus senticosus extract SHE-3 were investigated. Their role on spontaneous and phytohemagglutinin (PHA)-induced proliferation of human peripheral blood lymphocytes (PBL) and on production of interferon-gamma (INF-gamma) and tumor necrosis factor-alpha (TNF-alpha) were determined in vitro. Proliferation of PBL induced by PHA was enhanced by co stimulation with andrographolide and Kan Jang. At the same time andrographolide and Kan Jang inhibit spontaneous proliferation of PBL in vitro. These preparations also have effect on the formation of INF-gamma, TNF-alpha and some immune activation markers such as neopterin (Neo), beta-2-microglobulin (beta2MG), and soluble receptor for interleukin-2 (sIL-2R or sCD25) in blood cells culture. Andrographolide and Kan Jang stimulate the INF-gamma, Neopterin and beta2MG formation, but do not have any significant effect on the production of INF-gamma and Neopterin in PHA stimulated blood cells. An opposite effect on these immune makers was observed in the PHA-stimulated blood cells: both andrographolide and Kan Jang increase the formation of TNF-alpha and beta2MG in cultivated whole blood cells. Thus, andrographolide and Kan Jang can have an in vitro effect on the activation and proliferation of immunocompetent cells as well on the production of key cytokines and immune activation markers. The results show an overall higher effect of the fixed combination as compared with the equivalent amount of the pure substance andrographolide. The data are consistent with results from clinical studies of Kan Jang and contributed to a better understanding of these results.     

Cardiac-Like Action Potentials Recorded from Spontaneously-Contracting Structures Induced in Post-Nodal Pieces of Chick Blastoderm Exposed to an RNA-Enriched Fraction from Adult Heart

In 19–20-h-old chick embryonic blastoderm, the portion anterior to Hensen's node contains the cells which are destined to give rise to the heart, whereas the cells in the portion of the blastoderm posterior to Hensen's node (the post-nodal piece, PNP) normally do not. However, when the PNP was isolated from the blastoderm by cutting 0.6 mm posterior to Hensen's node and the isolated PNP was exposed to an RNA-enriched extract obtained from adult chicken heart, a spontaneously contracting tubular structure was formed in vitro within 1 week. Typical cardiac action potentials, both spontaneous and evoked, were recorded by intracellular microelectrodes from cells within these contracting tubules. Actinomycin D or cycloheximide prevented the inducing effect of the RNA extract. Induction of tubule formation and the appearance of excitability did not occur in untreated controls or in PNPs treated with RNA-containing extracts from adult chicken liver. These results indicate that cells not originally destined to become myocardial cells can be induced to gain many of the properties of heart cells by an RNA-enriched fraction from adult heart, and that this induction is dependent upon protein synthesis. Although it is not known what substance is the active principle, it is likely to be RNA.