The differentiation of oval cells into hepatocytes during induced hepatic carcinogenesis in mice. An electron microscopic study

An electron microscopic study of murine oval cells, induced by a single injection of genotoxic agent dipin and by a partial hepatectomy, has shown that their ultrastructure and direction of differentiation depend on localization in the liver lobule. Oval cells around portal tracts go through three stages of development: low differentiated cells 4.40 +/- 0.51 mu in diameter with ovoid nuclei 3.43 +/- 0.44 mu, intermediate cells, and young hepatocytes. They form common ducts surrounded by a basal lamina, and produce bile canaliculi-like structures and intermediate junctions between them. Another part of the oval cell population is organized similar to the bile duct epithelium. It consists of cells 9.37 +/- 1.1 mu in diameter with nuclei 7.28 +/- 1.16 mu in diameter and form a system of branching and anastomosing ducts widespread along the parenchyma from the portal to the central veins. Our data indicate that the oval cells can differentiate into hepatocytes, and support a hypothesis according to which the cells of terminal bile ductules are liver epithelial stem cells which can differentiate into a hepatocyte or a bile duct cell lineage in periportal microenvironment.     

Alkylating Damage by Dipin of Hematopoietic and Stromal Cells of the Bone Marrow

Effect of alkylating agent dipin was studied on hematopoietic (CFU-S) and stromal (CFU-F) progenitor cells. Single administration of dipin (0.06 mg/g) to adult (CBA × C57Bl/6) F1 hybrid mice induced a long-term (2 years) oscillations in the numbers of day 7 CFU-S and day 11 CFU-S in the bone marrow and spleen. Dipin also damaged the hematopoietic stroma as indicated by decreased numbers of CFU-F which remained low for at least a year. The capacity of stromal cells to form ectopic hematopoietic foci was considerably decreased and also remained low for 10 months. The obtained data suggest high dipin sensitivity of the earliest hematopoietic and stromal cells. The dynamics of CFU-S numbers in the hematopoietic organs supports their functioning on the basis of clonal succession (Kay, 1965).__________Translated from Izvestiya Akademii Nauk, Seriya Biologicheskaya, No. 3, 2005, pp. 267–272.Original Russian Text Copyright © 2005 by Domaratskaya, Bueverova, Payushina, Starostin.     

Development of resistance in Fisher lymphadenosis cells to dipin and bruneomycin when these preparations are used separately and in combination]

It was shown that dipin and bruneomycin resistant tumor cells appeared in mice with transplanted lymphadenosis after 10 passages on single use of the drugs. When the drugs were used in combination, no lymphadenosis cells resistant either to bruneomycin, or to dipin and their combination were found. The combined use of the drugs prevented development of resistance to them in the lymphadenosis cells at least during 10 passages on mice. The data on the possible prevention of the resistance development in the mouse lymphadenosis cells by means of combined use of low doses of dipin and bruneomycin provided an assumption that it is expedient to test the combination in clinics.     

Response of the spermatogenic system to chemical mutagen dipin in SAMP1 senescence-accelerated mice

Specific features of spermatogenesis were studied in senescence-accelerated mice of the strain SAMP1 after one-time injection of the chemical mutagen dipin. Quantitative and histomorphological changes in the spermatogenic epithelium proved to develop gradually. Cell loss and disorganization of spermatogenesis reached the peak as late as on days 28 and 35 after the injection. Differentiating spermatogonia manifested increased sensitivity to dipin. In prophase I of meiosis, developing spermatocytes proved to be less sensitive to the cytotoxic action of dipin at the pachytene than at the preleptotene-leptotene stages. Spermatogenesis in most seminiferous tubules was restored by day 56 after dipin treatment. At the end of the experiment (day 100), both quantitative parameters and morphological pattern of spermatogenesis did not differ significantly from those in the control. Thus, the cytotoxic action of dipin does not lead to irreversible structural disorganization of the spermatogenic epithelium in SAMP1 mice. Radioautography revealed a large proportion of highly differentiated Sertoli cells with ³H-thymidine-labeled nuclei in experimental animals. In some cases, structures resembling embryonic seminiferous tubules were revealed in the vicinity of rete testis in histological sections of testes of experimental mice. These structures contained the cells morphologically similar to gonocytes and immature Sertoli cells.     

Estimation of the frequencies of induced mutations in spermatogenic cells of senescence-accelerated prone mice of the SAMP1 strain

The results obtained in this work demonstrate the dynamics of cytogenetic changes of spermatogenic cells in senescence-accelerated prone mice, strain SAMP1, after a single exposure to a chemical mutagen, dipin, at a genetically active dose of 30 mg/kg. In the time interval between days 3 and 28 the frequency of induced spermatogonial micronuclei does not significantly exceed the level of spontaneous mutagenesis. The lack of an experimental effect of micronuclei in this time interval is probably a consequence of mitotic delay and (or) of the death of a considerable part of genetically defective cells in the spermatogonial compartment. Different stages of meiosis exhibit different chemical sensibilities: the yield of round spermatids with micronuclei is maximum after treatment of early primary spermatocytes (preleptotene-leptotene stage) with dipin. The high sensibility of preleptotene and leptotene spermatocytes is confirmed by the sperm head shape abnormality assay. Chromosome damage caused by dipin in spermatogonial stem cells is irreversible, as evidenced by a sharp increase in the frequencies of spermatogonial and meiotic micronuclear aberrations within long periods after treatment. Increased genetic instability in the stem compartment does not lead to irreversible degradation of the system of development of male sex cells in senescence-accelerated SAMP1 mice.     

Interchangeability of mutagens during fractionated effects of unequal concentrations of alkylating agents

The ability of thiophosphamide and dipin to substitute for each other in "clastogenic adaptation" of human lymphocytes was investigated at Go phase. There were used 5 low concentrations of mutagens 2, 0.2, 2.10(-2), 2.10(-3), 2.10(-4) micrograms/ml and the high one of 20 micrograms/ml with which cells were treated 2 hr after the effect of low concentrations. The "protective" concentrations for both mutagens were 0.2, 2.10(-2), 2.10(-3) micrograms/ml. The pretreatment with thiophosphamide caused the decrease in chromatid aberrations in "challenge" treatment with dipin, the pretreatment with dipin caused the decrease in chromosome aberrations in "challenge" treatment with thiophosphamide.     

Agonist-induced 4-1BB activation prevents the development of Sjӧgren's syndrome-like sialadenitis in non-obese diabetic mice

Activation of costimulatory receptor 4-1BB enhances T helper 1 (Th1) and CD8 T cell responses in protective immunity, and prevents or attenuates several autoimmune diseases by increasing Treg numbers and suppressing Th17 or Th2 effector response. We undertook this study to elucidate the impact of enforced 4-1BB activation on the development of Sjögren's syndrome (SS)-like sialadenitis in non-obese diabetic (NOD) model of this disease. An anti-4-1BB agnostic antibody was intraperitoneally injected to female NOD mice aged 7 weeks, prior to the disease onset that occurs around 10–11 weeks of age, 3 times weekly for 2 weeks, and the mice were analyzed for SS pathologies at age 11 weeks. The salivary flow rate was markedly higher in the anti-4-1BB-treated NOD mice compared to the IgG-treated controls. Anti-4-1BB treatment significantly reduced the leukocyte infiltration of the submandibular glands (SMGs) and the levels of serum antinuclear antibodies. Flow cytometric analysis showed that the percentages of CD4 T cells, Th17 cells and plasmacytoid dendritic cells among SMG leukocytes were markedly reduced by anti-4-1BB treatment, in conjunction with a reduction in SMG IL-23p19 mRNA levels and serum IL-17 concentrations. Although the proportion of Tregs and IL-10 mRNA levels in SMGs were not altered by 4-1BB activation, IL-10 mRNA levels in salivary gland-draining lymph nodes and serum IL-10 concentrations were both markedly increased. While anti-4-1BB treatment did not affect the amount of Th1 cells and IFNγ mRNA in the SMGs, it increased these measurables in salivary gland-draining lymph nodes. Hence, agonistic activation of 4-1BB impedes the development of SS-like sialadenitis and hyposalivation.     

Isolation, Biochemical Characterization, Long-Term Culture, and Phenotype Modulation of Oval Cells from Carcinogen-Fed Rats

Oval cells are liver epithelial cells that proliferate during hepatocarcinogenesis and chemically induced severe liver injury. It has been suggested that these cells represent hepatic stem cells which might play an important role in the histogenesis of cholangiocellular as well as hepatocellular carcinomas. In order to test this hypothesis highly purified oval cell preparations and propagable oval cell lines are needed. In the present study the isolation, biochemical characterization, and longterm culture of oval cells from rats fed a choline-deficient/DL-ethionine-supplemented diet for 6, 14, or 22 weeks are described. The freshly isolated oval cells were γ-glutamyltranspeptidase-positive, cytokeratin 7-, 8-, 18-, and 19-positive, albumin-positive, peroxidase-negative, and α-fetoprotein-negative and expressed lactate dehydrogenase isoenzymes 1-5. In addition, low but clearly measurable glucose-6-phosphatase and high γ-glutamyltranspeptidase and alkaline phosphatase activities (when compared to activities in untreated liver parenchymal cells) were measured in oval cells. Three oval cell lines, OC/CDE 6, OC/CDE 14, and OC/CDE 22, were established. They contained small and large epithelial cells replicating to form uniform monolayers with a cobblestone appearance; furthermore, a very low number of mononucleated giant cells were also present in the three cell lines. OC/CDE 6, OC/CDE 14, and OC/CDE 22 cells were γ-glutamyltranspeptidase-negative, were transiently albumin-positive, maintained the glucose-6-phosphatase activity levels measured in freshly isolated oval cells, and expressed lactate dehydrogenase isoenzymes 2-5. After exposure of the cultured oval cells to dimethyl sulfoxide or sodium butyrate, 35-40% of the cells reexpressed albumin, and glucose-6-phosphatase activity was enhanced; in addition, sodium butyrate strongly increased γ-glutamyltranspeptidase and alkaline phosphatase activities. In conclusion, oval cells express phenotypic markers of liver parenchymal as well as bile duct epithelial cells and possess a certain intrinsic plasticity. In order to test if the oval cells indeed represent an intermediate step in the differentiation of certain cells within the bile duct and ductular epithelial cell compartment to parenchymal cells, the three cell lines described herein will be transformed in vitro and their potential to give rise to cholangiocellular and/or hepatocellular carcinomas will be verified in vivo.     

Sca-1+ endothelial cells (SPECs) reside in the portal area of the liver and contribute to rapid recovery from acute liver disease

The liver has a high potential to regenerate however, the relation between oval cells and endothelial cells in the portal area during liver regeneration has not been adequately described. We have focused on sca-1+ endothelial cells (SPEC: sca-1+CD31+CD45− cells) and analyzed their localization, growth potential, and the role of these cells in damaged liver. SPECs are localized in the portal area and comprise approximately 20-30% of CD31+CD45− cells. These cells have higher growth potential than sca-1− endothelial cells and grow aggressively when the liver is severely damaged on the lateral side of the oval cells. In an in vivo study we show that when the liver is severely damaged in the presence of a VEGF (vascular endothelial growth factor)-inhibitor, the frequency of SPECs decreased and the recovery of liver volume was also delayed. These results strongly suggest that SPECs play important roles in the recovery of severely damaged liver.     

The effect of fractionated exposure to unequal concentrations of chemical mutagens

The effect of unequally fractionated concentrations of dipin and thiophosphamid on chromosomes of human lymphocytes was investigated at Go phase. There were used five low concentrations of mutagens 2, 0.2, 2.10(-2), 2.10(-3), 2.10(-4) mcg/ml and one high concentration 20 mcg/ml by which cells have been treated. Decrease of chromosome breaks and exchanges were observed, but the level of the aberration cell was stable. The "protective" levels for dipin were in concentrations of 0.2, 2.10(-2), 2.10(-3) mcg/ml. Only one "protective" concentration was 2.10(-2) mcg/ml.     

Effect of the alkylating agent, dipin, on induced hepatocyte proliferation. II. An increase in the DNA synthesis period]

The alkylating drug dipin was injected to mice 2 hours before a partial hepatectomy. The duration of DNA synthesis of proliferating hepatocytes was determined by means of cytophotometry of DNA mass in the nuclei, labeled with 3H-thymidine 30 hours after the operation. The DNA mass was doubled simultaneously in diploid and tetraploid nuclei within 18-26 hours. The DNA in labeled nuclei of the control mice was doubled in 8 hours. The kinetics of 3H-thymidine incorporation at different stages of S-period was similar in alkylated and normal cells.     

Transient expression of bile-duct-specific cytokeratin in fetal mouse hepatocytes

The differentiation of hepatocytes and biliary epithelial cells has been histochemically analyzed with anti-calf cytokeratin antiserum in the fetal mouse liver. Almost all young fetal hepatocytes transiently express bile-duct-specific cytokeratin; subsequently, the strong staining of the cytokeratin is confined to progenitor cells of intrahepatic biliary epithelial cells around portal veins. These results suggest that all fetal hepatocytes are bi-potent in terms of the differentiation of mature hepatocytes and intrahepatic bile-duct cells, and that the microenvironment around portal veins plays an important role in bile-duct differentiation. Large periportal hepatocytes continue to stain weakly for cytokeratin until 2 weeks after birth, although the number of positive hepatocytes decreases with development. The differentiation of bile ducts from periportal hepatocytes may continue for 2 weeks after birth.     

Hepatogenic differentiation of mesenchymal stem cells in a rat model of thioacetamide-induced liver cirrhosis

Implantation of bone-marrow-derived MSCs (mesenchymal stem cells) has emerged as a potential treatment modality for liver failure, but in vivo differentiation of MSCs into functioning hepatocytes and its therapeutic effects have not yet been determined. We investigated MSC differentiation process in a rat model of TAA (thioacetamide)-induced liver cirrhosis. Male Sprague-Dawley rats were administered 0.04% TAA-containing water for 8 weeks, MSCs were injected into the spleen for transsplenic migration into the liver, and liver tissues were examined over 3 weeks. Ingestion of TAA for 8 weeks induced micronodular liver cirrhosis in 93% of rats. Injected MSCs were diffusely engrafted in the liver parenchyma, differentiated into CK19 (cytokeratin 19)- and thy1-positive oval cells and later into albumin-producing hepatocyte-like cells. MSC engraftment rate per slice was measured as 1.0-1.6%. MSC injection resulted in apoptosis of hepatic stellate cells and resultant resolution of fibrosis, but did not cause apoptosis of hepatocytes. Injection of MSCs treated with HGF (hepatocyte growth factor) in vitro for 2 weeks, which became CD90-negative and CK18-positive, resulted in chronological advancement of hepatogenic cellular differentiation by 2 weeks and decrease in anti-fibrotic activity. Early differentiation of MSCs to progenitor oval cells and hepatocytes results in various therapeutic effects, including repair of damaged hepatocytes, intracellular glycogen restoration and resolution of fibrosis. Thus, these results support that the in vivo hepatogenic differentiation of MSCs is related to the beneficial effects of MSCs rather than the differentiated hepatocytes themselves.     

Effect of the alkylating agent dipin on the induced proliferation of hepatocytes. I. The kinetics of the cell population]

The alkylating drug dipin was injected to mice 2 hours before a partial hepatectomy. Liver regeneration was characterized by a decrease of the intensity of 3H-thymidine label, an increase of the labeled cell index, absence of mitoses, constant number of binuclear cells. The analysis of these data has shown that dipin causes a sharp (more than by 2 times) increase of the S-period and prolonged (up to 6--20 days) blocking of cells in the G2-period. No phenomenon of unbalanced growth was recorded. No changes in duration of prereplicative period, or in the volume of proliferative pool were recorded. The increase of mitotic cycle periods resulted in the cell population synchronization: by the end of the second ay more than a half of hepatocytes were in S-period, by the end of the third day about 80% of cells passed to G2-period.     

绿豆根边缘细胞发育特性

以东北绿豆为试验材料,采用琼脂悬空培养法,研究了绿豆边缘细胞的发育特性。结果表明:绿豆根尖发育初期根边缘细胞呈球形,随着根尖伸长逐渐发育形成椭圆形、长椭圆形和长条形;发育过程中,根边缘细胞具有较高的存活率,在根长大于10mm后根边缘细胞的存活率均在70%～80%之间并趋于稳定;在根长为25～30mm时根边缘细胞数目达到最大值(约13 000个);根冠果胶甲基酯酶(PME)活性在根长5mm时达到最高值(1.486H+μmol.root cap-1.h-1),此后随着根的伸长,根冠PME活性在1.107～1.256H+μmol.root cap-1.h-1间变化并趋于稳定。     

The life cycle of Toxocara vitulorum in Asian buffalo (Bubalus bubalis)

A procedure for labelling hatched Toxocara vitulorum larvae with 75 selenium is described. Labelled larvae are infective when administered into the small intestine and portal vein of buffalo of all ages. Only very young calves are infected after oral administration. The labelled larvae are used with an enhanced fluorographic autoradiographic procedure to study the dynamics of the infection in non-pregnant and pregnant buffalo. Larvae penetrate the wall of the small intestine between 2 and 8 h after administration. Most larvae go straight to the liver via the portal vein but a few enter the mesenteric lymph nodes. Over the next 90 h some larvae migrate to the lung and a few to muscle, brain, kidney and peripheral lymph nodes. Most remain in the liver. Over the next 3-7 weeks the larvae grow by about 10% and no moulting is observed. In a pregnant host larvae grow to 500-600 microns in liver and lung 1-8 days before parturition and migrate to the mammary gland around the time of parturition. In the mammary gland they grow to about 1200 microns and pass into the milk during the 7 days after parturition.     

Regionalisation of the endoderm progenitors and morphogenesis of the gut portals of the mouse embryo

This fate-mapping study reveals that the progenitors of all major parts of the embryonic gut are already present in endoderm of the early-head-fold to early-somite stage (1-9 somites) mouse embryo. The anterior endoderm contributes primarily to the anterior intestinal portal of the early-organogenesis stage (16-19 somites) embryo. Endoderm cells around and lateral to the node are allocated to the open “midgut” region of the embryonic gut. The posterior (post-nodal) endoderm contributes not only to the posterior intestinal portal but also the open “midgut”. Descendants of the posterior endoderm span a length of the gut from the level of the 3rd-5th somites to the posterior end of the embryonic gut. The formation of the anterior and posterior intestinal portals is accompanied by similar repertoires of morphogenetic tissue movement. We also discovered that cells on contralateral sides of the anterior endoderm are distributed asymmetrically to the dorsal and ventral sides of the anterior intestinal portal, heralding the acquisition of laterality by the embryonic foregut.     

The formation of oval-cell ducts during hepatic carcinogenesis in mice. Its relationship to the pre-existing canals of Hering]

It has been shown that a population of the oval cells is formed in mouse liver during the dipin-induced carcinogenesis (Radaeva, Factor, 1990b). This paper deals with the origin of the oval cells and their proliferation potential depending on localization in the liver lobule. Series of semithin liver sections were studied under the light microscope and detected labeled cells analyzed under electron microscope on serial ultrathin sections. We found that proliferation of cells of terminal bile ductules (Hering [correction of Gering] canals) takes place at the early stages of liver carcinogenesis. These cells and first labeled oval cells had similar size and morphology and jointly formed the ducts. Oval cell population was heterogeneous in terms of proliferative potential. Proportion of proliferating cells (38-45%) in the oval cells of Hering [correction of Gering] canals and small ducts surrounding portal tracts remained similar throughout the period of formation of the oval cell population. In the oval cells infiltrating the parenchyma, the proportion of proliferating cells appeared to depend on the intensity of the oval cell response: it attained the maximum (62%) on intermediate stage and decreased to the minimum (22%) at the peak of the reaction. These data suggest that Hering [correction of Gering] canals probably give origin to the ducts formed by oval cells.