Stem cells giving rise to extraembryonic tissues.

The review is devoted to characterization of stem cells involved in the formation of extraembryonic tissues during the early development of mammalian embryos. Here we present our results of characterization of stem cells from the trophoblast and extraembryonic endoderm of voles and comparative analysis of these cells and the corresponding mouse cells and discuss possible signal pathways maintaining these cells in undifferentiated state.

[Generation of rat induced pluripotent stem cells: the analysis of reprogramming and culturing media].

The rat represents very important, superior in many respects to the mous, animal model for studying pharmacology, physiology, ageing, cardiovascular etc. However, numerous attempts to derive rat ES cells necessary to carry out loss-of-gene-function studies have not been successful thus far. Therefore rat induct pluripotent stem cells (or riPS) should provide a notable alternative to ES cell, allowing to study gene functions in this valuable animal model. Here we report an improved lentivirus-based riPS derivation protocol that makes use of small inhibitors of MEK and GSK3. We show that the excision of proviruses does not affect neither karyotype and pluripotency state of these cells. Also, we propose genetic tool for an improvement of the quality of riPS cells in culture. These data may prompt further iPS-based gene targeting in rat as well as the development iPS-based gene therapies, using this animal model.

[Localization and composition of renal immunodeposits in mice developing HgCl2-induced autoimmune process].

A characteristic feature of systemic autoimmune diseases along with appearance of autoantibodies targeting self-antigenes is deposition of immunoglobulins and components of the complement system in kidneys. However, mechanisms of the deposit formation and their cytotoxic effects still remain poorly studied. To elucidate these questions, we used SJL/J mice which are known to develop autoimmune process accompanied by the appearance of anti-fibrillarin antibodies following regular administrations of sublethal dozes of HgCl2. Using antibodies to the total murine ummunoglobulins we showed that immunodeposits were present in glomeruli of autoimmune and control (not-autoimmune) animals, but their intensity was directly correlated with the titer of anti-fibrillarin autoantibodies and was minimal in control mice. By confocal microscopy and conventional fluorescence microscopy it was defined that immunodeposits deeply penetrate glomeruli and are the most likely located within mesangial cells. In autoimmune animals, ummunoglobulins completely colocolized with the C3--component of complement, but not with the major autoantigen--the protein fibrillarin. We failed to determine the signs of cell proliferation or death in glomeruli. The most prominent difference between control and autoimmune mice was the presence if immunodeposits in renal blood vessels. These observations argue in favor of the idea that destructive and disfunctional renal lesions accompanying development of autoimmune diseases can be caused, in part, by accumulation of immunodeposits in blood vessels.

[Theoretical and applied aspects of epigenetic reprogramming in mammalian development].

Epigenetic reprogramming implies changes in germ and somatic cells of an embryo, which are the consequences of gene activity regulation by means of DNA methylation, histone modification, and altered chromatin compaction. This suggests that epigenetic changes in mammalian cell nucleus occur during gametogenesis and toti-potent zygote formation. Epigenetic changes proceed during morphological and inductive interactions between cleaving blastomeres and subsequent interactions between the inner cell contents and trophoectoderm, as well as when the germinal layers (blastophyllums) and their derivatives appear, i.e., during the embryonic histogenesis. Some authors assume that in vitro fertilization and consequent human zygote cultivation lead to defects of genomic imprinting. This leads to abnormal embryonic and fetal development and increased incidence of hereditary diseases-Beckwith-Wiederman or Angelman syndromes. The present review, critically considers the facts on which the above hypothesis is based.

[Characteristics of morphogenesis of rudimentary rat gonads transplanted into the testis]. / Osobennosti morfogeneza transplantirovannykh v semennik zachatkov gonad zarodyshei krys.

The rat embryo (13 and 15 days of development) gonad germs of both sex, as well as isolated primary germ cells (PGC) have been transplanted into testes of mature animals of the same strain and investigated for 1.5 years. The isolated PGC are not able for further development and subjected to reduction. The gonad germs form analogues of the gonads with formation of definitive gonadal cells in 30 days. Further, degeneration of mature gonadal cells takes place. For realization of PGC ++cyto-differential processes and their stage-to-stage transformation into mature gametes certain interactions are necessary with concrete surrounding tissues that are at a strictly synchronized (with germ cells) stages of development.

[The differentiation characteristics of neural derivatives in rat teratomas]. / Osobennosti differentsirovki neiral'nykh proizvodnykh v teratomakh krys.

Peculiarities of differentiation in teratomas obtained by transplantation of 9- and 11-day-old Wistar rat embryos under testicle capsule of adult rats. The main portion of the teratoma is represented by neural derivatives, i.e. by neural tissue foci with pyramidal, stellate and fusiform neural cells, glial cells, and a developed capillary net. Nervous fibers and ganglia-like structures have also been revealed. At the same time, cytoarchitectonics of cerebral cortex does not form in neural areas.

[Morphogenesis of neural derivates after transplantation of the rat embryo telencephalon to the testis of sexually mature animals]. / Morfogenez neiral'nykh proizvodnykh pri transplantatsii konechnogo mozga émbriona krysi v semennik polovozrelykh zhivotnykh.

Some aspects of proliferation (the number of DNA synthesizing cells) and differentiation of forebrain (telencephalon) isolated from a 14.5 day old rat embryo and then transplanted into the testis of syngenetic animals were studied for 90 days. The embryonic cells proliferated for 12 days then their differentiation started and many types of differentiated neuronal and glial cells of the definitive brain were found in the transplant. Histological structure of the transplant was without cytoarchitecture of cerebral cortex. The comparison of the obtained data and literature allows us to make a conclusion that realization of morphogenetic potencies of the rat forebrain anlage are the same not only after transplantation into definitive brain, but after being transplanted into different ectopic places; including testis.

[Transformation of the cells of transplantable teratocarcinoma CBA9H6 by a bacterial dihydrofolate reductase gene]. / Transformatsiia kletok perevivnoi teratokartsinomy CBA9H6 bakterial'nym genom digidrofolatreduktazy.

A plasmid containing the bacterial gene of methotrexate-resistant dihydrofolate reductase (dhfr), under the control of early SV40 promoter, was introduced into murine teratocarcinoma CBA9H6 cells. From the whole pool of teratocarcinoma cells, which survived after transient methotrexate selection in vivo, the individual cells were isolated to give rise to 15 clones of tumors. Six of the 15 clones displayed nucleotide sequences of the original vector containing pBR322 sequences and the early SV40 promoter region; however, the bacterial dhfr gene was absent from the transformant clones. Possible causes of the loss of introduced dhfr gene from teratocarcinoma cells under non-selective conditions are discussed.

[Features of the growth and differentiation of the spinal ganglion of the embryonic rat in diffusion chambers]. / Osobennosti rosta i differentsirovki spinnomozgovykh gangliev émbrionov krys v diffuzionnykh kamerakh.

Cultivation peculiarities in diffusive chambers on nucleoporous and milliporous filters of the rat embryo spinal ganglia have been investigated on the 18th-20th days of development. Optimal variants have been obtained on the nucleoporous filters. Growth and differentiation of the sensitive neurons and cells of the peripheral glia are realized quicker in the diffusive chambers on the nucleoporous filters, than under in vitro cultivation. In 7-12 days in the diffusive chambers there are already mature pseudounipolar and multipolar neurons, and lemmocyte--axonal interrelations are of definitive character. The disadvantage of the method is its limited time of cultivation, since a connective tissue capsule is formed around the diffusive chamber.

[Characteristics of the growth and differentiation of teratocarcinoma OC15S1 in syngeneic and allogeneic mice]. / Osobennosti rosta i differentsirovki teratokartsinomy OC15S1 v singennykh i allogennykh myshakh.

Ascites teratocarcinoma OC15S1 represented by embryoid bodies contains two populations of embryocarcinoma cells with different rates of proliferation and levels of differentiation: diffusely dispersed cells (mitotic cycle 11 h, isotope exchange index 32.7%) and cavity-forming cells (14 h and 23.9%, respectively). Upon transplantation of embryoid bodies to allogeneic C57BL mice the growth of teratocarcinoma is inhibited: ascites tumor does not transform to solid; the number of embryoid bodies is 40 times less than in syngeneic mice; the number of necrotic embryoid bodies increases; the animals do not die. Retransplantation of teratocarcinoma cells from allogeneic C57BL mice to syngeneic 129 mice has demonstrated the presence of stem embryocarcinoma cells which retain their oncogenic properties and the mode of differentiation.

[Development of preimplantation mouse embryos in diffusion chambers]. / Razvitie doimplantatsionnykh zarodyshei myshei v diffuzionnykh kamerakh.

The diffusion chambers with the preimplantation CBA X C57BL mouse embryos (at the stages of blastomeres, morula, blastocyst) were placed in the abdominal cavity of males. Developmental peculiarities of the embryos, level of mitotic activity in their separate parts, as well as dynamics of the number of gigantic trophoblast cells and the volume of their nuclei have been studied. The development of the embryos in the diffusion chambers was shown to depend on the type of membranous filters: they developed from an earlier stage (stage of 4 blastomeres) on the nucleopore filters than on the millipore ones. The morphogenetic transformation proceeded in the preimplantation embryos at a slower rate resulting in the formation of two -and (in single cases) three-layered embryonic cylinder. Later the embryos were disorganized. The repeated passage of the disorganized embryo cells (six passages during the year) allowed to obtain structures similar with yolk sac carcinoma.

[Immunomorphological analysis of G2-chalone localization during the process of rat epidermis histogenesis]. / Immunomorfologicheskii analiz lokalizatsii G2-keilona v protsesse gistogeneza épidermisa krys.

The appearance of G2-chalone in the cytoplasm of the intermediate cell layer and partly in the periderm of 17-day-old rat embryo epidermis has been demonstrated by the indirect method of Coons using a monospecific antiserum. G2-chalone was absent from the basal cell layer of 17--21-day-old embryos and of the newborn rats. It was found in all the epidermal layers in 2--5-day-old postnatal rats, while in 6--9-day-old animals it was primarily detected in the cytoplasm of spinous and basal cells. Thus the localization of epidermal G2-chalone typical for defined tissue becomes stabilized at the end of epidermis histogenesis.

[Anomalous relationships between oocyte growth and the proliferative activity of the follicular epithelium in the ovaries of LT/Sv strain mutant mice]. / Anomal'nye vzaimootnosheniia mezhdu rostom ootsitov i proliferativnoi aktivnost'iu follikuliarnogo épiteliia v iaichnikakh myshei linii LT/Sv.

A comparison of the number of follicular epithelium cells and its proliferative activity with the oocyte diameter upon normal (ICR mice) and abnormal (LT/Sv mice) oogenesis was carried out. Two populations of monolayered follicles were found in the LT/Sv mice. The population of normal follicles providing for reproductive function is characterized by a distinct correlation between the follicle size, number of DNA-synthesizing follicular cells and oocyte diameter. The group of abnormal follicles contains large oocytes the growth of which is ahead of the development of granulosa cells. The number of DNA-synthesizing cells of monolayered follicular epithelium is 6 times less than in the normal follicles. 96% of parthenogenetically cleaving eggs were found in such abnormal monolayered follicles as well, the number of DNA-synthesizing cells being 14 to 17 times less than that in the normal follicles. Possible mechanism of this phenomenon are discussed.

[Morphologic characteristics of successive transformations of mouse blastocysts into teratoids during development under the testicular capsule]. / Morfologicheskaia kharakteristika posledovatel'nykh preobrazovanii blastotsist myshei v teratoidy pri ikh razvitii pod kapsuloi semennika.

Blastocysts of the CBAXC57Bl mouse strain transplanted under the testicular albuminous membrane normally develop (but delay in time) during 7-9 days forming embryos at the stage of the deep neural groove. The data obtained demonstrate that there is a definite autonomy in the development of early embryos not depending on any influence from the uterus at this stage. Desorganization of the embryo occurs 9-11 days after transplantation of the blastocyst. Further (14-20 days after transplantation) as a result of active histogenesis and atypical organogenesis, immature teratoid is forming. Secondary giant cells of the trophoblast develop in the presence of the internal cellular mass derivatives.

[Comparative study of mouse teratoids developed from blastocysts and embryos at the three germ layer stage]. / Sravnitel'noe izuchenie teratoidov myshi, razvivshikhsia iz blastotsist i iz émbrionov na stadii trekh zarodyshevykh listkov.

Germs of three stages: blasocyst, three layer germ cylinder, as well as head-fold and neural plate were implanted under the testicular capsule of mice, CBAXC57Bl line. Teratoids developed at the place of implantation are represented by the derivatives of all germ layers, as well as by some organic structures. The method for an experimental production of teratoids by means of transplantation of developing embryos is discussed as one of the methods for experimental histology. At the same time, possible tissue determination in embryos which do not undergo normal gastrulation is considered.

[Effect of hydrocortisone on DNA synthesis by urinary bladder epithelial cells and renal cortical substance and on the concentration of glucocorticoids in the blood]. / Deistvie gidrokortizona na sintez DNK kletok épiteliia mochevogo puzyria i korkovogo veshchestva pochki i na soderzhanie gliukokortikoidov v krovi.

Single administration of hydrocortisone (10 mg per 100 g of body weight) led at first to increase, and then to statistically significant reduction of the peripheral blood plasma corticosteroid level in rats. An increase of glucocorticosteroids was accompanied by a reduction in number of DNA-synthesizing cells of the urinary bladder epithelium leading later to the fall in the number of mitoses, whereas reduction of the hormone--to increase of the cell change from G1 to S period. At the same time inhibition of the proliferative processes in the kidney epithelium lasted during the whole experiment (48 hours). Possible causes of different sensitivity of the epithelial tissues to hydrocortisone are discussed.

[The effect of hydrocortisone on the proliferative activity ureteral epithelial cells]. / Deistvie gidrokortizona na proliferativnuiu aktivnost' kletok épiteliia mochetochnika

Autoradiography has demonstrated that a single injection of hydrocortisone (10mg/100 gm b.w.) reduced statistically significant the number of DNA synthesizing cells and the intensity of 3H-thymidine incorporation in these cells, as well as reduced the number of mitotically dividing epithelial cells in the rat ureter. 16 hours after hydrocortisone administration, the amount of proliferating cells increases statistically, but in 48 hours it reaches the control level.

[Renewal time, proliferative pool and duration of the mitotic cycle of the epithelial cells of the common bile duct]. / Vremia obnovleniia, proliferativnyi pul i prodolzhitel'nost' mitoticheskogo tsikla kletok épiteliia obshchego zhelchnogo protoka

Using cholchicine and H3-thymidine the following parameters of the mitotic cycle (in hours) were calculated: T=56.6; tm=0.9; tg2=1.2; ts=6; tg1=48.5 The proliferative pool was 7.5% and the time of epithelium renewal--754.5 hours. The common bile duct epithelium should be referred to the tissue systems with slow renewal.