Embryonic stem cell/fibroblast hybrid cells with near-tetraploid karyotype provide high yield of chimeras.

Ten primary clones of hybrid cells were produced by the fusion of diploid embryonic stem (ES) cells, viz., line E14Tg2aSc4TP6.3 marked by green fluorescent protein (GFP), with diploid embryonic or adult fibroblasts derived from DD/c mice. All the hybrid clones had many characteristics similar to those of ES cells and were positive for GFP. Five hybrid clones having ploidy close to tetraploidy (over 80% of cells had 76-80 chromosomes) were chosen for the generation of chimeras via injection into C57BL blastocysts. These hybrid clones also contained microsatellites marking all ES cell and fibroblast chromosomes judging from microsatellite analysis. Twenty chimeric embryos at 11-13 days post-conception were obtained after injection of hybrid cells derived from two of three clones. Many embryos showed a high content of GFP-positive descendents of the tested hybrid cells. Twenty one adult chimeras were generated by the injection of hybrid cells derived from three clones. The contribution of GFP-labeled hybrid cells was significant and comparable with that of diploid E14Tg2aSc4TP6.3 cells. Cytogenetic and microsatellite analyses of cell cultures derived from chimeric embryos or adults indicated that the initial karyotype of the tested hybrid cells remained stable during the development of the chimeras, i.e., the hybrid cells were mainly responsible for the generation of the chimeras. Thus, ES cell/fibroblast hybrid cells with near-tetraploid karyotype are able to generate chimeras at a high rate, and many adult chimeras contain a high percentage of descendants of the hybrid cells.

["Chromosome memory" of parental genomes in embryonic hybrid cells]. / "Khromosomnaia pamiat'" roditel'skikh genomov v émbrional'nykh gibridnykh kletkakh.

In the hybrid cells obtained by fusion of embryonic stem cells with adult differentiated cells, homologous chromosomes are in two ontogenetic configurations: pluripotent and differentiated. In order to assess the role of cis- and trans-regulation in the maintenance of these states, we studied a set of clones of hybrid cells of the type embryonic stem cells-splenocytes and used two approaches: segregation of parental chromosomes and comparison of pluripotency of the past hybrid cells and embryonic stem cells. The segregation test showed that the hybrid cells lost only the homologs of the somatic partner and this process was sharply accelerated when the cells were cultivated in nonselective conditions, thus suggesting the full or partial preservation of the initial differences in the organization of parental homologs. The descendants of the former hybrid cells, which had the karyotype similar to that of embryonic stem cells, demonstrated the level of pluripotency, comparable with that of embryonic stem cells despite the long-term effect of trans-acting factors from the somatic partner in the genome of hybrid cells. The data obtained are interpreted in the framework of the concept of "chromosome memory", in the maintenance of which the key role is played by cis-regulatory factors.

[The interaction of homotypic and heterotypic embryonic stem cells with developing embryos]. / Vzaimodeistvie gomo- i geterotipcheskikh émbrional'nykh stvolovykh kletok s razvivaiushchimisia zarodyshami.

It has been demonstrated that embryonic stem cells form adhesive contacts with external blastomeres of mouse morula, while there is no such contact with blastocysts. The development of morula in the blastocysts is delayed inside a dense layer of such cells; however, in some cases, external blastomeres of the morula begin to differentiate into trophoblastic cells. The introduction of an excessive number of embryonic stem cells (15-20) into a 4-8-cell embryo results in abnormal development. When heterotypic embryonic mink stem cells are co-cultivated, they show only very weak adhesion with mouse blastomeres and are displaced as a result of compactization. When blastocysts are formed after the injection of heterotypic embryonic stem cells, such cells remain in the perivitelline space. In some cases, heterotypic embryonic stem cells continue to be determined in the trophoblastic direction and produce trophoblastic vesicles autonomously. The role of cell interaction in the determination of cells during early mammalian development is discussed.

In vitro and in vivo study of pluripotency in intraspecific hybrid cells obtained by fusion of murine embryonic stem cells with splenocytes.

Hypoxanthine phosphoribosyltransferase-deficient (HPRT-) mouse embryonic stem (ES) cells, HM-1 cells (genotype XY), were fused with adult female DD/c mouse spleen cells. As a result, a set of HAT-resistant clones was isolated. Four hybrid clones most similar in morphology and growth characteristics to the HM-1 cells were studied in detail with respect to their pluripotency. Of these, three clones contained 41-43 chromosomes, and one clone was nearly tetraploid. All the clones had the XXY set of sex chromosomes and expressed the HPRT of the somatic partner only. The hybrid clones shared features with the HM-1 cells, indicating that they retained their pluripotent properties: (1) embryonic ECMA-7 antigen, not TROMA-1 antigen, was present in most cells; (2) the hybrid cells showed high activity of endogenous alkaline phosphatase (AP); (3) all the hybrid clones were able to form complex embryoid bodies containing derivatives of all the embryonic germinal layers; (4) the hybrid cells contained synchronously replicating X chromosomes, indicating that they were in an active state; and (5) a set of chimeric animals was generated by injecting hybrid cells into BALB/c and C57BL/6J mouse blastocysts. Evidence for chimerism was provided by the spotted coat derived from 129/Ola mice and identification of 129/Ola glucose phosphate isomerase (GPI) in many organs. Thus the results obtained demonstrated that the hybrid cells retain their high pluripotency level despite the close contact of the "pluripotent" HM-1 genome with the "somatic" spleen cell genome during hybrid cell formation and the presence of the "somatic" X chromosome during many cell generations. The presence of HPRT of the somatic partner in many organs and tissues, including the testes in chimeric animals, shows that the "somatic" X chromosome segregates weakly, if at all, during development of the chimeras. There were no individuals with the 129/Ola genotype among the more than 50 offspring from chimeric mice. The lack of the 129/Ola genotype is explained by the imbalance of the sex chromosomes in the hybrid cells rendering the passage of hybrid cell descendants through meiosis in chimeras impossible. As a result, chimeras become unable to produce gametes of the hybrid cell genotype.

[The visualization of the pronuclei in zygotes of the American mink]. / Vizualizatsiia pronukleusov v zigotakh amerikanskoi korki.

Visualization of the pronuclei in optically nontransparent mink zygotes was achieved after centrifugation at 15,000 g. Under these conditions, the lipid fraction was concentrated in the light hemisphere of the zygote, while the pronuclei were localized in the equatorial area. Centrifugation did not affect the viability of zygotes, and no reliable differences in the rate of birth were found after transplantation of the native and centrifuge zygotes to the recipient females. Treatment of the zygotes with cytochalasin B before centrifugation leads to the condensation of lipids in the light area and allows distinct visualization of the pronuclei in the free zone. However, when these zygotes are cultivated, cleavage is irregular. Organization of the cytoskeleton in the mink zygotes is discussed.

Phenotypic expression of the fused (Fu) gene in chimaeric mice.

The dominant gene Fused (Fu) produces skeletal abnormalities during embryonic development. It was previously shown that C57BL/6 mice contain a suppressor of Fu, which acts after fertilization. Chimaeras were used to study whether this gene would suppress the Fu phenotype after the 8-cell stage of embryo development. We found no effect of the suppressor gene on Fu phenotype (its degree and frequency of expression) in chimaeric mice. We conclude that either the suppressor gene from C57BL/6 mice can only influence Fu expression at the intracellular level or Fu expression is determined before the 8-cell embryonic stage.

[The preimplantation embryonic development of 2 species of mammals from the family Mustelidae (Mustela erminea and Mustela vison)]. / Doimplantatsionnoe razvitie zarodyshei dvukh vidov mlekopitaiushchikh iz semeistva kunitseobraznykh (Mustela erminea i Mustela vison).

The preimplantation development of common weasel and American mink embryos was studied using light microscopy. Oocytes and blastomeres of these embryos are rich with lipids synthesized during oogenesis. Apparently, most lipids are utilized during the trophoblast formation. Large spherical blastomeres protecting zona pellucida are formed by the stage of implantation. The shape of the blastocyst, as well as central superficial implantation, are typical for carnivores and distinguish the studied order from other ones (e.g., from rodents or artiodactyls). Several aspects of evolution of mammals are discussed. A suggestion is made that differences between orders in the shape of the blastocyst and ways of their implantation reflect poly-phyletic origin of mammals.

Embryonic stem cells derived from morulae, inner cell mass, and blastocysts of mink: comparisons of their pluripotencies.

A characterization of cell lines that we derived from morulae (three lines), blastocysts (two lines), and the inner cell mass (ICM) is given. The karyotype of all the lines was normal; the genotype of four lines was XX, and four lines were genotypically XY. The pluripotencies and commitment status of the derived lines were estimated. First, there were not less than two-thirds of cells in the populations of the lines derived from morulae and the ICM with both Xs active; 70-100% of cells of the blastocyst-derived lines had one of the Xs in an inactive state. The activity of glucose-6-phosphate dehydrogenase (G6PD) in the lines (genotype XX) derived from morulae and ICM was found to be twofold higher than in lines with genotype XY, and G6PD activity was the same in the blastocyst-derived XX lines and XY lines. Second, when injected intraperitoneally into athymic mice, morulae- and ICM-derived cells gave rise to simple and complex embryoid bodies (EB) resembling to typical "cystic" mouse EBs. Third, when injected subcutaneously to athymic mice, the ICM- or morula-derived cells gave rise to typical teratomas containing derivatives of the three germ layers and components of organogenesis. Comparisons of cell lines of different derivations demonstrated that the pluripotencies of the ES cells derived from morulae or the ICM are higher than those of blastocyst derivation.

[Analysis of chimeric mice bearing the fused mutation]. / Analiz khimernykh myshei, nesushchikh mutatsiiu fused.

Interaction of cells with different genotypes and expression of Fused (Fu) gene in chimaeric mice were studied. Genetic analysis of 20 chimaeras showed that gonads of chimaeras in our experiments consisted of white component cells. The analysis of Fu gene expression in chimaeric mice supported the conclusions of the previous papers that mutations of Fu gene appeared not in the result of the hypofunction of normal allele. Two hypotheses were proposed: 1) the product of Fu gene migrates out of the mutant cells and changes the phenotype of the cells with normal allele; 2) suppressors from C57BL/6 mice influence on the penetrance of Fu gene only on the intracellular level.

Isolation and cultivation of blastocyst-derived stem cell lines from American mink (Mustela vison).

Ten embryonic stem (ES) cell lines from mink blastocysts were isolated and characterized. All the lines had a normal diploid karyotype; of the ten lines studied, five had the XX and five had the XY constitution. Testing of the pluripotency of the ES-like cells demonstrated that 1) among four lines of genotype XX, and X was late-replicating in three; both Xs were active in about one-third of cells of line MES8, and analysis of glucose-6-phosphate dehydrogenase revealed no dosage compensation for the X-linked gene; 2) when cultured in suspension, the majority of lines were capable of forming "simple" embryoid bodies (EB), and two only showed the capacity for forming "cystic" multilayer EBs. However, formation of ectoderm or foci of yolk sac hematopoiesis, a feature of mouse ES cells, was not observed in the "cystic" EB; 3) when cultured as a monolayer without feeder, the ES cells differentiated into either vimentin-positive fibroblast-like cells or cytokeratin-positive epithelial-like cells (less frequently); neural cells appeared in two lines; 4) when injected into athymic mice, only one of the four tested lines gave rise to tumors. These were fibrosarcomas composed of fibroblast-like cells, with an admixture of smooth muscular elements and stray islets of epithelial tissue; (5) when the ES cells of line MES1 were injected into 102 blastocyst cavities and subsequently transplanted into foster mothers, we obtained 30 offspring. Analysis of the biochemical markers and coat color did not demonstrate the presence of chimaeras among offspring. Thus the cell lines derived from mink blastocysts are true ES cells. However, their pluripotential capacities are restricted.

[Embryonic development of 2 strains of mice in the early cleavage period]. / Issledovanie razvitiia émbrionov myshei dvukh linii v rannii period drobleniia.

RNA metabolism at 1-, 2- and 8-celled stages was studied in C3H and C57Bl mice by means of detection of RNA content in individual embryos and microcolumnal chromatography of lysate of the embryos labelled with 3H-uridine. The increase of RNA content in the 8-celled embryos of the both strains is due to active synthesis of high and low molecular weight RNAs during this period. A comparison of 3H-uridine incorporation in RNA, and nucleotide fractions of 2-celled embryos has shown that the embryonic genome per se is activated earlier in C3H mice. The embryonic development and RNA changes in them are similar in the pure bred and hybrid embryos with common mothers. This serves as an additional evidence of the leading role of maternal factors in embryonic development during the first cleavage divisions.