Natalia M. Matveeva

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. Mol. Reprod. Dev. 50:128–138, 1998.

Characterization of a new hybrid mink-mouse clone panel: chromosomal and regional assignments of the GLO, ACY, NP, CKBB, ADH2, and ME1 loci in mink (Mustela vison)

To expand the mink map, we established a new panel consisting of 23 mink-mouse clones. On the basis of statistical criteria (Wijnen et al. 1977; Burgerhout 1978), we developed a computer program for choice of clones of the panel. Assignments of the following mink genes were achieved with the use of the hybrid panel: glyoxalase (GLO), Chromosome (Chr) 1; acetyl acylase (ACY), Chr 5; creatine phosphokinase B (CKBB), Chr 10; alcohol dehydrogenase-2 (subunit B) (ADH2), Chr 8. Using a series of clones carrying rearrangements involving mink Chr 1 and 8, we assigned the gene for ME1 to the short arm of Chr 1 and that for ADH2 to Chr 8, in the region 8p12-p24. Mapping results confirm the ones we previously obtained with a mink-Chinese hamster panel. However, by means of an improved electrophoretic technique, we revised the localization of the gene for purine nucleoside phosphorylase (NP), which has been thought to be on mink Chr 2. It is reassigned to mink Chr 10.

Dominance of parental genomes in embryonic stem cell/fibroblast hybrid cells depends on the ploidy of the somatic partner

Two dozen hybrid clones were produced by fusion of diploid embryonic stem (ES) cells positive for green fluorescent protein (GFP) with tetraploid fibroblasts derived from DD/c and C57BL-I(I)1RK mice. Cytogenetic analysis demonstrated that most cells from these hybrid clones contained near-hexaploid chromosome sets. Additionally, the presence of chromosomes derived from both parental cells was confirmed by polymerase chain reaction (PCR) analysis of polymorphic microsatellites. All hybrid cells were positive for GFP and demonstrated growth characteristics and fibroblast-like morphology. In addition, most hybrid cells were positive for collagen type I, fibronectin, and lamin A/C but were negative for Oct4 and Nanog proteins. Methylation status of the Oct4 and Nanog gene promoters was evaluated by bisulfite genomic sequencing analysis. The methylation sites (CpG-sites) of the Oct4 and Nanog gene promoters were highly methylated in hybrid cells, whereas the CpG-sites were unmethylated in the parental ES cells. Thus, the fibroblast genome dominated the ES genome in the diploid ES cell/tetraploid fibroblast hybrid cells. Immunofluorescent analysis of the pluripotent and fibroblast markers demonstrated that establishment of the fibroblast phenotype occurred shortly after fusion and that the fibroblast phenotype was further maintained in the hybrid cells. Fusion of karyoplasts and cytoplast derived from tetraploid fibroblasts with whole ES cells demonstrated that karyoplasts were able to establish the fibroblast phenotype of the reconstructed cells but not fibroblast cytoplasts. Thus, these data suggest that the dominance of parental genomes in hybrid cells of ES cell/somatic cell type depends on the ploidy of the somatic partner.

Allelic expression and DNA methylation profiles of promoters at the parental Oct4 and Nanog genes in Mus musculus ES cell/Mus caroli splenocyte hybrid cells

Expression of the parental Oct4 and Nanog alleles and DNA methylation of their promoters were studied in a set of Mus musculus embryonic stem (ES) cell/M. caroli splenocyte hybrid cells containing a variable ratio of parental chromosomes 6 and 17. The transcripts of the reactivated splenocyte Oct4 and Nanog genes were revealed in all hybrid cell clones positive for M. caroli chromosomes 6 and 17. We found that 11 CpG sites in the Oct4 promoter were heavily methylated in M. caroli splenocytes (>80%), whereas M. musculus ES cells were essentially unmethylated (<1%). Analysis of the methylation status of the Oct4 promoter in seven hybrid cell clones showed that the splenocyte-derived promoter sequence lost DNA methylation so that its methylation level was comparable with that of the ES cells. Additionally, no preferential de novo methylation was seen in the Oct4 promoters of M. musculus and M. caroli in teratomas developed from two independent hybrid clones. The upstream region of Nanog was heavily methylated in mouse embryonic fibroblasts (66%) and less methylated in M. caroli splenocytes (24%). The Nanog promoter region was completely unmethylated in M. musculus ES cells. We found that both parental alleles of the Nanog gene promoter were essentially unmethylated in five examined hybrid clones. Thus, we have demonstrated that (1) the Oct4 and Nanog genes of splenocytes are activated, and their promoters undergo demethylation in ES cell hybrids; (2) these events are independent of the number and ratio of parental chromosomes carrying these genes.

Genetic modification of Mammalian Genome at Chromosome level

The review is concerned with a progress in genetic modification of a mammalian genome in vitro and in vivo at chromosomal level. Recently three new approaches for the chromosome biotechnology have been developed: Using Cre/loxP-system a researcher is able to produce targeted rearrangements of whole chromosomes or their segments or particular genes within the genome, and therefore to modify the set, position and copy number of the endogenous elements of the genome. Mammalian artificial chromosomes (MACs) provide a possibility to introduce into genome relatively large segments of alien chromosome material, either artificially constructed or derived from the genome of different species. Using ES-somatic cell hybrids allows to transfer whole chromosomes or their fragments between different genomes within and between species. Advantages and limitations of these approaches are discussed.

Alternative dominance of the parental genomes in hybrid cells generated through the fusion of mouse embryonic stem cells with fibroblasts

For the first time, two types of hybrid cells with embryonic stem (ES) cell-like and fibroblast-like phenotypes were produced through the fusion of mouse ES cells with fibroblasts. Transcriptome analysis of 2,848 genes differentially expressed in the parental cells demonstrated that 34–43% of these genes are expressed in hybrid cells, consistent with their phenotypes; 25–29% of these genes display intermediate levels of expression, and 12–16% of these genes maintained expression at the parental cell level, inconsistent with the phenotype of the hybrid cell. Approximately 20% of the analyzed genes displayed unexpected expression patterns that differ from both parents. An unusual phenomenon was observed, namely, the illegitimate activation of Xist expression and the inactivation of one of two X-chromosomes in the near-tetraploid fibroblast-like hybrid cells, whereas both Xs were active before and after in vitro differentiation of the ES cell-like hybrid cells. These results and previous data obtained on heterokaryons suggest that the appearance of hybrid cells with a fibroblast-like phenotype reflects the reprogramming, rather than the induced differentiation, of the ES cell genome under the influence of a somatic partner.