Induced Pluripotent Stem Cells of Microtus levis x Microtus arvalis Vole Hybrids: Conditions Necessary for Their Generation and Self-Renewal.

Every year, the list of mammalian species for which cultures of pluripotent stem cells (PSCs) are generated increases. PSCs are a unique tool for extending the limits of experimental studies and modeling different biological processes. In this work, induced pluripotent stem cells (iPSCs) from the hybrids of common voles Microtus levis and Microtus arvalis, which are used as model objects to study genome organization on the molecular-genetic level and the mechanisms of X-chromosome inactivation, have been generated. Vole iPSCs were isolated and cultured in a medium containing cytokine LIF, basic fibroblast growth factor (bFGF), ascorbic acid, and fetal bovine serum. Undifferentiated state of vole iPSCs is maintained by activation of their endogenous pluripotency genes - Nanog, Oct4, Sox2, Sall4, and Esrrb. The cells were able to maintain undifferentiated state for at least 28 passages without change in their morphology and give rise to three germ layers (ectoderm, mesoderm and endoderm) upon differentiation.

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.

[Induced pluripotent stem cells].

Induced pluripotent stem cells (iPS) result from a reprogramming of somatic cells via transduction with viral vectors expressing the Oct4, Sox2, c-Myc, Klf4, Nanog, and Lin28 genes, which are essential for the establishment and maintenance of the pluripotent state. In properties, iPS are almost fully similar to embryonic stem cells (ESC). To date, iPS have been obtained from various differentiated cells of mice and humans. Along with ESC, iPS are highly promising for research and medicine.

[OCT4 and NANOG are the key genes in the system of pluripotency maintenance in mammalian cells].

Embryonic stem cells are able to give rise after differentiation to derivatives of three germinal layers (ectoderm, endoderm, and mesoderm) and to functional gametes. This property of cells is referred to as pluripotency. The pluripotent status of preimplantation embryo cells and embryonic stem cells is maintained by a complicated system of molecular signaling pathways and transcription factors. The key regulators in this system are the transcription factors OCT4 and NANOG. The role and place of these factors in the pluripotency-sustaining system and their interaction with other factors are considered in the review. Data are presented on the structure, chromosomal location, expression, and regulation of the Oct4 and Nanog genes in mammals.

[Extraembryonic endoderm stem cell lines from common voles of the genus Microtus].

Twenty-eight independent extraembryonic endoderm (XEN) stem cell lines have been obtained from morula and blastocyst cells of common voles. Most cell lines form very few cell-cell contacts when growing and morphologically correspond to the XEN that were earlier described in mice. In addition, XEN cell lines with atypical morphology forming colonies have been obtained for the first time. Both types of XEN lines rapidly proliferate, retain their morphology and karyotype during more than 25 passages in cell culture, and express genes characteristic of XEN. One of two X chromosomes in XEN lines with karyotype XX has been shown to be inactive and associated with the Xist gene transcript. It has been demonstrated that the paternal X chromosome is inactive.

[Isolation of ES-like lines of common voles of the genus Microtus from blastocysts and germ cells and as a result of the fusion of somatic cells with mouse embryonic stem cells]. / Poluchenie ES-podobnykh linii obyknovennykh polevok roda Microtus iz blastotsist, germinal'nykh kletok i ot sliianiia somaticheskikh kletok s émbryonal'nymi stvolovymi kletkami myshi.

Three and four independent cell lines with limited pluripotency were obtained from the inner cell mass cells of blastocysts and primordial germ cells of common voles, respectively. The results of cytogenetic analysis suggest that all these lines originated from the embryos of F1 Microtus rossiaemeridionalis x M. arvalis males and had a great number of near-triploid cells already during the early passages. The cells of these lines, like those of the inner cell mass, were characterized by the alkaline phosphatase activity. Nine independent cell lines were obtained as a result of hybridization of the mouse embryonic stem cells and vole splenocytes: eight lines and one line from hybridization with the M. kirgisorum and M. rossiaemeridionalis splenocytes, respectively. The cells of these lines expressed some properties of embryonic stem lines had a chromosome complement similar to the sum of two initial diploid sets of the mouse and vole.

Reorganization of the X chromosome in voles of the genus Microtus.

Comparative chromosomal analysis is a powerful tool in the investigation of the mechanisms of chromosomal evolution. The accuracy of the analysis depends on the availability of region-specific markers to follow the fate of the particular chromosomal region through the evolution of species. We have assigned 12 unique sequences to the euchromatic part of the vole X chromosome, which serve as reliable markers of chromosomal segments. Together with region-specific libraries and GTG banding, these markers allow us to delineate the homologous regions of the X chromosomes in five species of the genus Microtus. We found that X chromosomes of these species differ by numerous rearrangements and all rearrangements are clustered at specific breakpoints. Moreover, these breakpoints were found to colocalise with repetitive and/or duplicated DNA sequences. We suggest that clusters of repeated and/or duplicated DNA sequences have played a crucial role in the formation of rearrangement hot spots during evolution of the X chromosome in the subgenus Microtus.

[Genomic organization and chromosomal localization of a new repeat MS7, specific for heterochromatin of sex chromosomes in Microtus species voles of the arvalis group]. / Genomnaia organizatsiia i khoromosomnaia lokalizatsiia novogo povtora MS7, spetsifichnogo dlia geterokhromatina polovykh khromosom u polevok roda Microtus gruppy arvalis.

The tandemly arranged MS4 repeat with monomeric units of 4.1 kb is species-specifically distributed in heterochromatin of sex chromosomes of four common vole species of genus Microtus, group arvalis [1, 2]. In this work, we studied the genomic organization of the MS4 homolog in euchromatin of the X chromosome of M. arvalis. It has been shown by analyzing the phage genomic clones that one MS4 copy makes a part of a monomeric unit exceeding 8.5 kb that also includes a new MS7 repeat and, possibly, LINE fragments. MS7 is located together with MS4 in heterochromatin of common vole sex chromosomes, but in a substantially lesser amount. Probably, as a result of an evolutionary transition of an original repeat from euchromatin of the X chromosome to heterochromatin of the Y chromosome, MS4 underwent multiple amplification, and MS7 spread throughout heterochromatin, being surrounded by the MS4 tandem arrays.

Comparative chromosome and mitochondrial DNA analyses and phylogenetic relationships within common voles (Microtus, Arvicolidae).

The four species of common voles within the genus Microtus--M. kirgisorum, M. transcaspicus, M. arvalis, and M. rossiaemeridionalis--are so closely related that neither morphological features nor paleontological evidence allow clarification of their phylogeny. Analysis of vole karyotypes and mitochondrial DNA sequences, therefore, is essential for determining their phylogenetic relationships. A comparison of high resolution GTG-banding patterns allows us to ascertain the similarity between the karyotypes of these species, revealing that they are composed of rearrangements of the same chromosomal elements. Based on this analysis, we propose possible routes of chromosomal divergence involved in speciation within this group of voles and construct a phylogenetic tree of their karyotypes. We suggest that two different karyotypic variants existed during the course of vole evolution--one resulting in M. rossiaemeridionalis and M. transcaspicus, the other, M. kirgisorum and M. arvalis. As an alternative approach FITCH and KITSCH computer programs were used to construct a phylogenetic tree of vole molecular evolution based on a pairwise comparison of mitochondrial cytochrome b sequences and the divergence time of the species was determined. The correlation between the trees constructed using karyologic and molecular approaches is discussed in the context of other available data.

[High-resolution GTG-banding and nucleolar organizer regions of chromosomes of two vole species: Microtus rossiaemeridonionalis and M. transcaspicus (Rodentia, Arvicolidae)]. / GTG-okraska vysokogo razresheniia i iadryshkoobrazuiushchie raiony khromosom dvukh vidov obyknovennykh polevok: Microtus rossiaemeridionalis i M. transcaspicus (Rodentia, Arvicolidae).

With the use of the GTG-banding of prometaphase chromosomes, 503 and 402 segments were revealed in haploid chromosome sets of voles Microtus rossiaemeridionalis and M. transcaspicus, respectively. Based on a detailed study of chromosomes at different condensation levels, idiograms of M. rossiaemeridionalis and M. transcaspicus chromosomes were constructed. Sequential Ag-staining and GTG-banding allowed nucleolar organizer regions (NORs) to be localized in 16 and 11 chromosome pairs of M. rossiaemeridionalis and M. transcaspicus, respectively.

Comparative mapping of X chromosomes in vole species of the genus Microtus.

Comparative mapping of X-linked genes has progressed rapidly since Ohno's prediction that genes on the X chromosome should be conserved as a syntenic group in all mammals. Although several conserved blocks of homology between human and mouse have been discovered, rearrangements within the X chromosome have also been characterized. More recently, some exceptions to Ohno's law have been reported. We have used fluorescence in situ hybridization (FISH) to map five genes, Gla, G6pd, Hprt, Pgk1 and Xist, to two of the largest conserved segments of X material in five members of the genus Microtus (grey vole) and show that vole X chromosomes demonstrate greater homology to human than to mouse. Cytogenetic analysis indicates a relatively high frequency of rearrangement during vole evolution, although certain blocks of homology appear to be highly conserved in all species studied to date. On this basis we were able to predict the probable location of the rat X inactivation centre (Xic) based solely on high-resolution G-banding. Our prediction was then confirmed by mapping the rat Xist gene by FISH. The possible significance of conserving long-range chromosome structure in the vicinity of the Xic is discussed with respect to the mechanism of X inactivation.

High-resolution G-banding of chromosones in the common vole Microtus arvalis (Rodentia, Arvicolidae)

We have applied the G-banding technique to early metaphase chromosomes of the vole Microtus arvalis, form 'arvalis'. Fifty metaphase spreads were analysed from spleen and primary fibroblast cell cultures from six voles. We were able to distinguish 435 bands in the haploid set. An idiogram was constructed from the chromosomes at various stages of condensation.

[High resolution GTG banding and nucleolus organizer regions of chromosomes from the vole Microtus kirgisorum]. / GTG-okraska vysokogo razresheniia i iadryshkoobrazuiushchie raiony khromosom polevki Microtus kirgisorum.

The use of GTG-banding of chromosomes in combination with the pipette method of chromosome preparation at the early metaphase made it possible to distinguish about 520 segments in the haploid chromosome set of vole Microtus kirgisorum. The ideogram of M. kirgisorum chromosomes was obtained on the basis of detailed investigation of chromosomes at different condensation levels. Data of the localization and the number of nucleolus-organizer regions are given.

High-resolution G-banding of chromosomes in Microtus subarvalis (Rodentia, Arvicolidae)

Karyotypes of six females and four males of the East European vole (Microtus subarvalis) were prepared from spleen cell cultures. G-banding of early metaphase chromosomes has allowed us to distinguish 488 bands in the haploid set of chromosomes of M. subarvalis. Based on the detailed study of chromosomes at various levels of condensation, an idiogram has been built up for the chromosomes of the East European vole.

High resolution G-banding of chromosomes in Microtus kirgisorum (Muridae, Rodentia).

The use of G-banding in conjunction with the pipette method of chromosome preparation has allowed 524 band resolution in chromosomes of Microtus kirgisorum. After detailed analysis of M. kirgisorum chromosomes at different stages of condensation, an ideogram has been prepared.