Three-dimensional genome architecture persists in a 52,000-year-old woolly mammoth skin sample.

Analyses of ancient DNA typically involve sequencing the surviving short oligonucleotides and aligning to genome assemblies from related, modern species. Here, we report that skin from a female woolly mammoth (†Mammuthus primigenius) that died 52,000 years ago retained its ancient genome architecture. We use PaleoHi-C to map chromatin contacts and assemble its genome, yielding 28 chromosome-length scaffolds. Chromosome territories, compartments, loops, Barr bodies, and inactive X chromosome (Xi) superdomains persist. The active and inactive genome compartments in mammoth skin more closely resemble Asian elephant skin than other elephant tissues. Our analyses uncover new biology. Differences in compartmentalization reveal genes whose transcription was potentially altered in mammoths vs. elephants. Mammoth Xi has a tetradic architecture, not bipartite like human and mouse. We hypothesize that, shortly after this mammoth's death, the sample spontaneously freeze-dried in the Siberian cold, leading to a glass transition that preserved subfossils of ancient chromosomes at nanometer scale.

Effects of forskolin on cryopreservation and embryo development in the domestic cat.

One of the approaches to improve cryotolerance in lipid-rich embryos is to modify their lipidome in vitro. This work is aimed to study the effects of forskolin exposure on the in vitro embryo development of the domestic cat and to evaluate how the change in lipid content affects the cryopreservation results. In vitro-derived embryos were cultured with 10 µM forskolin from the 2-cell stage for 24 h or 96/168 h to the morula/blastocyst stage. Some of the embryos treated with forskolin for 24 h were cryopreserved with slow freezing, the other ones were used to characterize their developmental rates and the amount of intracellular lipids. The in vitro exposure to forskolin had a positive effect on the embryo development, as more embryos developed to the morula stage in the forskolin-treated group (92.9%) compared to the controls (64.7%) after 120 h of in vitro culture (IVC). Nile Red staining revealed a reduced amount of intracellular lipids in the forskolin-treated embryos. The percentage of embryos developed to the morula stage was lower in the frozen-thawed embryos not treated with forskolin (54.5%), but not in the frozen-thawed forskolin-treated group (63.6%) as compared to non-frozen controls (80.8%). Thus, the exposure of embryos to forskolin in vitro reduced the level of intracellular lipids and affected embryo development before and after cryopreservation.

Chromosome Asynapsis Is the Main Cause of Male Sterility in the Interspecies Hybrids of East Asian Voles (Alexandromys, Rodentia, Arvicolinae).

Closely related mammalian species often have differences in chromosome number and morphology, but there is still a debate about how these differences relate to reproductive isolation. To study the role of chromosome rearrangements in speciation, we used the gray voles in the Alexandromys genus as a model. These voles have a high level of chromosome polymorphism and substantial karyotypic divergence. We investigated testis histology and meiotic chromosome behavior in the captive-bred colonies of Alexandromys maximowiczii, Alexandromys mujanensis, two chromosome races of Alexandromys evoronensis, and their interracial and interspecies hybrids, to explore the relationship between karyotypic differences and male hybrid sterility. We found that the seminiferous tubules of the males of the parental species and the interracial hybrids, which were simple heterozygotes for one or more chromosome rearrangements, contained germ cells at all stages of spermatogenesis, indicating their potential fertility. Their meiotic cells displayed orderly chromosome synapsis and recombination. In contrast, all interspecies male hybrids, which were complex heterozygotes for a series of chromosome rearrangements, showed signs of complete sterility. Their spermatogenesis was mainly arrested at the zygotene- or pachytene-like stages due to the formation of complex multivalent chains, which caused extended chromosome asynapsis. The asynapsis led to the silencing of unsynapsed chromatin. We suggest that chromosome asynapsis is the main cause of meiotic arrest and male sterility in the interspecies hybrids of East Asian voles.

Mitomycin-Treated Endothelial and Smooth Muscle Cells Suitable for Safe Tissue Engineering Approaches.

In our previous study, we showed that discarded cardiac tissue from the right atrial appendage and right ventricular myocardium is an available source of functional endothelial and smooth muscle cells for regenerative medicine and tissue engineering. In the study, we aimed to find out what benefits are given by vascular cells from cardiac explants used for seeding on vascular patches engrafted to repair vascular defects in vivo. Additionally, to make the application of these cells safer in regenerative medicine we tested an in vitro approach that arrested mitotic division to avoid the potential tumorigenic effect of dividing cells. A tissue-engineered construction in the form of a patch based on a polycaprolactone-gelatin scaffold and seeded with endothelial and smooth muscle cells was implanted into the abdominal aorta of immunodeficient SCID mice. Aortic patency was assessed using ultrasound, MRI, immunohistochemical and histological staining. Endothelial and smooth muscle cells were treated with mitomycin C at a therapeutic concentration of 10 µg/ml for 2 h with subsequent analysis of cell proliferation and function. The absence of the tumorigenic effect of mitomycin C-treated cells, as well as their angiogenic potential, was examined by injecting them into immunodeficient mice. Cell-containing patches engrafted in the abdominal aorta of immunodeficient mice form the vessel wall loaded with the appropriate cells and extracellular matrix, and do not interfere with normal patency. Endothelial and smooth muscle cells treated with mitomycin C show no tumorigenic effect in the SCID immunodeficient mouse model. During in vitro experiments, we have shown that treatment with mitomycin C does not lead to a decrease in cell viability. Despite the absence of proliferation, mitomycin C-treated vascular cells retain specific cell markers, produce specific extracellular matrix, and demonstrate the ability to stimulate angiogenesis in vivo. We pioneered an approach to arresting cell division with mitomycin C in endothelial and smooth muscle cells from cardiac explant, which prevents the risk of malignancy from dividing cells in vascular surgery. We believe that this approach to the fabrication of tissue-engineered constructs based on mitotically inactivated cells from waste postoperative material may be valuable to bring closer the development of safe cell products for regenerative medicine.

Reproductive Isolation Between Taxonomically Controversial Forms of the Gray Voles (Microtus, Rodentia; Arvicolinae): Cytological Mechanisms and Taxonomical Implications.

The formation of hybrid sterility is an important stage of speciation. The voles of the genus Microtus, which is the most speciose genus of rodents, provide a good model for studying the cytological mechanisms of hybrid sterility. The voles of the "mystacinus" group of the subgenus Microtus (2n = 54) comprising several recently diverged forms with unclear taxonomic status are especially interesting. To resolve the taxonomic status of Microtus mystacinus and Microtus kermanensis, we crossed both with Microtus rossiaemeridionalis, and M. kermanensis alone with Microtus arvalis "obscurus" and M. transcaspicus and examined the reproductive performance of their F1 hybrids. All interspecies male hybrids were sterile. Female M. kermanensis × M. arvalis and M. kermanensis × M. transcaspicus hybrids were sterile as well. Therefore, M. mystacinus, M. kermanensis, and M. rossiaemeridionalis could be considered valid species. To gain an insight into the cytological mechanisms of male hybrid sterility, we carried out a histological analysis of spermatogenesis and a cytological analysis of chromosome synapsis, recombination, and epigenetic chromatin modifications in the germ cells of the hybrids using immunolocalization of key meiotic proteins. The hybrids showed wide variation in the onset of spermatogenesis arrest stage, from mature (although abnormal) spermatozoa to spermatogonia only. Chromosome asynapsis was apparently the main cause of meiotic arrest. The degree of asynapsis varied widely across cells, individuals, and the crosses-from partial asynapsis of several small bivalents to complete asynapsis of all chromosomes. The asynapsis was accompanied by a delayed repair of DNA double-strand breaks marked by RAD51 antibodies and silencing of unpaired chromatin marked by γH2A.X antibodies. Overall, the severity of disturbances in spermatogenesis in general and in chromosome synapsis in particular increased in the hybrids with an increase in the phylogenetic distance between their parental species.

Homogeneously Staining Regions (HSR) in Chromosome 1 of the House Mouse: Synapsis and Recombination at Meiosis.

Amplified sequences constitute a large part of mammalian genomes. A chromosome 1 containing 2 large (up to 50 Mb) homogeneously staining regions (HSRs) separated by a small inverted euchromatic region is present in many natural populations of the house mouse (Mus musculus musculus). The HSRs are composed of a long-range repeat cluster, Sp100-rs, with a repeat length of 100 kb. In order to understand the organization and function of HSRs in meiotic chromosomes, we examined synapsis and recombination in male mice hetero- and homozygous for the HSR-carrying chromosome using FISH with an HSR-specific DNA probe and immunolocalization of the key meiotic proteins. In all homozygous and heterozygous pachytene nuclei, we observed fully synapsed linear homomorphic bivalents 1 marked by the HSR FISH probe. The synaptic adjustment in the heterozygotes was bilateral: the HSR-carrying homolog was shortened and the wild-type homolog was elongated. The adjustment was reversible: desynapsis at diplotene was accompanied by elongation of the HSRs. Immunolocalization of H3K9me2/3 indicated that the HSRs in the meiotic chromosome retained the epigenetic modification typical for C-heterochromatin in somatic cells. MLH1 foci, marking mature recombination nodules, were detected in the proximal HSR band in heterozygotes and in both HSR bands of homozygotes. Unequal crossing over within the long-range repeat cluster can cause variation in size of the HSRs, which has been detected in the natural populations of the house mouse.

Generation of GABAergic striatal neurons by a novel iPSC differentiation protocol enabling scalability and cryopreservation of progenitor cells.

Cell models are promising tools for studying hereditary human neurodegenerative diseases. Neuronal derivatives of pluripotent stem cells provide the opportunity to investigate different stages of the neurodegeneration process. Therefore, easy and large-scale production of relevant cell types is a crucial barrier to overcome. In this work, we present an alternative protocol for iPSC differentiation into GABAergic medium spiny neurons (MSNs). The first stage involved dual-SMAD signalling inhibition through treatment with SB431542 and LDN193189, which results in the generation of neuroectodermal cells. Moreover, we used bFGF as a neuronal survival factor and dorsomorphin to inhibit BMP signalling. The combined treatment of dorsomorphin and SB431542 significantly enhanced neuronal induction, which was confirmed by the increased expression of the telencephalic-specific markers SOX1 and OTX2 as well as the forebrain marker PAX6. The next stage involved the derivation of actively proliferating MSN progenitor cells. An important feature of our protocol at this stage is the ability to perform prolonged cultivation of precursor cells at a high density without losing phenotypic properties. Moreover, the protocol enables multiple expansion steps (> 180 days cultivation) and cryopreservation of MSN progenitors. Therefore, this method allows quick production of a large number of neurons that are relevant for basic research, large-scale drug screening, and toxicological studies.

Germline-restricted chromosome (GRC) in the sand martin and the pale martin (Hirundinidae, Aves): synapsis, recombination and copy number variation.

All songbirds studied to date have an additional Germline Restricted Chromosome (GRC), which is not present in somatic cells. GRCs show a wide variation in genetic content and little homology between species. To check how this divergence affected the meiotic behavior of the GRC, we examined synapsis, recombination and copy number variation for GRCs in the closely related sand and pale martins (Riparia riparia and R. diluta) in comparison with distantly related estrildid finches. Using immunolocalization of meiotic proteins and FISH with GRC-specific DNA probes, we found a striking similarity in the meiotic behavior of GRCs between martins and estrildid finches despite the millions of years of independent evolution. GRCs are usually present in two copies in female and in one copy in male pachytene cells. However, we detected polymorphism in female and mosaicism in male martins for the number of GRCs. In martin and zebra finch females, two GRCs synapse along their whole length, but recombine predominately at their ends. We suggest that the shared features of the meiotic behavior of GRCs have been supported by natural selection in favor of a preferential segregation of GRCs to the eggs.

Effects of slow freezing and vitrification on embryo development in domestic cat.

Cryopreservation of gametes and embryos is used to maintain genetic diversity of domestic and wild felids. However, felid oocytes and preimplantation embryos contain large amount of intracellular lipids, which affect their cryosensitivity. The objective was to compare the effects of slow freezing and vitrification and to study lipid phase transition (LPT) during cooling in cat embryos. In vitro-derived embryos were cultured 48 hr up to 4-8 cell stage, thereafter were either slow frozen or vitrified. Propylene glycol (PG) alone was used as a cryoprotective agent (CPA) for slow freezing, and a mixture of PG and dimethyl sulfoxide (DMSO) were used as CPAs for vitrification. After thawing/warming, embryos were in vitro cultured additionally for 72 hr. The total time of in vitro culture was 120 hr for all the groups including non-frozen controls. Effects of both cryopreservation procedures on the subsequent embryo development and nuclear fragmentation rate in embryonic cells were compared. There was no significant differences among the percentages of embryos achieved morula and early blastocyst stage in frozen-thawed group (36.4% and 20.0%), in vitrified-warmed group (34.3% and 28.6%) and in controls (55.6% and 25.9%). Cell numbers as well as nuclear fragmentation rate did not differ in these three groups. Average lipid phase transition (LPT) temperature (T*) was found to be relatively low (-2.2 ± 1.3°C) for the domestic cat embryos. It is supposed that the low LPT of LDs may provide a good background for successful application of slow freezing to domestic cat embryos. Generally, our study indicates that slow freezing and vitrification are both applicable for domestic cat embryo cryopreservation.

Germline-restricted chromosome (GRC) is widespread among songbirds.

An unusual supernumerary chromosome has been reported for two related avian species, the zebra and Bengalese finches. This large, germline-restricted chromosome (GRC) is eliminated from somatic cells and spermatids and transmitted via oocytes only. Its origin, distribution among avian lineages, and function were mostly unknown so far. Using immunolocalization of key meiotic proteins, we found that GRCs of varying size and genetic content are present in all 16 songbird species investigated and absent from germline genomes of all eight examined bird species from other avian orders. Results of fluorescent in situ hybridization of microdissected GRC probes and their sequencing indicate that GRCs show little homology between songbird species and contain a variety of repetitive elements and unique sequences with paralogs in the somatic genome. Our data suggest that the GRC evolved in the common ancestor of all songbirds and underwent significant changes in the extant descendant lineages.

Targeted genomic integration of EGFP under tubulin beta 3 class III promoter and mEos2 under tryptophan hydroxylase 2 promoter does not produce sufficient levels of reporter gene expression.

Neuronal tracing is a modern technology that is based on the expression of fluorescent proteins under the control of cell type-specific promoters. However, random genomic integration of the reporter construct often leads to incorrect spatial and temporal expression of the marker protein. Targeted integration (or knock-in) of the reporter coding sequence is supposed to provide better expression control by exploiting endogenous regulatory elements. Here we describe the generation of two fluorescent reporter systems: enhanced green fluorescent protein (EGFP) under pan-neural marker class III ß-tubulin (Tubb3) promoter and mEos2 under serotonergic neuron-specific tryptophan hydroxylase 2 (Tph2) promoter. Differentiation of Tubb3-EGFP embryonic stem (ES) cells into neurons revealed that though Tubb3-positive cells express EGFP, its expression level is not sufficient for the neuronal tracing by routine fluorescent microscopy. Similarly, the expression levels of mEos2-TPH2 in differentiated ES cells was very low and could be detected only on messenger RNA level using polymerase chain reaction-based methods. Our data shows that the use of endogenous regulatory elements to control transgene expression is not always beneficial compared with the random genomic integration.

Sperm cryopreservation in the Far-Eastern wildcat (Prionailurus bengalensis euptilurus).

The Far-Eastern wildcat (Prionailurus bengalensis euptilurus) is a rare and poorly investigated nondomestic felid species. An attempt of freezing and cryopreserving Far-Eastern wildcat spermatozoa in CaniPlus Freeze (CPF) medium is reported. Sperm was collected by electroejaculation from five adult Far-Eastern wildcat captive-born males. Epididymal spermatozoa from five adult randomly bred domestic cat males were used as a reference. The viability of frozen-thawed spermatozoa evaluated by double staining with SYBR Green I and PI followed by the subsequent confocal laser scanning microscopy (CLSM) was 38.2% ± 3.0% for the domestic cat and 38.0% ± 10.2% for the Far-Eastern wildcat. The motility of frozen-thawed spermatozoa was 30.8% ± 9.8% for the domestic cat and 33.7% ± 15.1% for the Far-Eastern wildcat. Sperm morphology was assessed by light microscopy. The total percentage of normal spermatozoa after freezing and thawing was 51.9 ± 5.9 for the domestic cat and 55.0% ± 6.4% for the Far-Eastern wildcat. Defects of flagella were the most frequently observed abnormalities in both species (32.2% ± 4.8% and 30.8% ± 4.4% of all reported anomalies for the domestic cat and Far-Eastern wildcat, respectively). Domestic cat epididymal and Far-Eastern ejaculatory spermatozoa fertilized in vitro-matured oocytes of the domestic cat (30.0% ± 5.5% and 35.5% ± 15.0%, respectively). Taken together, these results suggest that the freezing of Far-Eastern wildcat spermatozoa with CPF medium is a suitable method for Felidae cryopreservation.

Chromosome Synapsis and Recombination in Male-Sterile and Female-Fertile Interspecies Hybrids of the Dwarf Hamsters (Phodopus, Cricetidae).

Hybrid sterility is an important step in the speciation process. Hybrids between dwarf hamsters Phodopus sungorus and P.campbelli provide a good model for studies in cytological and genetic mechanisms of hybrid sterility. Previous studies in hybrids detected multiple abnormalities of spermatogenesis and a high frequency of dissociation between the X and Y chromosomes at the meiotic prophase. In this study, we found that the autosomes of the hybrid males and females underwent paring and recombination as normally as their parental forms did. The male hybrids showed a significantly higher frequency of asynapsis and recombination failure between the heterochromatic arms of the X and Y chromosomes than the males of the parental species. Female hybrids as well as the females of the parental species demonstrated a high incidence of centromere misalignment at the XX bivalent and partial asynapsis of the ends of its heterochromatic arms. In all three karyotypes, recombination was completely suppressed in the heterochromatic arm of the X chromosome, where the pseudoautosomal region is located. We propose that this recombination pattern speeds up divergence of the X- and Y-linked pseudoautosomal regions between the parental species and results in their incompatibility in the male hybrids.

Mitochondria structural reorganization during mouse embryonic stem cell derivation.

Mouse embryonic stem (ES) cells are widely used in developmental biology and transgenic research. Despite numerous studies, ultrastructural reorganization of inner cell mass (ICM) cells during in vitro culture has not yet been described in detail. Here, we for the first time performed comparative morphological and morphometric analyses of three ES cell lines during their derivation in vitro. We compared morphological characteristics of blastocyst ICM cells at 3.5 and 4.5 days post coitum on feeder cells (day 6, passage 0) with those of ES cells at different passages (day 19, passage 2; day 25, passage 4; and passage 15). At passage 0, there were 23-36% of ES-like cells with various values of the medium cross-sectional area and nucleocytoplasmic parameters, 55% of fibroblast-like (probably trophoblast derivatives), and ~ 19% of dying cells. ES-like cells at passage 0 contained autolysosomes and enlarged mitochondria with reduced numerical density per cell. There were three types of mitochondria that differed in matrix density and cristae width. For the first time, we revealed cells that had two and sometimes three morphologically distinct mitochondria types in the cytoplasm. At passage 2, there were mostly ES cells with a high nucleocytoplasmic ratio and a cytoplasm depleted of organelles. At passage 4, ES cell morphology and morphometric parameters were mostly stable with little heterogeneity. According to our data, cellular structures of ICM cells undergo destabilization during derivation of an ES cell line with subsequent reorganization into the structures typical for ES cells. On the basis of ultrastructural analysis of mitochondria, we believe that the functional activity of these organelles changes during early stages of ES cell formation from the ICM.

Characteristics and fertility of domestic cat epididymal spermatozoa cryopreserved with two different freezing media.

The study represents a comparison of cryopreservation of domestic cat epididymal spermatozoa with two commercially available freezing media: CaniPlus Freeze (CPF) and SpermFreeze (SF). The viability of nonfrozen spermatozoa evaluated by the VitalScreen test was 68.7 ±â€¯3.0%. These figures were lower for the frozen-thawed spermatozoa: 51.2 ±â€¯6.3% for CPF group and 54.4 ±â€¯3.1% for SF group. The motility of nonfrozen spermatozoa was 57.2 ±â€¯4.5%. These figures were reduced in both frozen-thawed groups; however, there was no significant difference in these parameters between CPF (30.8 ±â€¯7.1%) and SF (27.4 ±â€¯8.1%) groups. The percentage of nonprogressively moving motile spermatozoa after freezing-thawing was decreased in both frozen-thawed groups (23.5 ±â€¯5.9 and 12.0 ±â€¯2.4 for CPF and SF frozen correspondingly) as compared with nonfrozen controls (42.1 ±â€¯4.1%). Morphology of spermatozoa was assessed by light microscopy. The mean percentages of normal spermatozoa were 28.5 ±â€¯4.1% for nonfrozen group, 26.0 ±â€¯2.3% for CPF frozen group, and 23.9 ±â€¯1.9% for SF frozen group. The most frequent anomalies in all the three groups were flagella and combined defects. In vitro fertilization (IVF) of domestic cat oocytes with nonfrozen and frozen-thawed spermatozoa produced developing embryos. The percentage of in-vitro-derived embryos was 43.6% after using nonfrozen spermatozoa. Frozen-thawed spermatozoa developed at a similar rate (44.0%) after using SF. However, the rate of embryo development was lower (20.1%) when CPF was used. The in-vitro-derived embryos in the nonfrozen group consisted of 46.9 ±â€¯2.5 cells after 5-day culturing. After cryopreservation with SF and CPF the cell numbers per embryo were 39.9 ±â€¯2.7 and 31.8 ±â€¯3.4 correspondingly. In CPF group these numbers were lower than in nonfrozen controls. Cryopreservation of spermatozoa with either of two freezing media led to a decrease in post-thaw viability and motility of spermatozoa but did not affect the rate or spectrum of their morphological anomalies. The use of CPF, but not SF led to a decrease of sperm fertilizing abilities.

Hemozoin is a product of heme detoxification in the gut of the most medically important species of the family Opisthorchiidae.

Many species of trematodes such as Schistosoma spp., Fasciola hepatica and Echinostoma trivolvis are blood-feeding parasites. Nevertheless, there is no consensus on the feeding habits of the family Opisthorchiidae (Opisthorchis felineus, Opisthorchis viverrini and Clonorchis sinensis). Previously, histological studies of O. felineus and C. sinensis revealed some dark stained material in their gut lumen. In this study we conducted a comprehensive analysis of the gut contents of three members of the family Opisthorchiidae (O. felineus, O. viverrini and C. sinensis). Using transmission electron microscopy, we demonstrated for the first known time the presence of disintegrating blood cells in the gut of O. felineus as well as electron-dense crystals in the gut of O. felineus and C. sinensis. Electron energy loss spectroscopy revealed iron atoms in these crystals, and mass spectrometry of the purified pigment demonstrated the presence of heme. Fourier-transform infrared spectroscopy identified the signature peaks of the common iron-carboxylate bond characteristic in crystals isolated from O. felineus and C. sinensis. Scanning electron microscopy showed layered ovoid crystals of various sizes from 50 nm to 2 µm. Morphological, chemical and paramagnetic properties of these crystals were similar to those of hemozoin from Schistosoma mansoni. Crystal formation occurs on the surface of lipid droplets in O. felineus and C. sinensis guts. Our results suggest that the diet of O. felineus and C. sinensis includes blood. Detoxification of the free heme produced during the digestion proceeds via formation of insoluble crystals that contain iron and heme dimers, i.e. crystals of hemozoin. Furthermore, we believe that biocrystallisation of hemozoin takes place on the surface of the lipid droplets, similar to S. mansoni. Hemozoin was not detected in the closely related species O. viverrini.

Comparison of American mink embryonic stem and induced pluripotent stem cell transcriptomes.

BACKGROUND: Recently fibroblasts of many mammalian species have been reprogrammed to pluripotent state using overexpression of several transcription factors. This technology allows production of induced pluripotent stem (iPS) cells with properties similar to embryonic stem (ES) cells. The completeness of reprogramming process is well studied in such species as mouse and human but there is not enough data on other species. We produced American mink (Neovison vison) ES and iPS cells and compared these cells using transcriptome analysis. RESULTS: We report the generation of 10 mink ES and 22 iPS cell lines. The majority of the analyzed cell lines had normal diploid chromosome number. The only ES cell line with XX chromosome set had both X-chromosomes in active state that is characteristic of pluripotent cells. The pluripotency of ES and iPS cell lines was confirmed by formation of teratomas with cell types representing all three germ layers. Transcriptome analysis of mink embryonic fibroblasts (EF), two ES and two iPS cell lines allowed us to identify 11831 assembled contigs which were annotated. These led to a number of 6891 unique genes. Of these 3201 were differentially expressed between mink EF and ES cells. We analyzed expression levels of these genes in iPS cell lines. This allowed us to show that 80% of genes were correctly reprogrammed in iPS cells, whereas approximately 6% had an intermediate expression pattern, about 7% were not reprogrammed and about 5% had a "novel" expression pattern. We observed expression of pluripotency marker genes such as Oct4, Sox2 and Rex1 in ES and iPS cell lines with notable exception of Nanog. CONCLUSIONS: We had produced and characterized American mink ES and iPS cells. These cells were pluripotent by a number of criteria and iPS cells exhibited effective reprogramming. Interestingly, we had showed lack of Nanog expression and consider it as a species-specific feature.

Transcriptome Characteristics and X-Chromosome Inactivation Status in Cultured Rat Pluripotent Stem Cells.

Rat pluripotent stem cells, embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs) as mouse and human ones have a great potential for studying mammalian early development, disease modeling, and evaluation of regenerative medicine approaches. However, data on pluripotency realization and self-renewal maintenance in rat cells are still very limited, and differentiation protocols of rat ESCs (rESCs) and iPSCs to study development and obtain specific cell types for biomedical applications are poorly developed. In this study, the RNA-Seq technique was first used for detailed transcriptome characterization in rat pluripotent cells. The rESC and iPSC transcriptomes demonstrated a high similarity and were significantly different from those in differentiated cells. Additionally, we have shown that reprogramming of rat somatic cells to a pluripotent state was accompanied by X-chromosome reactivation. There were two active X chromosomes in XX rESCs and iPSCs, which is one of the key attributes of the pluripotent state. Differentiation of both rESCs and iPSCs led to X-chromosome inactivation (XCI). The dynamics of XCI in differentiating rat cells was very similar to that in mice. Two types of facultative heterochromatin described in various mammalian species were revealed on the rat inactive X chromosome. To explore XCI dynamics, we established a new monolayer differentiation protocol for rESCs and iPSCs that may be applied to study different biological processes and optimized for directed derivation of specific cell types.

Generation of mouse chimeras with high contribution of tetraploid embryonic stem cells and embryonic stem cell-fibroblast hybrid cells.

The in vitro long-term cultivation of embryonic stem (ES) cells derived from pre-implantation embryos offers the unique possibility of combining ES cells with pre-implantation embryos to generate chimeras, thus facilitating the creation of a bridge between in vitro and in vivo investigations. Genomic manipulation using ES cells and homologous recombination is one of the most outstanding scientific achievements, resulting in the generation of animals with desirable genome modifications. As such, the generation of ES cells with different ploidy via cell fusion also deserves much attention because this approach allows for the production of chimeras that contain somatic cells with various ploidy. Therefore, this is a powerful tool that can be used to study the role of polyploidy in the normal development of mammals.

Embryo cryopreservation and in vitro culture of preimplantation embryos in Campbell's hamster (Phodopus campbelli).

The aims of this study were to compare different protocols of Campbell's hamster (Phodopus campbelli) embryos freezing-thawing and to explore the possibilities of their in vitro culture. First, the embryos were flushed from the reproductive ducts 2 days post coitum at the two-cell stage and cultured in rat one-cell embryo culture medium (R1ECM) for 48 hours. Most (86.7%) of the two-cell embryos developed to blastocysts in R1ECM. Second, the embryos at the two- to eight-cell stages were flushed on the third day post coitum. The eight-cell embryos were frozen in 0.25 mL straws according to standard procedures of slow cooling. Ethylene glycol (EG) was used either as a single cryoprotectant or in a mixture with sucrose. The survival of frozen-thawed embryos was assessed by double staining with fluorescein diacetate and propidium iodide. The use of EG as a single cryoprotectant resulted in fewer alive embryos when compared with control (fresh embryos), but combined use of EG and sucrose improved the survival rate after thawing. Furthermore, granulocyte-macrophage colony-stimulating factor rat (2 ng/mL) improved the rate of the hamster frozen-thawed embryo development in vitro by increasing the final cell number and alleviating nuclear fragmentation. Our data show the first attempt in freezing and thawing Campbell's hamster embryos and report the possibility of successful in vitro culture for this species in R1ECM supplemented with granulocyte-macrophage colony-stimulating factor.