Differential expression and methylation patterns of NFATC1, NADSYN1 and JAK3 gene in equine chondrocytes expanded in monolayer culture.

Ex vivo expansion of chondrocytes in monolayer (ML) culture for therapeutic purposes is burdened with difficulties related to the loss of cartilaginous phenotype. Epigenetic mechanisms responsible for regulation of gene expression are believed to underlie chondrocyte dedifferentiation. We have inspected the relevance of DNA methylation alterations for passage-related differential expression of NFATC1 gene involved in hard connective tissue turnover and development, NADSYN1 influencing redox metabolism, and JAK3 - an important driver of inflammation. We have assessed relative amount of transcript abundance and performed DNA bisulfite sequencing of upstream located elements. It seems that anabolic-like effects of chondrogenic differentiation were observed in form of NFATC1 and NADSYN1 upregulation in chondrocytes at the earlier stages of passaging whereas JAK3 upregulation at the 11th passage was the sign of chondrocytes dedifferentiation. Summarizing the inversely correlated DNA methylation and expression patterns in NFATC1 and JAK3 locus might be relevant for cellular dedifferentiation during chondrocyte expansion in monolayer. Obtained results are supportive for further studies on the role of encoded proteins in regenerative biology of articular cartilage using in vitro expanded chondrocytes.

Intrinsic and extrinsic molecular determinants or modulators for epigenetic remodeling and reprogramming of somatic cell-derived genome in mammalian nuclear-transferred oocytes and resultant embryos.

The efficiency of somatic cell cloning in mammals remains disappointingly low. Incomplete and aberrant reprogramming of epigenetic memory of somatic cell nuclei in preimplantation nuclear- transferred (NT) embryos is one of the most important factors that limit the cloning effectiveness. The extent of epigenetic genome-wide alterations, involving histone or DNA methylation and histone deacetylation, that are mediated by histone-lysine methyltransferases (HMTs) or DNA methyltransferases (DNMTs) and histone deacetylases (HDACs) can be modulated/reversed via exogenous inhibitors of these enzymes throughout in vitro culture of nuclear donor cells, nuclear recipient oocytes and/or cloned embryos. The use of the artificial modifiers of epigenomically-conditioned gene expression leads to inhibition of both chromatin condensation and transcriptional silencing the genomic DNA of somatic cells that provide a source of nuclear donors for reconstruction of enucleated oocytes and generation of cloned embryos. The onset of chromatin decondensation and gene transcriptional activity is evoked both through specific/selective inactivating HMTs by BIX-01294 and through non-specific/non-selective blocking the activity of either DNMTs by 5-aza-2'-deoxycytidine, zebularine, S-adenosylhomocysteine or HDACs by trichostatin A, valproic acid, scriptaid, oxamflatin, sodium butyrate, m-carboxycinnamic acid bishydroxamide, panobinostat, abexinostat, quisinostat, dacinostat, belinostat and psammaplin A. Epigenomic modulation of nuclear donor cells, nuclear recipient cells and/or cloned embryos may facilitate and accelerate the reprogrammability for gene expression of donor cell nuclei that have been transplanted into a host ooplasm and subsequently underwent dedifferentiating and re-establishing the epigenetically dependent status of their transcriptional activity during pre- and postimplantation development of NT embryos. Nevertheless, a comprehensive additional work is necessary to determine whether failures in the early-stage reprogramming of somatic cell-inherited genome are magnified downstream in development of cloned conceptuses and neonates.

In vitro development and cytological quality of inter-species (porcine→bovine) cloned embryos are affected by trichostatin A-dependent epigenomic modulation of adult mesenchymal stem cells.

Artificial epigenomic modulation of in vitro cultured mesenchymal stem cells (MSCs) by applying a non-selective HDAC inhibitor, termed TSA, can facilitate more epigenetic reprogramming of transcriptional activity of the somatic cell-descended nuclear genome in NT pig embryos. The results of the present investigation showed that TSA-dependent epigenomic modulation of nuclear donor MSCs highly affects both the in vitro developmental capability and the cytological quality of inter-species (porcine→bovine) cloned embryos. The developmental competences to reach the blastocyst stage among hybrid (porcine→bovine) nuclear-transferred embryos that had been reconstructed with bovine ooplasts and epigenetically modulated porcine MSCs were maintained at a relatively high level. These competences were higher than those noted in studies by other authors, but they were still decreased compared to those of intra-species (porcine) cloned embryos that had been reconstituted with porcine ooplasts and either the cell nuclei of epigenetically transformed MSCs or the cell nuclei of epigenetically non-transformed MSCs. In conclusion, MSCs undergoing TSA-dependent epigenetic transformation were used for the first time as a source of nuclear donor cells not only for inter-species somatic cell cloning in pigs but also for inter-species somatic cell cloning in other livestock species. Moreover, as a result of the current research, efficient sequential physicochemical activation of inter-species nuclear-transferred clonal cybrids derived from bovine ooplasm and porcine MSC nuclei was developed.

Characterization of mesenchymal stem cells and their application in experimental embryology.

The efficiency of somatic cell cloning (somatic cell nuclear transfer; SCNT) as well as in vitro fertilization/in vitro embryo production (IVF/IVP) in mammals stay at relatively same level for over a decade. Despite plenty of different approaches none satisfactory break-through took place. In this article, we briefly summarize the implementation of mesenchymal stem cells (MSCs) for experimental embryology. The advantages of using MSCs as nuclear donors in somatic cell cloning and in vitro embryo culture are described. The description of results obtained with these cells in mammalian embryo genomic engineering is presented.

Pseudophysiological transcomplementary activation of reconstructed oocytes as a highly efficient method used for producing nuclear-transferred pig embryos originating from transgenic foetal fibroblast cells.

The completely new strategy of pseudophysiological transcomplementary (transcytoplasmic) activation (PP-TCA) of nuclear-transferred oocytes, which had been derived from pWAPhGH-GFPBsd transfected foetal fibroblast cells, was recently applied to the somatic cell cloning of pigs. It resulted in the considerable enhancing not only the cleavage activity of cultured cloned embryos, but also their morula and blastocyst formation rates as compared to the use of standard simultaneous fusion and electrical activation of reconstituted oocytes (77% vs. 57%, 63% vs. 46% and 40% vs. 27%, respectively). Altogether, the use of cytosolic components descended from heterologous (rabbit) zygotes as the agents for stimulation of porcine clonal cytoplasmic hybrids (cybrids) turned out to be reliable and feasible strategy for the generation of transgenic blastocysts by somatic cell nuclear transfer (SCNT). Furthermore, to our knowledge, no previous study has reported the preimplantation developmental outcome of transgenic nuclear-transferred pig embryos following the PP-TCA that was developed and optimised in our laboratory.

Roscovitine is a novel agent that can be used for the activation of porcine oocytes reconstructed with adult cutaneous or fetal fibroblast cell nuclei.

The present study was undertaken to investigate the preimplantation developmental competence of cloned pig embryos that were derived from fibroblast cell nuclei by different methods for the activation of reconstructed oocytes. In subgroups IA and IB, nuclear-transferred (NT) oocytes derived from either adult cutaneous or fetal fibroblast cells that had been classified as nonapoptotic by intra vitam analysis for programmed cell death using the YO-PRO-1 DNA fluorochrome underwent sequential physical (i.e., electrical) and chemical activation (SE-CA). This novel method of SE-CA, which was developed and optimized in our laboratory, involves treatment of reconstituted oocytes with direct current pulses and subsequent exposure to 7.5 µM calcium ionomycin, followed by incubation with 30 µM R-roscovitine (R-RSCV), 0.7 mM 6-dimethylaminopurine and 3.5 µg/mL cycloheximide. In subgroups IIA and IIB, NT oocytes were subjected to the standard method of simultaneous fusion and activation mediated by direct current pulses. The proportion of cloned embryos in subgroup IA that reached the morula and blastocyst stages was 145/248 (58.5%) and 78/248 (31.5%), respectively. The proportions of cloned embryos in subgroup IB that reached the morula and blastocyst stages were 186/264 (70.5%) and 112/264 (42.4%), respectively. In turn, subgroup IIA yielded proportions at the morula and blastocyst stages of 110/234 (47.0%) and 49/234 (20.9%), respectively. Subgroup IIB yielded proportions at the morula and blastocyst stages of 144/243 (59.3%) and 74/243 (30.5%), respectively. In summary, the SE-CA of NT oocytes reconstructed from either type of nonapoptotic/nonnecrotic (i.e., YO-PRO-1-negative) fibroblast cell resulted in porcine cloned embryos with considerably better in vitro developmental outcomes than those of cloned embryos generated using the simultaneous fusion and activation approach. To our knowledge, this is the first report of the successful stimulation of porcine NT oocytes using electric pulses followed by an additional activation with a higher dose (1.5 times) of calcium ionomycin and subsequent exposure to a combination of 30 µM R-RSCV and lower concentrations (by 3 times) of 6-dimethylaminopurine and cycloheximide. Moreover, we report here the first use of R-RSCV, a novel meiosis-promoting factor-related p34(cdc2) kinase inhibitor, in the oocyte activation protocol for the somatic cell cloning of pigs.

Transgenic mammalian species, generated by somatic cell cloning, in biomedicine, biopharmaceutical industry and human nutrition/dietetics--recent achievements.

Somatic cell cloning technology in mammals promotes the multiplication of productively-valuable genetically engineered individuals, and consequently allows also for standardization of transgenic farm animal-derived products, which, in the context of market requirements, will have growing significance. Gene farming is one of the most promising areas in modern biotechnology. The use of live bioreactors for the expression of human genes in the lactating mammary gland of transgenic animals seems to be the most cost-effective method for the production/processing of valuable recombinant therapeutic proteins. Among the transgenic farm livestock species used so far, cattle, goats, sheep, pigs and rabbits are useful candidates for the expression of tens to hundreds of grams of genetically-engineered proteins or xenogeneic biopreparations in the milk. At the beginning of the new millennium, a revolution in the treatment of disease is taking shape due to the emergence of new therapies based on recombinant human proteins. The ever-growing demand for such pharmaceutical or nutriceutical proteins is an important driving force for the development of safe and large-scale production platforms. The aim of this paper is to present an overall survey of the state of the art in investigations which provide the current knowledge for deciphering the possibilities of practical application of the transgenic mammalian species generated by somatic cell cloning in biomedicine, the biopharmaceutical industry, human nutrition/dietetics and agriculture.

The possibilities of practical application of transgenic mammalian species generated by somatic cell cloning in pharmacology, veterinary medicine and xenotransplantology.

Cloning of genetically-modified mammals to produce: 1) novel animal bioreactors expressing human genes in rens, urinary bladder and the male accessory sex glands, as well as 2) porcine organs suitable in pig-to-human xenotransplantology, could offer new advantages for biomedical purposes. So too does the generation and/or multiplication of genetically-engineered cloned animals in order to produce: 3) physiologically-relevant animal models of serious monogenic human diseases and 4) prion disease-resistant small as well as large animals (i.e., rodents, ruminants). The basic purpose of this paper is to overview current knowledge deciphering the possibilities of using transgenic specimens created by somatic cell nuclear transfer in medical pharmacology, veterinary medicine, agriculture, transplantational medicine and immunology.

Development of porcine transgenic nuclear-transferred embryos derived from fibroblast cells transfected by the novel technique of nucleofection or standard lipofection.

The aim of our study was to determine the in vitro developmental potential of porcine nuclear-transferred (NT) embryos that had been reconstructed with Tg(pWAPhGH-GFPBsd) transgene-expressing fibroblast cells. The gene construct was introduced into fibroblast cells by the novel method of nucleofection or standard lipofection. NT oocytes derived from foetal and adult dermal fibroblast cells were stimulated by either simultaneous fusion and electrical activation (Groups IA and IB) or sequential electrical and chemical activation (Groups IIA and IIB). The percentages of cloned embryos that reached the morula and blastocyst stages were 152/254 (59.8%) and 77/254 (30.3%) or 139/276 (50.4%) and 45/276 (16.3%) in Groups IA or IB, respectively. The rates of NT embryos that developed to the morula and blastocyst stages were 103/179 (57.5%) and 41/179 (22.9%) or 84/193 (43.5%) and 27/193 (14.0%) in Groups IIA and IIB, respectively. In conclusion, the in vitro developmental competences of porcine transgenic NT embryos that had been reconstructed with the Tg(pWAPhGH-GFPBsd) gene-transfected fibroblast cells were relatively high. Further, the nucleofection efficiency of all the porcine fibroblast cell lines as estimated by intra-vitam fluorescent evaluation based on the index of reporter eGFP transgene expression was nearly 100%. However, PCR analysis for transgene screening confirmed the absence of Tg(pWAPhGH-GFPBsd) fusion gene in some of the nucleofected cell lines. To our knowledge, the novel method of nucleofection is the first to transfect nuclear donor cells in the production of transgenic cloned embryos.

Generation of transgenic rabbits by the novel technique of chimeric somatic cell cloning.

A novel technique of chimeric somatic cell cloning was applied to produce a transgenic rabbit (NT20). Karyoplasts of transgenic adult skin fibroblasts with Tg(Wap-GH1) gene construct as a marker were microsurgically transferred into one, previously enucleated, blastomere of 2-cell non-transgenic embryos, while the second one remained intact. The reconstructed embryos either were cultured in vitro up to the blastocyst stage (Experiment I) or were transferred into recipient-females immediately after the cloning procedure (Experiment II). In Experiment I, 25/102 (24.5%) embryos formed blastocysts from whole embryos and 46/102 (44.12%) embryos developed to the blastocyst stage from single non-operated blastomeres, while the reconstructed blastomeres were damaged and degenerated. Thirteen (12.7%) embryos did not exceed 3- to 4-cell stages and 18 (17.7%) embryos were inhibited at the initial 2-cell stage. Out of 14 blastocysts which were subjected to molecular analysis, the transgene was detected in the cells of 4 blastocysts. In Experiment II, 163/217 (75.0%) embryos were transferred into 9 pseudopregnant recipient-rabbits (an average of 18 embryos per recipient). Four recipient-females (44.4%) became pregnant and delivered a total of 24 (14.7%) pups. Molecular analysis confirmed that two pups (1.2%), one live and one stillborn, showed a positive transgene signal. Live transgenic rabbit NT20 appeared healthy and anatomically as well as physiologically normal. The results of our experiments showed that transgenic adult skin fibroblast cell nuclei, which have been introduced into the cytoplasmic microenvironment of single enucleated blastomeres from 2-cell stage rabbit embryos, are able to direct the development of chimeric embryos not only to the blastocyst stage but also up to term.