Pluripotency-Associated microRNAs in Early Vertebrate Embryos and Stem Cells.

MicroRNAs (miRNAs), small non-coding RNA molecules, regulate a wide range of critical biological processes, such as proliferation, cell cycle progression, differentiation, survival, and apoptosis, in many cell types. The regulatory functions of miRNAs in embryogenesis and stem cell properties have been extensively investigated since the early years of miRNA discovery. In this review, we will compare and discuss the impact of stem-cell-specific miRNA clusters on the maintenance and regulation of early embryonic development, pluripotency, and self-renewal of embryonic stem cells, particularly in vertebrates.

The Effects of Freezing Media on the Characteristics of Male and Female Chicken Primordial Germ Cell Lines.

Recently, in vitro gene preservation has gained ground thanks to its lower cost and higher stability compared to in vivo techniques. One of the methods that can preserve female-specific W chromosome-linked genes is primordial germ cell (PGC) freezing. PGCs can be isolated from Hamburger-Hamilton stage 14-16 embryos via blood sampling. In our experiment, we used two newly established Black Transylvanian naked neck chicken cell lines and four cell lines from our gene bank. We compared two different freezing media (FAM1 and FAM2) in this study. The cell number and viability of the PGCs were measured before freezing (BF) and after thawing on Day 0, Day 1, and Day 7 of cultivation. We analyzed the germ cell-specific chicken vasa homologue (CVH) expression profile in PGCs using RT-qPCR. We found that on Day 0, immediately after thawing, the cell number in cell lines frozen with the FAM2 medium was significantly higher than in the FAM1-treated ones. On Day 1 and Day 7, the cell number and viability were also higher in most cell lines frozen with FAM2, but the difference was insignificant. The freezing also affected the chicken vasa homologue gene expression in male lines treated with both freezing media.

Successful cryopreservation and regeneration of a partridge colored Hungarian native chicken breed using primordial germ cells.

Primordial germ cells (PGCs) are the precursors of germline cells that generate sperm and ova in adults. Thus, they are promising tools for gene editing and genetic preservation, especially in avian species. In this study, we established stable male and female PGC lines from 6Hungarian indigenous chicken breeds with derivation rates ranging from 37.5 to 50 percent. We characterized the PGCs for expression of the germ cell-specific markers during prolonged culture in vitro. An in vivo colonization test was performed on PGCs from four Hungarian chicken breeds and the colonization rates were between 76 and 100%. Cryopreserved PGCs of the donor breed (Partridge color Hungarian) were injected into Black Transylvanian Naked Neck host embryos to form chimeric progeny that, after backcrossing, would permit reconstitution of the donor breed. For 24 presumptive chimeras 13 were male and 11 were female. In the course of backcrossing, 340 chicks were hatched and 17 of them (5%) were pure Partridge colored. Based on the backcrossing 1 hen and 3 roosters of the 24 presumptive chimeras (16.6%) have proven to be germline chimeras. Therefore, it was proven that the original breed can be recovered from primordial germ cells which are stored in the gene bank. To our knowledge, our study is a first that applied feeder free culturing conditions for both male and female cell lines successfully and used multiple indigenous chicken breeds to create a gene bank representing a region (Carpathian Basin).

Effect of Post-Hatch Heat-Treatment in Heat-Stressed Transylvanian Naked Neck Chicken.

Although numerous studies reported the effects of heat stress in chickens, it was not investigated in the Transylvanian Naked Neck breed. In our research, Transylvanian Naked Neck chickens, 24 h after hatching, were heat-treated at 38.5 °C for 12 h. We compared the control and heat-treated adult chickens' productivity parameters following 12 weeks of heat-stress at 30 °C. We found that the heat-treated layers had significantly higher egg production in heat stress, but in cockerels, the sperm quality did not differ significantly between the two groups. To detect the effect of heat-treatment on a molecular level, the expression of two heat-shock proteins and four heat-shock factors were analysed in the gonads of control and heat-treated chickens. We found that the expression level of HSP90 and HSF4 increased significantly in heat-treated female chicken gonads. Still, in adult females, the expression of HSF2 and HSF3 were substantially lower compared to the control. In adult heat-treated males, the HSP70, HSF1 and HSF3 expression levels showed a significant increase in both gonads compared to the control. We think that the presented significant differences in egg production might be related to the increased expression level of HSP90 and HSF4 in heat-treated female gonads.

Effect of miR-302b MicroRNA Inhibition on Chicken Primordial Germ Cell Proliferation and Apoptosis Rate.

The primordial germ cells (PGCs) are the precursors for both the oocytes and spermatogonia. Recently, a novel culture system was established for chicken PGCs, isolated from embryonic blood. The possibility of PGC long-term cultivation issues a new advance in germ cell preservation, biotechnology, and cell biology. We investigated the consequence of gga-miR-302b-5P (5P), gga-miR-302b-3P (3P) and dual inhibition (5P/3P) in two male and two female chicken PGC lines. In treated and control cell cultures, the cell number was calculated every four hours for three days by the XLS Imaging system. Comparing the cell number of control and treated lines on the first day, we found that male lines had a higher proliferation rate independently from the treatments. Compared to the untreated ones, the proliferation rate and the number of apoptotic cells were considerably reduced at gga-miR-302b-5P inhibition in all PGC lines on the third day of the cultivation. The control PGC lines showed a significantly higher proliferation rate than 3P inhibited lines on Day 3 in all PGC lines. Dual inhibition of gga-miR-302b mature miRNAs caused a slight reduction in proliferation rate, but the number of apoptotic cells increased dramatically. The information gathered by examining the factors affecting cell proliferation of PGCs can lead to new data in stem cell biology.

The effect of dual inhibition of Ras-MEK-ERK and GSK3ß pathways on development of in vitro cultured rabbit embryos.

Dual inhibition (2i) of Ras-MEK-ERK and GSK3ß pathways enables the derivation of embryo stem cells (ESCs) from refractory mouse strains and, for permissive strains, allows ESC derivation with no external protein factor stimuli involvement. In addition, blocking of ERK signalling in 8-cell-stage mouse embryos leads to ablation of GATA4/6 expression in hypoblasts, suggesting fibroblast growth factor (FGF) dependence of hypoblast formation in the mouse. In human, bovine or porcine embryos, the hypoblast remains unaffected or displays slight-to-moderate reduction in cell number. In this study, we demonstrated that segregation of the hypoblast and the epiblast in rabbit embryos is FGF independent and 2i treatment elicits only a limited reinforcement in favour of OCT4-positive epiblast populations against the GATA4-/6-positive hypoblast population. It has been previously shown that TGFß/Activin A inhibition overcomes the pervasive differentiation and inhomogeneity of rat iPSCs, rat ESCs and human iPSCs while prompting them to acquire naïve properties. However, TGFß/Activin A inhibition, alone or together with Rho-associated, coiled-coil containing protein kinase (ROCK) inhibition, was not compatible with the viability of rabbit embryos according to the ultrastructural analysis of preimplantation rabbit embryos by electron microscopy. In rabbit models ovulation upon mating allows the precise timing of progression of the pregnancy. It produces several embryos of the desired stage in one pregnancy and a relatively short gestation period, making the rabbit embryo a suitable model to discover the cellular functions and mechanisms of maintenance of pluripotency in embryonic cells and the embryo-derived stem cells of other mammals.

Investigation of the Guinea fowl and domestic fowl hybrids as potential surrogate hosts for avian cryopreservation programmes.

In the last decade, avian gene preservation research has focused on the use of the early precursors of the reproductive cells, the primordial germ cells (PGCs). This is because avian PGCs have a unique migration route through the vascular system which offers easy accessibility. Furthermore, culturing of the cells in vitro, freezing/thawing, reintegration into a recipient embryo and the development of the germ cells can be carried out in well-defined laboratory circumstances. The efficient recovery of the donor genotype and the frequency of germline transmission from the surrogate host animals are still areas which need further development. Thus, the aim of the present study was to investigate an infertile interspecific hybrid (recipient) as an appropriate host for primordial germ cells from native poultry breeds. Guinea fowl × chicken hybrids were produced, the crossing was repeated inversely. The phenotype, the hatching time, the hatching rate, the sex ratio, the presence of own germ cells, the fertility and the phenotype of viable hybrids and the incidence of chromosomal abnormalities of dead hybrid embryos were described. 6.65% viable offspring was obtained with crossing of Guinea fowl females with domestic fowl males. Crossing of domestic fowl hens with Guinea fowl male resulted in lower fertility, 0.14% viable offspring. Based on the investigations, the observed offspring from the successful crossing were sterile male hybrids, thus an extreme form of Haldane's rule was manifested. The sterile hybrid male embryos were tested by injecting fluorescently labeled chicken PGCs. The integration rate of labeled PGCs was measured in 7.5-day, 14.5-day and 18.5-day old embryonic gonads. 50%, 5.3% and 2.4% of the injected hybrid embryos survived and 40%, 5.3% and 2.4% of the examined gonads contained fluorescent labeled donor PGCs. Therefore, these sterile hybrid males may be suitable recipients for male PGCs and possibly for female PGCs although with lower efficiency. This research work shows that the sterility of hybrids can be used in gene conservation to be a universal host for PGCs of different avian species.

CRISPR/Cas9-Mediated Knock-Out of dUTPase in Mice Leads to Early Embryonic Lethality.

Sanitization of nucleotide pools is essential for genome maintenance. Deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) is a key enzyme in this pathway since it catalyzes the cleavage of 2'-deoxyuridine 5'-triphosphate (dUTP) into 2'-deoxyuridine 5'-monophosphate (dUMP) and inorganic pyrophosphate. Through its action dUTPase efficiently prevents uracil misincorporation into DNA and at the same time provides dUMP, the substrate for de novo thymidylate biosynthesis. Despite its physiological significance, knock-out models of dUTPase have not yet been investigated in mammals, but only in unicellular organisms, such as bacteria and yeast. Here we generate CRISPR/Cas9-mediated dUTPase knock-out in mice. We find that heterozygous dut +/- animals are viable while having decreased dUTPase levels. Importantly, we show that dUTPase is essential for embryonic development since early dut -/- embryos reach the blastocyst stage, however, they die shortly after implantation. Analysis of pre-implantation embryos indicates perturbed growth of both inner cell mass (ICM) and trophectoderm (TE). We conclude that dUTPase is indispensable for post-implantation development in mice.

Trophoblastic microRNAs are downregulated in a diabetic pregnancy through an inhibition of Drosha.

MicroRNAs are promising biological markers for prenatal diagnosis. They regulate placental development and are present in maternal plasma. Maternal metabolic diseases are major risk factors for placental deterioration. We analysed the influence of a maternal insulin-dependent diabetes mellitus on microRNA expression in maternal plasma and in blastocysts employing an in vivo rabbit diabetic pregnancy model and an in vitro embryo culture in hyperglycaemic and hypoinsulinaemic medium. Maternal diabetes led to a marked downregulation of Dicer protein in embryoblast cells and Drosha protein in trophoblast cells. MiR-27b, miR-141 and miR-191 were decreased in trophoblast cells and in maternal plasma of diabetic rabbits. In vitro studies indicate, that maternal hyperglycaemia and hypoinsulinaemia partially contribute to the downregulation of trophoblastic microRNAs. As the altered microRNA expression was detectable in maternal plasma, too, the plasma microRNA signature could serve as an early biological marker for the prediction of trophoblast function during a diabetic pregnancy.

Enhancement of chicken primordial germ cell in vitro maintenance using an automated cell image analyser.

Primordial germ cells (PGCs) were isolated from blood samples of chicken embryos. We established four PGC lines: two males (FS-ZZ-101, GFP-ZZ-4ZP) and two females (FS-ZW-111, GFP-ZW-5ZP). We could not detect a significant difference in the marker expression profile, but there was a remarkable difference between the proliferation rates of these PGC lines. We monitored the number of PGCs throughout a three-day period using a high-content screening cell imaging and analysing system (HCS). We compared three different initial cell concentrations in the wells: ~1000 cells (1×, ~4000 (4× and ~8000 (8×. For the GFPZW- 5ZP, FS-ZZ-101 and FS-ZW-111 PGC lines the lowest doubling time was observed at 4× concentration, while for GFP-ZZ-4ZP we found the lowest doubling time at 1× concentration. At 8× initial concentration, the growth rate was high during the first two days for all cell lines, but this was followed by the appearance of cell aggregates decreasing the cell growth rate. We could conclude that the difference in proliferation rate could mainly be attributed to genotypic variation in the established PGC lines, but external factors such as cell concentration and quality of the culture medium also affect the growth rate of PGCs.

Comparison of the MicroRNA Expression Profiles of Male and Female Avian Primordial Germ Cell Lines.

Primordial germ cells (PGCs) are the precursors of adult germ cells, and among the embryonic stem-like cells in the bird embryo, only they can transmit the genetic information to the next generation. Despite the wide range of applications, very little is known about the mechanism that governs primordial germ cell self-renewal and differentiation. As a first step, we compared 12 newly established chicken PGC lines derived from two different chicken breeds, performing CCK-8 proliferation assay. All of the lines were derived from individual embryos. A significant difference was found among the lines. As microRNAs have been proved to play a key role in the maintenance of pluripotency and the cell cycle regulation of stem cells, we continued with a complex miRNA analysis. We could discover miRNAs expressing differently in PGC lines with high proliferation rate, compared to PGC lines with low proliferation rate. We found that gga-miR-2127 expresses differently in female and male cell lines. The microarray analysis also revealed high expression level of the gga-miR-302b-3p strand (member of the miR-302/367 cluster) in slowly proliferating PGC lines compared to the gga-miR-302b-5p strand. We confirmed that the inhibition of miR-302b-5p significantly increases the doubling time of the examined PGC lines. In conclusion, we found that gga-miR-181-5p, gga-miR-2127, and members of the gga-miR-302/367 cluster have a dominant role in the regulation of avian primordial germ cell proliferation.

Glomerulosclerosis in transgenic rabbits with ubiquitous Venus protein expression.

Focal segmental glomerulosclerosis (FSGS) is a potential cause of nephrotic syndrome both in humans and pet mammals. Glomerulopathy was reported earlier in green fluorescent protein (GFP) transgenic (TG) mice, but glomerulosclerosis has not been examined in GFP TG rabbits so far. In the present study, the potential manifestation of FSGS was investigated in both Venus TG rabbits generated by Sleeping Beauty (SB) transposition and age-matched control New Zealand White (NZW) rabbits. Venus protein fluorescence was detected by confocal microscopy and quantified by microplate reader. Urinalysis, haematology, serum biochemistry and renal histology were performed to assess the signs of FSGS. Higher levels of Venus fluorescence were determined in renal cortex samples than in the myocardium by both methods. Urinalysis revealed proteinuria in Venus heterozygote TG bucks, while Venus homozygote TG bucks developed microscopic haematuria. Supporting the urinalysis data, the histological findings of FSGS (glomerulomegaly and sclerotic glomeruli) were observed in renal cortex sections of Venus TG rabbits. Taken together, Venus TG bucks were diagnosed with FSGS; thus, this type of glomerulopathy could be a common disease in TG animals overexpressing GFP.

Secretion of a recombinant protein without a signal peptide by the exocrine glands of transgenic rabbits.

Transgenic rabbits carrying mammary gland specific gene constructs are extensively used for excreting recombinant proteins into the milk. Here, we report refined phenotyping of previously generated Venus transposon-carrying transgenic rabbits with particular emphasis on the secretion of the reporter protein by exocrine glands, such as mammary, salivary, tear and seminal glands. The Sleeping Beauty (SB) transposon transgenic construct contains the Venus fluorophore cDNA, but without a signal peptide for the secretory pathway, driven by the ubiquitous CAGGS (CAG) promoter. Despite the absence of a signal peptide, the fluorophore protein was readily detected in milk, tear, saliva and seminal fluids. The expression pattern was verified by Western blot analysis. Mammary gland epithelial cells of SB-CAG-Venus transgenic lactating does also showed Venus-specific expression by tissue histology and fluorescence microscopy. In summary, the SB-CAG-Venus transgenic rabbits secrete the recombinant protein by different glands. This finding has relevance not only for the understanding of the biological function of exocrine glands, but also for the design of constructs for expression of recombinant proteins in dairy animals.

Reprogramming of rabbit induced pluripotent stem cells toward epiblast and chimeric competency using Krüppel-like factors.

Rabbit induced pluripotent stem cells (rbiPSCs) possess the characteristic features of primed pluripotency as defined in rodents and primates. In the present study, we reprogrammed rbiPSCs using human Krüppel-like factors (KLFs) 2 and 4 and cultured them in a medium supplemented with fetal calf serum and leukemia inhibitory factor. These cells (designated rbEKA) were propagated by enzymatic dissociation for at least 30 passages, during which they maintained a normal karyotype. This new culturing protocol resulted in transcriptional and epigenetic reconfiguration, as substantiated by the expression of transcription factors and the presence of histone modifications associated with naïve pluripotency. Furthermore, microarray analysis of rbiPSCs, rbEKA cells, rabbit ICM cells, and rabbit epiblast showed that the global gene expression profile of the reprogrammed rbiPSCs was more similar to that of rabbit ICM and epiblast cells. Injection of rbEKA cells into 8-cell stage rabbit embryos resulted in extensive colonization of ICM in 9% early-blastocysts (E3.5), epiblast in 10% mid-blastocysts (E4.5), and embryonic disk in 1.4% pre-gastrulae (E6). Thus, these results indicate that KLF2 and KLF4 triggered the conversion of rbiPSCs into epiblast-like, embryo colonization-competent PSCs. Our results highlight some of the requirements to achieve bona fide chimeric competency.

Placenta-specific gene manipulation in rabbits.

Lentiviral gene constructs can be efficiently and specifically delivered to trophoblast cell lineages in rodents. In vivo genetic manipulation of trophoblast cell lines enables functional and developmental studies in the placenta. In this report we show that genetic modification can be produced in the extraembryonic tissues of rabbits by lentiviral gene constructs. When 8-16 cell stage embryos were injected with lentiviral particles, strong reporter gene expression resulted in the rabbit placenta. The expression pattern displayed some mosaicism. A strikingly high degree of mosaic GFP expression was detected in some parts of the yolk sac, which is a hypoblast-derived tissue. Whereas expression of the reporter gene construct was detected in placentas and yolk sacs, fetuses never expressed the transgene. As rabbits are an ideal model for functional studies in the placenta, our method would open new possibilities in rabbit biotechnology and placentation studies.

A Panel of Embryonic Stem Cell Lines Reveals the Variety and Dynamic of Pluripotent States in Rabbits.

Conventional rabbit embryonic stem cell (ESC) lines are derived from the inner cell mass (ICM) of pre-implantation embryos using methods and culture conditions that are established for primate ESCs. In this study, we explored the capacity of the rabbit ICM to give rise to ESC lines using conditions similar to those utilized to generate naive ESCs in mice. On single-cell dissociation and culture in fibroblast growth factor 2 (FGF2)-free, serum-supplemented medium, rabbit ICMs gave rise to ESC lines lacking the DNA-damage checkpoint in the G1 phase like mouse ESCs, and with a pluripotency gene expression profile closer to the rabbit ICM/epiblast profiles. These cell lines can be converted to FGF2-dependent ESCs after culture in conventional conditions. They can also colonize the rabbit pre-implantation embryo. These results indicate that rabbit epiblast cells can be coaxed toward different types of pluripotent stem cells and reveal the dynamics of pluripotent states in rabbit ESCs.

Induced pluripotent stem cells derived from rabbits exhibit some characteristics of naïve pluripotency.

Not much is known about the molecular and functional features of pluripotent stem cells (PSCs) in rabbits. To address this, we derived and characterized 2 types of rabbit PSCs from the same breed of New Zealand White rabbits: 4 lines of embryonic stem cells (rbESCs), and 3 lines of induced PSCs (rbiPSCs) that were obtained by reprogramming adult skin fibroblasts. All cell lines required fibroblast growth factor 2 for their growth and proliferation. All rbESC lines showed molecular and functional properties typically associated with primed pluripotency. The cell cycle of rbESCs had a prolonged G1 phase and a DNA damage checkpoint before entry into the S phase, which are the 2 features typically associated with the somatic cell cycle. In contrast, the rbiPSC lines exhibited some characteristics of naïve pluripotency, including resistance to single-cell dissociation by trypsin, robust activity of the distal enhancer of the mouse Oct4 gene, and expression of naïve pluripotency-specific genes, as defined in rodents. According to gene expression profiles, rbiPSCs were closer to the rabbit inner cell mass (ICM) than rbESCs. Furthermore, rbiPSCs were capable of colonizing the ICM after aggregation with morulas. Therefore, we propose that rbiPSCs self-renew in an intermediate state between naïve and primed pluripotency, which represents a key step toward the generation of bona fide naïve PSC lines in rabbits.

Discovery of pluripotency-associated microRNAs in rabbit preimplantation embryos and embryonic stem-like cells.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate multiple biological processes. Increasing experimental evidence implies an important regulatory role of miRNAs during embryonic development and in embryonic stem (ES) cell biology. In the current study, we have described and analyzed the expression profile of pluripotency-associated miRNAs in rabbit embryos and ES-like cells. The rabbit specific ocu-miR-302 and ocu-miR-290 clusters, and three homologs of the human C19MC cluster (ocu-miR-512, ocu-miR-520e, and ocu-miR-498) were identified in rabbit preimplantation embryos and ES-like cells. The ocu-miR-302 cluster was highly similar to its human homolog, while ocu-miR-290 revealed a low level of evolutionary conservation with its mouse homologous cluster. The expression of the ocu-miR-302 cluster began at the 3.5 days post-coitum early blastocyst stage and they stayed highly expressed in rabbit ES-like cells. In contrast, a high expression level of the ocu-miR-290 cluster was detected during preimplantation embryonic development, but a low level of expression was found in rabbit ES-like cells. Differential expression of the ocu-miR-302 cluster and ocu-miR-512 miRNA was detected in rabbit trophoblast and embryoblast. We also found that Lefty has two potential target sites in its 3'UTR for ocu-miR-302a and its expression level increased upon ocu-miR-302a inhibition. We suggest that the expression of the ocu-miR-302 cluster is characteristic of the rabbit ES-like cell, while the ocu-miR-290 cluster may play a crucial role during early embryonic development. This study presents the first identification, to our knowledge, of pluripotency-associated miRNAs in rabbit preimplantation embryos and ES-like cells, which can open up new avenues to investigate the regulatory function of ocu-miRNAs in embryonic development and stem cell biology.

Positional identity of murine mesenchymal stem cells resident in different organs is determined in the postsegmentation mesoderm.

Although mesenchymal stem cells (MSCs) of distinct tissue origin have a large number of similarities and differences, it has not been determined so far whether tissue-resident MSCs are the progenies of one ancestor cell lineage or the results of parallel cell developmental events. Here we compared the expression levels of 177 genes in murine MSCs derived from adult and juvenile bone marrow and adult adipose tissue, as well as juvenile spleen, thymus, and aorta wall by quantitative real-time polymerase chain reaction and the results were partially validated at protein level. All MSC lines uniformly expressed a large set of genes including well-known mesenchymal markers, such as α-smooth muscle actin, collagen type I α-chain, GATA6, Mohawk, and vimentin. In contrast, pluripotency genes and the early mesodermal marker T-gene were not expressed. On the other hand, different MSC lines consistently expressed distinct patterns of Hox genes determining the positional identity of a given cell population. Moreover, MSCs of different origin expressed a few other transcription factors also reflecting their topological identity and so the body segment or organ to which they normally contributed in vivo: (1) thymus-derived cells specifically expressed Tbx5 and Pitx2; (2) spleen-derived MSCs were characterized with Tlx1 and Nkx2.5; (3) Pitx1 designated femoral bone marrow cells and (4) En2 appeared in aorta wall-derived MSCs. Thus, MSCs exhibited topographic identity and memory even after long-term cultivation in vitro. On the basis of these results, we suggest that postnatal MSCs isolated from different anatomical sites descend from precursor cells developing in the postsegmentation mesoderm.

Establishment of an embryonic stem cell line from blastocyst stage mouse embryos.

Embryonic stem cells have the ability to remain undifferentiated and proliferate in vitro while maintaining the potential to differentiate into derivatives of all three embryonic germ layers. The aim of the present study was to establish mouse ES lines from blastocyst stage embryos obtained after CD1/EGFP mice superovulation. We isolated, cultured and determined the characteristics of mouse embryonic stem cells in early passages, which were first described by Evans M and Kaufman M. Therefore, we evaluated the morphological criteria for the approval of ES cells in early expansion stage. Two cell lines were isolated (CDE1 and CDE2) and analyzed. They showed similar characteristics to those reported earlier for blastocyst-derived ES cell lines.