Telomere reprogramming and silencing of exogenous genes have been demonstrated in mouse and human induced pluripotent stem cells (iPS cells). Pigs have the potential to provide xenotransplant for ...humans, and to model and test human diseases. We investigated the telomere length and maintenance in porcine iPS cells generated and cultured under various conditions. Telomere lengths vary among different porcine iPS cell lines, some with telomere elongation and maintenance, and others telomere shortening. Porcine iPS cells with sufficient telomere length maintenance show the ability to differentiate in vivo by teratoma formation test. IPS cells with short or dysfunctional telomeres exhibit reduced ability to form teratomas. Moreover, insufficient telomerase and incomplete telomere reprogramming and/or maintenance link to sustained activation of exogenous genes in porcine iPS cells. In contrast, porcine iPS cells with reduced expression of exogenous genes or partial exogene silencing exhibit insufficient activation of endogenous pluripotent genes and telomerase genes, accompanied by telomere shortening with increasing passages. Moreover, telomere doublets, telomere sister chromatid exchanges and t-circles that presumably are involved in telomere lengthening by recombination also are found in porcine iPS cells. These data suggest that both telomerase-dependent and telomerase-independent mechanisms are involved in telomere reprogramming during induction and passages of porcine iPS cells, but these are insufficient, resulting in increased telomere damage and shortening, and chromosomal instability. Active exogenes might compensate for insufficient activation of endogenous genes and incomplete telomere reprogramming and maintenance of porcine iPS cells. Further understanding of telomere reprogramming and maintenance may help improve the quality of porcine iPS cells.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Induced pluripotent stem (iPS) cells can be obtained by the introduction of defined factors into somatic cells. The combination of Oct4 (also known as Pou5f1), Sox2 and Klf4 (which we term OSK) ...constitutes the minimal requirement for generating iPS cells from mouse embryonic fibroblasts. These cells are thought to resemble embryonic stem cells (ESCs) on the basis of global gene expression analyses; however, few studies have tested the ability and efficiency of iPS cells to contribute to chimaerism, colonization of germ tissues, and most importantly, germ-line transmission and live birth from iPS cells produced by tetraploid complementation. Using genomic analyses of ESC genes that have roles in pluripotency and fusion-mediated somatic cell reprogramming, here we show that the transcription factor Tbx3 significantly improves the quality of iPS cells. iPS cells generated with OSK and Tbx3 (OSKT) are superior in both germ-cell contribution to the gonads and germ-line transmission frequency. However, global gene expression profiling could not distinguish between OSK and OSKT iPS cells. Genome-wide chromatin immunoprecipitation sequencing analysis of Tbx3-binding sites in ESCs suggests that Tbx3 regulates pluripotency-associated and reprogramming factors, in addition to sharing many common downstream regulatory targets with Oct4, Sox2, Nanog and Smad1. This study underscores the intrinsic qualitative differences between iPS cells generated by different methods, and highlights the need to rigorously characterize iPS cells beyond in vitro studies.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Preimplantation embryos undergo zygotic genome activation and lineage specification resulting in three distinct cell types in the late blastocyst. The molecular mechanisms underlying this progress ...are largely unknown in bovines. Here, we sought to analyze an extensive set of regulators at the single-cell level to define the events involved in the development of the bovine blastocyst. Using a quantitative microfluidics approach in single cells, we analyzed mRNA levels of 96 genes known to function in early embryonic development and maintenance of stem cell pluripotency in parallel in 384 individual cells from bovine preimplantation embryos. The developmental transitions can be distinguished by distinctive gene expression profiles and we identified NOTCH1, expressed in early developmental stages, while T-box 3 (TBX3) and fibroblast growth factor receptor 4 (FGFR4), expressed in late developmental stages. Three lineages can be segregated in bovine expanded blastocysts based on the expression patterns of lineage-specific genes such as disabled homolog 2 (DAB2), caudal type homeobox 2 (CDX2), ATPase H+/K+ transporting non-gastric alpha2 subunit (ATP12A), keratin 8 (KRT8), and transcription factor AP-2 alpha (TFAP2A) for trophectoderm; GATA binding protein 6 (GATA6) and goosecoid homeobox (GSC) for primitive endoderm; and Nanog homeobox (NANOG), teratocarcinoma-derived growth factor 1 (TDGF1), and PR/SET domain 14 (PRDM14) for epiblast. Moreover, some lineage-specific genes were coexpressed in blastomeres from the morula. The commitment to trophectoderm and inner cellmass lineages in bovines occurs later than in the mouse, and KRT8 might be an earlier marker for bovine trophectoderm cells. We determined that TDGF1 and PRDM14 might play pivotal roles in the primitive endoderm and epiblast specification of bovine blastocysts. Our results shed light on early cell fate determination in bovine preimplantation embryos and offer theoretical support for deriving bovine embryonic stem cells. Summary Sentence Gene expression analysis of single blastomeres from zygote to blastocyst sheds light on the early cell fate determination in bovine preimplantation embryos and offers theoretical support for deriving bovine embryonic stem cells.
Microbially induced carbonate precipitation (MICP) has been utilized as a new method to improve loess soil strength. In this study, we investigated the influence of the main parameters on the shear ...strength of MICP-treated loess specimens. Initially, culture media with different formulas and pH values were examined to identify the most efficient medium for loess soil. To explore the shear behavior of MICP-treated loess under general stress levels, unconfined compressive strength (UCS) tests and triaxial tests relevant to the compression strength and vertical loads were performed on MICP-treated loess with different calcium sources, cementation concentrations, and curing periods. Subsequently, calcium chloride was selected as the optimal calcium source based on the ultimate strength of the MICP-treated loess. The effective cementation concentration in the loess soil was between 1.0 and 1.25 M. The ultimate strength of the MICP-treated loess was 3.6 times of the untreated loess. The stress-strain curves indicate that a higher cementing effect can be expected with an increase in the curing period. The formation process of calcium carbonate and the micromorphology of the MICP-treated loess samples were examined using scanning electron microscopy. In this study, we present an environmentally friendly technique for improving loess soil strength.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Spatially ordered embryo-like structures self-assembled from blastocyst-derived stem cells can be generated to mimic embryogenesis in vitro. However, the assembly system and developmental potential ...of such structures needs to be further studied. Here, we devise a nonadherent-suspension-shaking system to generate self-assembled embryo-like structures (ETX-embryoids) using mouse embryonic, trophoblast and extra-embryonic endoderm stem cells. When cultured together, the three cell types aggregate and sort into lineage-specific compartments. Signaling among these compartments results in molecular and morphogenic events that closely mimic those observed in wild-type embryos. These ETX-embryoids exhibit lumenogenesis, asymmetric patterns of gene expression for markers of mesoderm and primordial germ cell precursors, and formation of anterior visceral endoderm-like tissues. After transplantation into the pseudopregnant mouse uterus, ETX-embryoids efficiently initiate implantation and trigger the formation of decidual tissues. The ability of the three cell types to self-assemble into an embryo-like structure in vitro provides a powerful model system for studying embryogenesis.
Pipe jacking is a commonly used trenchless technology to install pipelines especially in congested urban areas or river crossings. However, the estimation of the jacking force is often heavily ...dependent on empirical calculations. The jacking force needs to be greater than the combined frictional resistance and face resistance. This investigation proposes to use a modified Protodyakonov’s arch model to compute the face resistance. A series of direct shear tests is performed to provide data of interface friction coefficient between different types of soil and pipe. The influence of slurry lubricant is also considered. A two-dimensional plane strain numerical model is conducted, where the surrounding soil is simulated as discrete particles and the lining is simplified as a single big particle. The novel modeling technique enables the evaluation of the normal force acting on the pipe. The friction resistance is then determined by multiplying the interface friction coefficient by the normal force. A ‘wavy’ shaped pipeline model is proposed to define an angular deviation influence factor to scale up the calculated jacking force due to pipe misalignment. In the end, comparison between calculated and field measured jacking force is conducted for three different drives in a pipe jacking project to illustrate the effectiveness of the proposed analysis framework.
Somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs) with the introduction of Oct4, Sox2, Klf4, and c-Myc. Among these four factors, Oct4 is critical in inducing pluripotency ...because no transcription factor can substitute for Oct4, whereas Sox2, Klf4, and c-Myc can be replaced by other factors. Here we show that the orphan nuclear receptor Nr5a2 (also known as Lrh-1) can replace Oct4 in the derivation of iPSCs from mouse somatic cells, and it can also enhance reprogramming efficiency. Sumoylation mutants of Nr5a2 with enhanced transcriptional activity can further increase reprogramming efficiency. Genome-wide location analysis reveals that Nr5a2 shares many common gene targets with Sox2 and Klf4, which suggests that the transcription factor trio works in concert to mediate reprogramming. We also show that Nr5a2 works in part through activating Nanog. Together, we show that unrelated transcription factors can replace Oct4 and uncovers an exogenous Oct4-free reprogramming code.
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► Nuclear receptor Nr5a2 enhances reprogramming of mouse fibroblasts ► Nr5a2 can replace Oct4 in the reprogramming cocktail ► Nr5a2 with enhanced transcriptional activity more effective in reprogramming ► DNA binding links Nr5a2 to the core pluripotency network
Proper follicle development is very important for the production of mature oocytes, which is essential for the maintenance of female fertility. This complex biological process requires precise gene ...regulation. The most abundant modification of mRNA, N
-methyladenosine (m
A), is involved in many RNA metabolism processes, including RNA splicing, translation, stability, and degradation. Here, we report that m
A plays essential roles during oocyte and follicle development. Oocyte-specific inactivation of the key m
A methyltransferase Mettl3 with Gdf9-Cre caused DNA damage accumulation in oocytes, defective follicle development, and abnormal ovulation. Mechanistically, combined RNA-seq and m
A methylated RNA immunoprecipitation sequencing (MeRIP-seq) data from oocytes revealed, that we found METTL3 targets Itsn2 for m
A modification and then enhances its stability to influence the oocytes meiosis. Taken together, our findings highlight the crucial roles of mRNA m
A modification in follicle development and coordination of RNA stabilization during oocyte growth.
Because few studies exist to describe the unique molecular network regulation behind pig pre-implantation embryonic development (PED), genetic engineering in the pig embryo is limited. Also, this ...lack of research has hindered derivation and application of porcine embryonic stem cells and porcine induced pluripotent stem cells (iPSCs).
We identified and analyzed the genome wide transcriptomes of pig in vivo-derived and somatic cell nuclear transferred (SCNT) as well as mouse in vivo-derived pre-implantation embryos at different stages using mRNA deep sequencing. Comparison of the pig embryonic transcriptomes with those of mouse and human pre-implantation embryos revealed unique gene expression patterns during pig PED. Pig zygotic genome activation was confirmed to occur at the 4-cell stage via genome-wide gene expression analysis. This activation was delayed to the 8-cell stage in SCNT embryos. Specific gene expression analysis of the putative inner cell mass (ICM) and the trophectoderm (TE) revealed that pig and mouse pre-implantation embryos share regulatory networks during the first lineage segregation and primitive endoderm differentiation, but not during ectoderm commitment. Also, fatty acid metabolism appears to be a unique characteristic of pig pre-implantation embryonic development. In addition, the global gene expression patterns in the pig SCNT embryos were different from those in in vivo-derived pig embryos.
Our results provide a resource for pluripotent stem cell engineering and for understanding pig development.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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