Isolation and characterization of embryonic stem cell-like cells from in vitro-produced buffalo (Bubalus bubalis) embryos

This study was carried out to isolate and characterize buffalo embryonic stem (ES) cell-like cells from in vitro-produced embryos. Inner cell mass (ICM) cells were isolated either mechanically or by enzymatic digestion from 120 blastocysts whereas 28 morulae were used for the isolation of blastomeres mechanically. The ICM cells/ blastomeres were cultured on mitomycin-C-treated feeder layer. Primary cell colony formation was higher (P < 0.05) for hatched blastocysts (73.1%, 30/41) than that for early/expanded blastocysts (25.3%, 20/79). However, no primary cell colonies were formed when blastomeres obtained from morulae were cultured. Primary colonies were formed in 14.1% (12/85) of intact blastocyst culture, which was significantly lower (P < 0.05) than that of 41.6% for ICM culture. These colonies were separated by enzymatic or mechanical disaggregation. Using mechanical disaggregation method, the cells remained undifferentiated and two buffalo ES cell-like cell lines (bES1, bES2) continued to grow in culture up to eight passages. However, disassociation through enzymatic method resulted in differentiation. Undifferentiated cells exhibited stem cell morphological features, normal chromosomal morphology, and expressed specific markers such as alkaline phosphatase (AP) and Oct-4. Cells formed embryoid bodies (EBs) in suspension culture; extended culture of EBs resulted in formation of cystic EBs. Following prolonged in vitro culture, these cells differentiated into several types of cells including neuron-like and epithelium-like cells. Furthermore, the vitrified-thawed ES cell-like cells also exhibited typical stem cell characteristics. In conclusion, buffalo ES cell-like cells could be isolated from in vitro-produced blastocysts and maintained in vitro for prolonged periods of time.     

Differential expression of the Wnt and Frizzled genes in Flk1+ cells derived from mouse ES cells

Embryonic stem (ES) cells have the potential to develop into various cell lineages including hemangioblasts (Flk1+), a common progenitor for hematopoietic and vascular endothelial cells. Previous studies indicate that Flk1+ cells, a marker for hemangioblast, can be derived from ES cell and that Flk1+ can be differentiated into hematopoietic or endothelial cells depending on culture conditions. We developed an improved in vitro system to generate Flk1+-enriched cultures from mouse ES cells and used this in vitro system to study the role of Wnt signalling in early endothelial progenitor cells. We determined the expression of the Wnt and Frizzled genes in Flk1+ cells derived from mouse ES cells. RT-PCR analyses identified significantly higher expression of non-canonical Wnt5a and Wnt11 genes in Flk1+ cells compared to Flk1- cells. In contrast, expression of canonical Wnt3a gene was reduced in Flk1+ cells. In addition, Frizzled2, Frizzled5 and Frizzled7 genes were also expressed at a higher level in Flk1+ cells. The differential expression of Wnt and Frizzled genes in Flk1+ cells provides a novel insight into the role of non-canonical Wnt signalling in vascular endothelial fate determination.     

Cell-autonomous and signal-dependent expression of liver and intestine marker genes in pluripotent precursor cells from Xenopus embryos

Early regulatory events in respect to the embryonic development of the vertebrate liver are only poorly defined. A better understanding of the gene network that mediates the formation of hepatocytes from pluripotent embryonic precursor cells may help to establish in vitro protocols for hepatocyte differentiation. Here, we describe our first attempts to make use of early embryonic explants from the amphibian Xenopus laevis in order to address these questions. We have identified several novel embryonic liver and intestine marker genes in a random expression pattern screen with cDNA libraries derived from the embryonic liver anlage and from the adult liver of Xenopus laevis. Based on their embryonic expression characteristics, these genes, together with the previously known ones, can be categorized into four different groups: the liver specific group (LS), the liver and intestine group A (LIA), the liver and intestine group B (LIB), and the intestine specific group (IS). Dissociation of endodermal explants isolated from early neurula stage embryos reveals that all genes in the LIB and IS groups are expressed in a cell-autonomous manner. In contrast, expression of genes in the LS and LIA groups requires cell-cell interactions. The regular temporal expression profile of genes in all four groups is mimicked in ectodermal explants from early embryos, reprogrammed by co-injection of VegT and beta-catenin mRNAs. FGF signaling is found to be required for the induction of liver specific marker (LS group) gene expression in the same system.     

Endocrine cells develop within pancreatic bud-like structures derived from mouse ES cells differentiated in response to BMP4 and retinoic acid

We have examined factors affecting the in vitro differentiation of Pdx1^GFP/w ESCs to pancreatic endocrine cells. Inclusion of Bone Morphogenetic Protein 4 (BMP4) during the first four days of differentiation followed by a 24-hour pulse of retinoic acid (RA) induced the formation of GFP⁺ embryoid bodies (EBs). GFP expression was restricted to E-cadherin⁺ tubes and GFP bright (GFP^br) buds, reminiscent of GFP⁺ early foregut endoderm and GFP^br pancreatic buds observed in Pdx1^GFP/w embryos. These organoid structures developed without further addition of exogenous factors between days 5 and 12, suggesting that day 5 EBs contained a template for the subsequent phase of development. EBs treated with nicotinamide after day 12 of differentiation expressed markers of endocrine and exocrine differentiation, but only in cells within the GFP^br buds. Analysis of Pdx1^GFP/w ESCs modified by targeting a dsRed1 gene to the Ins1 locus (Pdx1^GFP/wIns1^RFP/w ESCs) provided corroborating evidence that insulin positive cells arose from GFP^br buds, mirroring the temporal relationship between pancreatic bud development and the formation of endocrine cells in the developing embryo. The readily detectable co-expression of GFP and RFP in grafts derived from transplanted EBs demonstrated the utility of Pdx1^GFP/wIns1^RFP/w ESCs for investigating pancreatic differentiation in vitro and in vivo.     

ES cells derived from cloned embryos in monkey--a jump toward human therapeutic cloning

Therapeutic cloning refers to the derivation of embryonic stem cells （ntESC） from embryos derived from somatic cell nuclear transfer （SCNT） also known as cloning. Cloning involves transplanting a differentiated cell into an oocyte that has had its nucleus （DNA） removed. The reconstructed oocyte can be activated to divide and develop into an embryo. The process that allows this to happen is termed nuclear reprogramming, and is defined as the mechanism through which a differentiated cell de-differentiates or returns to a totipotent state （capable of giving rise to any cell type, including extra-embryonic） and directs embryonic development [1]. Cells from blastocyst stage cloned embryos can be used to generate ntESC lines. Such cell lines can differentiate into any adult cell type, and have tremendous potential for patient-specific disease therapy [2].     

Transfer of inner cell mass cells derived from bovine nuclear transfer embryos into the trophoblast of bovine in vitro-produced embryos

Presence of placental tissues from more normal noncloned embryos could reduce the pregnancy failure of somatic cloning in cattle. In this study, inner cell mass (ICM) cells of in vitro-produced (IVP) embryos was replaced with those of nuclear transfer (NT) embryos to reconstruct bovine blastocysts with ICM and trophoblast cells from NT and IVP embryos, respectively. A total of 65 of these reconstructed embryos were nonsurgically transferred to 20 recipient beef females. Of those, two females were diagnosed pregnant by ultrasonography on day 30 of gestation. One pregnancy was lost at 60-90 days of gestation, and the other recipient cow remained pregnant at day 240 of gestation; however, this female died on day 252 of gestation. Gross pathology of the internal organs of the recipient female, a large fetus, and a large placental tissue mass suggested the massive size of the fetus and placental tissue were likely involved in terminating the life of the recipient female. Biopsy samples were harvested from the skin of the dead recipient cow, the fetus and from cotyledonary tissue. Microsatellite DNA analysis of these samples revealed that the genotype of the fetus was the same as that of the NT donor cells and different from that of the recipient cow. Correspondingly, neither the fetus nor recipient cow had the same genotype with that of the fetal cotyledonary tissue. These results present the first known documented case of a bovine somatic NT pregnancy with nonclone placental tissues after transfer of a blastocyst reconstructed by a microsurgical method to exchange of ICM cells and trophoblast tissue between NT and IVP blastocysts.     

Organogenesis of heart-vascular system derived from mouse 2 cell stage embryos and from early embryonic stem cells in vitro

Regenerative medical treatment with embryonic stem cells (an ES cell) is a goal for organ transplantation. Structures that are tubular in nature (i.e. blood capillaries) were induced from early embryonic stem (EES) cells in vitro using embryotrophic factor (ETFs). In addition, cardiac muscle cells could be identified as well. However, differentiation of EES cells into a complete cardiovascular system was difficult because 3 germ layer primordial organs are directed embryologically in various ways and it is not possible to guide only cardiovascular organs. Thus, we introduced ETFs after the formation of an embryoid body and were successful in cloning cell clusters that beat, thus deriving only cardiovascular organs. The application of this to the treatment of various cardiovascular diseases is promising.     

Effect of 5-azadeoxycytidine and retinoic acid on expression of genomic imprinting in parthenogenetic mouse embryos

The action of two types of substances has been studied: 5-azadeoxycytidine and retinoic acid, which have a demethylation effect on DNA in the development process of diploid parthenogenetic mouse embryos. The effect of 5-azadeoxycytidine on hybrid mice (CBA × C57BL/6)F1 in vitro for 6 h, in the presence of single cell parthenogenetic embryos during the S-phase of the cell cycle has been studied. After developing to the blastocyst stage in vitro, parthenogenetic embryos were transplanted into the uterus of false pregnant females. It has been determined that a concentration of 0.1 μM 5-azadeoxycytidine activates embryonic development in the preimplantation period until the blastocyst stage (69% in experiment; 61% in the control) and during postimplantation, it increases the number of available space in the uterus for implantation (76% in experiment; 63% in the control).     

Nanoparticles from culture media are internalized by in vitro-produced bovine embryos and its depletion affect expression of pluripotency genes

Extracellular vesicles are nanoparticles secreted by cell and have been proposed as suitable markers to identify competent embryos produced in vitro. Characterizing EVs secreted by individual embryos is challenging because culture medium itself contributes to the pool of nanoparticles that are co-isolated. To avoid this, culture medium must be depleted of nanoparticles that are present in natural protein source. The aim of this study was to evaluate if the culture medium subjected to nanoparticle depletion can support the proper in vitro development of bovine embryos. Zygotes were cultured in groups on depleted or control medium for 8 days. Nanoparticles from the medium were characterized by their morphology, size and expression of EVs surface markers. Isolated nanoparticles were labelled and added to depleted medium containing embryos at different developmental stages and evaluated after 24 hours at 2, 8-16 cells, morula and blastocyst stages. There were no statistical differences on blastocyst rate at day 7 and 8, total cell count neither blastocyst diameter between groups. However, morphological quality was better in blastocysts cultured in non-depleted medium and the expression of SOX2 was significantly lower whereas NANOG expression was significantly higher. Few nanoparticles from medium had a typical morphology of EVs but were positive to specific surface markers. Punctuated green fluorescence near the nuclei of embryonic cells was observed in embryos from all developmental stages. In summary, nanoparticles from culture medium are internalized by in vitro cultured bovine embryos and their depletion affects the capacity of medium to support the proper embryo development.     

METTL3对猪干细胞多能基因表达的调控作用

m~6A是真核生物m RNA中重要的转录后修饰,METTL3作为m~6A甲基转移酶复合物中的重要组分,在细胞重编程、胚胎干细胞和诱导多能干细胞的干性维持、胚胎发育等过程中发挥重要作用。为了揭示猪METTL3的表达模式,对不同物种METTL3蛋白序列进行了比对,用RT-PCR检测了METTL3基因在不同猪组织和细胞中的表达情况,并确认了METTL3的细胞核定位。为了研究METTL3对猪干细胞多能基因表达的调控作用,克隆了猪METTL3编码区序列,设计了METTL3干扰片段,并构建了相应的过表达和沉默载体。发现干扰METTL3的表达后,猪多能干细胞出现类似na?ve状态的细胞克隆,NANOG、OCT4和LIN28A表达水平显著升高。在猪多能干细胞培养基中添加m~6A甲基化抑制剂环亮氨酸培养细胞48 h后,试验结果与干扰METTL3表达的结果一致。本研究为优化猪多能干细胞的培养体系提供了新的方向和依据。     

Effects of retinoic acid on rat forebrain cells derived from embryonic and perinatal rats

All-trans retinoic acid (RA), a potent inducer of neural development in non-committed neuroectodermal precursors and also, a teratogenic agent for early prosencephalic development is reported to promote the survival and differentiation of embryonic forebrain neurons, in vitro. In cultures of embryonic (E13, E15) rat forebrain cells, long-term (2–5 days) treatment with RA increased the number of neurons and the overall neurofilament immunoreactivity. Treatment with RA for periods longer than 1 h resulted in enhanced binding of the non-competitive NMDA-receptor antagonist, TCP, by embryonic and fetal (E17, E18) cells, but not by cells derived from perinatal (E19, P0) forebrains. As TCP binding-sites are localised within the channel-complex, treatment with RA was thought to result in an opening of the NMDA receptor channel. In direct binding assays, however, RA had no detectable effect, while conditioned media taken from RA-treated embryonic or fetal cells increased the TCP-binding, immediately. Analyses on conditioned media taken from control cultures of cells with various in vivo or in vitro ages revealed a stable extracellular glutamate level ([Glu]_e) of 1–3 μM. This basal [Glu]_e was restored within 24 h after addition of 100 μM exogenous glutamate. In the presence of RA, however, [Glu]_e was stabilised at an approximately three-fold higher (4–10 μM) level by cells derived from embryonic and fetal brains. RA-treatment did not influence the [Glu]_e in cultures of perinatal cells. The RA-induced rise in the neurofilament-immunoreactivity of embryonic brain cell cultures was prevented by simultaneous treatment with APV, a competitive antagonist of NMDA-receptors. The data suggest that a RA-induced shift in the set-point of extracellular glutamate-balance plays an important role in the promotion of survival and maturation of developing neurons, in culture.     

Derivation and induction of the differentiation of animal ES cells as well as human pluripotent stem cells derived from fetal membrane

We succeeded in the derivation and maintenance of pluripotent embryonic stem (ES) cells from equine and bovine blastocysts. These cells expressed markers that are characteristics of mouse ES cells, namely, alkaline phosphatase, stage-specific embryonic antigen 1, STAT 3 and Oct 4. We confirmed the pluripotential ability of these cells, which were able to undergo somatic differentiation in vitro to neural progenitors and to endothelial or hematopoietic lineages. We were able to use bovine ES cells as a source of nuclei for nuclear transfer and we generated cloned cattle with a higher frequency of pregnancies to term than has been achieved with somatic cells. On the other hand, we established human fetal membrane derived stem cell lines by the colonial cloning techniques using MEMalpha culture medium containing 10 ng/ml of EGF, 10 ng/ml of LIF and 10% fetal bovine serum (FBS). These cells appeared to maintain normal karyotype in vitro and expressed markers characteristics of stem cells. Furthermore, these cells contributed to the formation of chimeric murine embryoid bodies and gave rise to all three germ layers in vitro. Results from animal ES cells and human fetal membrane derived stem cells clearly demonstrate that these cells might be used for providing different types of cells for regenerative medicine as well as used for targeted genetic manipulation of the genome.     

Gene expression patterns in bovine in vitro-produced and nuclear transfer-derived embryos and their implications for early development

Bovine in vitro-produced (IVP) and nuclear transfer (NT)-derived embryos differ from their in vivo-developed counterparts in a number of characteristics. A preeminent observation is the occurrence of the large offspring syndrome, which is correlated with considerable embryonic fetal and postnatal losses. We summarize here results from our studies in which we compared gene expression patterns from IVP and NT-derived embryos with those from their IVP counterparts. Numerous aberrations were found in IVP and NT-derived embryos, including a complete lack of expression, an induced expression, or a significant up- or downregulation of a specific gene. These alterations may affect a number of physiological functions and are considered as a kind of stress response of the embryos to deficient environmental conditions. We hypothesize that the alterations are caused by epigenetic modifications, primarily by changes in the methylation patterns. Unravelling these epigenetic modifications is promising to reveal the underlying mechanisms of the large offspring syndrome.     

Selection of reference genes in mouse embryos and in differentiating human and mouse ES cells

Embryonic Stem (ES) cells have the potential to form every cell of the body and thus are of great promise for tissue transplantation. One of the rising techniques that allows studying the differentiation state of ES cells is quantitative RT-PCR (qRT-PCR). When relative quantification by qRT-PCR is applied, accurate normalization is necessary, since differentiated embryonic stem cells and developing embryos contain heterogeneous cell populations. Corrections for variations in the qRT-PCR reaction are needed to allow comparisons between different samples. We applied the normalization tools geNorm and Normfinder to ten reference genes identifying the most stable ones for relative quantification of gene expression during differentiation of human ES cells, as well as in differentiated mouse ES cells and in the developing mouse embryo. For relative quantification by qRT-PCR in these systems, we advise to use normalization factors based on multiple stable reference genes. However, when the use of several reference genes would be unpractical, a single reference gene in each experimental setup could be sufficient. When looking for single stable reference genes, beta-actin works best in both mouse embryo and ES cell experiments and glyceraldehyde-3-phosphate-dehydrogenase can be applied in both mouse and human ES cell experiments.     

Establishment of cell lines from somite stage mouse embryos and expression of major histocompatibility class I genes in these cells

To study the regulation of MHC class I gene expression during embryonic development, we have characterized a number of clonal cell lines derived from somite stage mouse embryos that were established with or without infection by several transforming retroviruses in combination with murine leukemia viruses. Unlike embryonal carcinoma (EC) cells that have been used as a model for early embryos, the cell lines derived from somite stage embryos are negative for stage specific embryonic Ag-1 and do not appear to differentiate after retinoic acid treatment. Morphology varies from clone to clone and is distinct from that of F9 and other EC cells. In agreement with previous findings in in vivo embryos, expression of surface MHC class I antigen in 57 new clones is either undetectable or low (with variability). All of the clones respond to the addition of interferons and express MHC class I antigens at high levels, but the kinetics of mRNA accumulation vary considerably. To examine the basis of the generally low or absent MHC class I gene expression in these cells, we tested promoter activity of a MHC class I gene by CAT assay after transient DNA transfection. Regardless of the basal levels of mRNA or surface Ag, CAT activity directed by various portions of the 5' flanking region of the MHC class I gene was uniformly low. The cells showed neither the negative nor the positive regulation of MHC class I genes that had been noted respectively for EC cells and for cells expressing the Ag constitutively. The pattern seen in the new cell lines suggests that there is an intermediate stage in the developmental regulation of MHC class I gene expression that may operate during the middle to late stage of fetal development.