Generation of rabbit pluripotent stem cell lines

Pluripotent stem cells have the capacity to divide indefinitely and to differentiate into all somatic cells and tissue lines. They can be genetically manipulated in vitro by knocking genes in or out, and therefore serve as an excellent tool for gene function studies and for the generation of models for some human diseases. Since 1981, when the first mouse embryonic stem cell (ESC) line was generated, many attempts have been made to generate pluripotent stem cell lines from other species. Comparative characterization of ESCs from different species would help us to understand differences and similarities in the signaling pathways involved in the maintenance of pluripotency and the initiation of differentiation, and would reveal whether the fundamental mechanism controlling self-renewal of pluripotent cells is conserved across different species. This report gives an overview of research into embryonic and induced pluripotent stem cells in the rabbit, an important nonrodent species with considerable merits as an animal model for specific diseases. A number of putative rabbit ESC and induced pluripotent stem cell lines have been described. All of them expressed stem cell-associated markers and maintained apparent pluripotency during multiple passages in vitro, but none have been convincingly proven to be fully pluripotent in vivo. Moreover, as in other domestic species, the markers currently used to characterize the putative rabbit ESCs are suboptimal because recent studies have revealed that they are not always specific to the pluripotent inner cell mass. Future validation of rabbit pluripotent stem cells would benefit greatly from a validated panel of molecular markers specific to pluripotent cells of the developing rabbit embryos. Using rabbit-specific pluripotency genes may improve the efficiency of somatic cell reprogramming for generating induced pluripotent stem cells and thereby overcome some of the challenges limiting the potential of this technology.     

myc maintains embryonic stem cell pluripotency and self-renewal

While endogenous Myc (c-myc) and Mycn (N-myc) have been reported to be separately dispensable for murine embryonic stem cell (mESC) function, myc greatly enhances induced pluripotent stem (iPS) cell formation and overexpressed c-myc confers LIF-independence upon mESC. To address the role of myc genes in ESC and in pluripotency generally, we conditionally knocked out both c- and N-myc using myc doubly homozygously floxed mESC lines (cDKO). Both lines of myc cDKO mESC exhibited severely disrupted self-renewal, pluripotency, and survival along with enhanced differentiation. Chimeric embryos injected with DKO mESC most often completely failed to develop or in rare cases survived but with severe defects. The essential nature of myc for self-renewal and pluripotency is at least in part mediated through orchestrating pluripotency-related cell cycle and metabolic programs. This study demonstrates that endogenous myc genes are essential for mESC pluripotency and self-renewal as well as providing the first evidence that myc genes are required for early embryogenesis, suggesting potential mechanisms of myc contribution to iPS cell formation.     

Derivation and characterization of human embryonic stem cell lines from the Chinese population

Human embryonic stem cells (hESCs) can self-renew indefinitely and differentiate into all cell types in the human body. Therefore, they are valuable in regenerative medicine, human developmental biology and drug discovery. A number of hESC lines have been derived from the Chinese population,but limited of them are available for research purposes. Here we report the derivation and characterization of two hESC lines derived from human blastocysts of Chinese origin. These hESCs express alkaline phosphatase and hESC-specific markers, including Oct4, Nanog, SSEA-3, SSEA-4,TRA-1-60 and TRA-1-81. They also have high levels of telomerase activity and normal karyotypes. These cells can form embryoid body in vitro and can be differentiated into all three germ layers in vivo by teratoma formation. The newly established hESCs will be distributed for research purposes.The availability of hESC lines from the Chinese population will facilitate studies on the differences in hESCs from different ethnic groups.     

Cell signalling pathways underlying induced pluripotent stem cell reprogramming

Induced pluripotent stem(i PS) cells, somatic cells reprogrammed to the pluripotent state by forced expression of defined factors, represent a uniquely valuable resource for research and regenerative medicine. However, this methodology remains inefficient due to incomplete mechanistic understanding of the reprogramming process. In recent years, various groups have endeavoured to interrogate the cell signalling that governs the reprogramming process, including LIF/STAT3, BMP, PI3 K, FGF2, Wnt, TGFβ and MAPK pathways, with the aim of increasing our understanding and identifying new mechanisms of improving safety, reproducibility and efficiency. This has led to a unified model of reprogramming that consists of 3 stages: initiation, maturation and stabilisation. Initiation of reprogramming occurs in almost all cells that receive the reprogramming transgenes; most commonly Oct4, Sox2, Klf4 and c Myc, and involves a phenotypic mesenchymal-to-epithelial transition. The initiation stage is also characterised by increased proliferation and a metabolic switch from oxidative phosphorylation to glycolysis. The maturation stage is considered the major bottleneck within the process, resulting in very few "stabilisation competent" cells progressing to the final stabilisation phase. To reach this stage in both mouse and human cells, pre-i PS cells must activate endogenous expression of the core circuitry of pluripotency, comprising Oct4, Sox2, and Nanog, and thus reach a state of transgene independence. By the stabilisation stage, i PS cells generally use the same signalling networks that govern pluripotency in embryonic stem cells. These pathways differ between mouse and human cells although recent work has demonstrated that this is context dependent. As i PS cell generation technologies move forward, tools are being developed to interrogate the process in more detail, thus allowing a greater understanding of this intriguing biological phenomenon.     

Derivation and characterization of human embryonic stem cell lines from the Chinese population

Human embryonic stem cells(hESCs) can self-renew indefinitely and differentiate into all cell types in the human body.Therefore,they are valuable in regenerative medicine,human developmental biology and drug discovery.A number of hESC lines have been derived from the Chinese population, but limited of them are available for research purposes.Here we report the derivation and characterization of two hESC lines derived from human blastocysts of Chinese origin.These hESCs express alkaline phosphatase and hE...     

鸡胚胎干细胞的分离、培养和鉴定

SNL cells (permanent line of irradiated mouse fibroblast cells), primary mice embryonic fibroblasts (PMEF) cells and primary chicken embryonic fibroblasts (PCEF) cells were respectively used as the feeder cells for chicken embryonic stem cell culture. The isolated blastoderm cells front the stage X embryos of chicken were cultured in Dulercco‘‘ s Modified Eagle Medium (DMEM) supplemented with leukemia inhibitory factor (LIF, 1 000 IU/ml), basic fibroblast growth factor (bFGF 10 ng/ml) and stem cell factor (SCF, 5 ng/ml). The alkaline phosphatase (AKP) test, differentiation experiment in vitro and chimeric chicken production were carried out. The resuts showed that culture on feeder layer of PMEF yielded high quality CES cell colonies. The shape of typical CES clone showed as follows: nested aggregation (clone) with clear edge and round surface as well as close arrangement within the clone. Strong positive AKP reactive cellswere observed. On the other hand, the fourth passage CES cells could differentiate into various cells in the absence of feeder layer cells and LIF in vitro. The third and fourth passage cells were injected into the subgerminal cavity of recipient embryos at stage X. The manipulated embryos were incubated until hatching. Of 269 Hailan embryos injected with CES cells of Shouguang Chickens, 8.2 % (22/269) survived to hatching, 3 feather chimeras had been produced, which suggests that an effective culture systems were established and it could promote the growth of CES cells and maintain them in an undifferentiated state .     

Underwater predatory behaviour of the American mink (Mustela vison)

Fishing behaviour of the American mink ( Mustela vison Schreber) was investigated in the laboratory. Data were recorded using ciné film and tape recorded commentaries. Three species of prey were presented to mink, namely, carp ( Cyprinus curpio ), goldfish ( Carassius auratus ) and minnows ( Phoxinus phoxinus ).
It was found to be necessary to train ranch-bred mink to enter water and catch fish; young mink appeared to be easier to train than adults. Mink spent 5–20 sec under water when fishing; prey had usually previously been located from an aerial vantage point. Predatory behaviour was highly organized sequentially whereas fish were more prone to indulge in unpredictable stratagems; the behaviour of mink and fish were highly correlated.
The mink's efficiency in catching fish was related to prey size (smaller individuals being more vulnerable to capture) and shoaling. Minnows, which form highly organized shoals, were less easily caught when present in large numbers; this was not true of a loosely shoaling species, the carp.
Of the three species of prey presented, vulnerability to capture took the form goldfish> carp> minnow; these differences, however, may have been influenced by the fish's previous experience of underwater predators.     

Mapping of 53 loci in American mink (Mustela vison)

Fifty-three genes were mapped in the American mink genome using polymerase chain reaction (PCR)-based analysis of a Chinese hamster-American mink somatic cell hybrid panel. Heterologous primers designed for cat gene mapping were used in this study. Forty-nine of these loci were localized into expected chromosome regions according to Zoo-FISH data, whereas four loci--ALPL, CDC20, ERF-2, and Fc(Mv)23617--were mapped out of expected conserved regions. PCR products amplified with primers corresponding to these four markers were partly sequenced and verified using BLAST. The results showed the homology to be more than 90% between mink and human or cat counterparts. At present, the gene map of American mink has expanded to 127 loci.     

Multivariate variation in feral American mink (Mustela vison) from Southern Norway

Multiple group principal component analysis was used to investigate the morphological relationships between feral American mink from three areas of Southern Norway based on 15 skull dimensions. The first principal axis calculated from the variance-covariance matrix of log-transformed data is usually interpreted as a growth axis. In the present analysis, the scores of the first principal axis of males were found to be uncorrelated with estimated age for specimens younger than seven months old, and was interpreted as a growth-free size axis. However, the second component of males was found to reflect mainly growth in the postorbital area. The first component of females was thought to reflect a mixing of growth-out and growth-in size variation. One of the reasons for separating size and size-free variation in the present work was to test whether growth-dependent size influenced the assessment of geographic variation within each sex. The scores on the components reflecting size variation (growthout and growth-in) were, however, not found to deviate significantly between the areas. Discriminant analyses run on the component scores, both with size variation included and excluded, gave the same result. In mean, the mink from Western Norway deviated significantly from the mink from Trondelag and Eastern Norway, while the mink from the two latter areas did not deviate morphometrically. The deviation found was in shape rather than in size of both sexes.     

Induction of pluripotency in mammalian fibroblasts by cell fusion with mouse embryonic stem cells

BackgroundCell fusion is a phenomenon that is observed in various tissues in vivo, resulting in acquisition of physiological functions such as liver regeneration. Fused cells such as hybridomas have also been produced artificially in vitro. Furthermore, it has been reported that cellular reprogramming can be induced by cell fusion with stem cells.MethodsFused cells between mammalian fibroblasts and mouse embryonic stem cells were produced by electrofusion methods. The phenotypes of each cell lines were analyzed after purifying the fused cells.ResultsColonies which are morphologically similar to mouse embryonic stem cells were observed in fused cells of rabbit, bovine, and zebra fibroblasts. RT-PCR analysis revealed that specific pluripotent marker genes that were never expressed in each mammalian fibroblast were strongly induced in the fused cells, which indicated that fusion with mouse embryonic stem cells can trigger reprogramming and acquisition of pluripotency in various mammalian somatic cells.ConclusionsOur results can help elucidate the mechanism of pluripotency maintenance and the establishment of highly reprogrammed pluripotent stem cells in various mammalian species.     

Quality of embryonic bodies and seeding density effects on neural differentiation of mouse embryonic stem cells

Mouse embryonic stem (ES) cells can be differentiated into neural lineage cells, but the differentiation efficiency remains low. This study revealed two important factors that influence the neural differentiation efficiency of mouse ES cells: the first is the quality of embryonic bodies (EBs); good quality of EBs consistently originated from a suspension culture of 1 × 10⁵ ES cells/ml serum-free chemically defined neural inducing medium and they exhibited a smooth round shape, with a dark central region surrounded by a light band. Such EBs are capable of attaining high neural differentiation efficiency. However, poor quality EBs originated from a suspension culture of 1 × 10⁶ ES cells/ml serum-free chemically defined neural inducing medium and exhibited an irregular shape or adhered to the bottom of the dish; they displayed low neural differentiation efficiency. The second factor is the seeding density of EBs: a low seeding density (5 EBs/cm²) induced cells to differentiate into a more caudalized subtypes compared to the cells obtained from high seeding density (20 EBs/cm²). These findings provided fresh insight into the neural induction of mouse ES cells.     

Generation and characterization of bat-induced pluripotent stem cells

Induced pluripotent stem cells (iPSCs) were first generated from mouse embryonic fibroblasts in the year 2006. These cells resemble the typical morphology of embryonic stem cells, express pluripotency markers, and are able to transmit through germlines. To date, iPSCs of many species have been generated, whereas generation of bat iPSCs (biPSCs) has not been reported. To facilitate in-depth study of bats at the molecular and cellular levels, we describe the successful derivation of biPSCs with a piggyBac (PB) vector that contains eight reprogramming factors Oct4, Sox2, Klf4, Nanog, cMyc, Lin28, Nr5a2, and miR302/367. These biPSCs were cultured in media containing leukemia inhibitory factor and three small molecule inhibitors (CHIR99021, PD0325901, and A8301). They retained normal karyotype, displayed alkaline phosphatase activity, and expressed pluripotency markers Oct4, Sox2, Nanog, TBX3, and TRA-1-60. They could differentiate in vitro to form embryoid bodies and in vivo to form teratomas that contained tissue cells of all three germ layers. Generation of biPSCs will facilitate future studies on the mechanisms of antiviral immunity and longevity of bats at the cellular level.