Search

The zebrafish species Danio rerio has become one of the major vertebrate model organisms used in biomedical research. However, there are aspects of the model that need to be improved. One of these is the ability to identify individual fish and fish lines by DNA profiling. Although many dinucleotide short tandem repeat (diSTR) markers are available for this and similar purposes, they have certain disadvantages such as an excessive polymerase slippage (“stutter”) that causes difficulties in automated genotyping and cross-laboratory comparisons. Here we report on the development of a 13-plex of tetranucleotide and pentanucleotide STRs (tetraSTRs and pentaSTRs, respectively) that have low stutter. The system uses an inexpensive universal primer labelling system, which can easily be converted to a direct labeling system if desired. This 13-plex was examined in three zebrafish lines (NHGRI-1, kca33Tg, and kca66Tg, originally obtained from ZIRC). The average observed heterozygosity (Ho) and expected heterozygosity (He) in these highly inbred lines were 0.291 and 0.359, respectively, which is very similar to what has been found with diSTRs. The probability of identity (PI) for all fish tested was 2.1 × 10−5 and the PI for siblings (PIsib) was 6.4 × 10−3, as calculated by the Genalex package. Ninety percent of the fish tested were correctly identified with their respective strains. It is also demonstrated that this panel can be used to confirm doubled-haploid cell lines. This multiplex should find multiple uses for improving the accuracy and reproducibility of studies using the zebrafish model.

The human genome with all its ethnic variations contributes to differences in human development, aging, disease, repair and response to medical treatments and is an exciting area of research and clinical study. The availability of well-characterized ethnically diverse stem cell lines is limited and has not kept pace with other advances in stem cell research. Here we derived xenofree ethnically diverse-human induced pluripotent stem cell (ED-iPSC) lines from fibroblasts obtained from individuals of African American, Hispanic-Latino, Asian and Caucasian ethnic origin and have characterized the lines under a uniform platform for comparative analysis. Derived ED-iPSC lines are low passage number and evaluated in vivo by teratoma formation and in vitro by high throughput microarray analysis of EB formation and early differentiation for tri-lineage commitment to endoderm, ectoderm and mesoderm. These new xenofree ED-iPSC lines represent a well-characterized valuable resource with potential for use in future research in drug discovery or clinical investigations.

Understanding the mechanisms of epigenetic remodeling that follow fertilization is a fundamental step toward understanding the bases of early embryonic development and pluripotency. Extensive and dynamic chromatin remodeling is observed after fertilization, including DNA methylation and histone modifications. These changes underlie the transition from gametic to embryonic chromatin and are thought to facilitate embryonic genome activation. In particular, trimethylation of histone 3 lysine 27 (H3K27me3) is associated with gene-specific transcription repression. Global levels of this epigenetic mark are high in oocyte chromatin and decrease to minimal levels at the time of embryonic genome activation. We provide evidence that the decrease in H3K27me3 observed during early development is cell-cycle independent, suggesting an active mechanism for removal of this epigenetic mark. Among H3K27me3-specific demethylases, Jumonji domain-containing protein 3 (JMJD3), but not ubiquitously transcribed tetratricopeptide repeat X (UTX), present high transcript levels in oocytes. Soon after fertilization JMJD3 protein levels increase, concurrent with a decrease in mRNA levels. This pattern of expression suggests maternal inheritance of JMJD3. Knockdown of JMJD3 by siRNA injection in parthenogenetically activated metaphase II oocytes resulted in inhibition of the H3K27me3 decrease normally observed in preimplantation embryos. Moreover, knockdown of JMJD3 in oocytes reduced the rate of blastocyst development. Overall, these results indicate that JMJD3 is involved in active demethylation of H3K27me3 during early embryo development and that this mark plays an important role during the progression of embryos to blastocysts.

The legends for Figure 7 and Figure 8 were switched in production; the legend for Figure 7 should be that of Figure 8 and vice versa. The figures themselves are correct.

Background Global activation of the embryonic genome (EGA), one of the most critical steps in early mammalian embryo development, is recognized as the time when interspecies somatic cell nuclear transfer (iSCNT) embryos fail to thrive. Methodology/Principal Findings In this study, we analyzed the EGA-related transcriptome of rhesus-bovine iSCNT 8- to 16-cell embryos and dissected the reprogramming process in terms of embryonic gene activation, somatic gene silencing, and maternal RNA degradation. Compared with fibroblast donor cells, two thousand and seven genes were activated in iSCNT embryos, one quarter of them reaching expression levels comparable to those found in in vitro fertilized (IVF) rhesus embryos. This suggested that EGA in iSCNT embryos had partially recapitulated rhesus embryonic development. Eight hundred and sixty somatic genes were not silenced properly and continued to be expressed in iSCNT embryos, which indicated incomplete nuclear reprogramming. We compared maternal RNA degradation in bovine oocytes between bovine-bovine SCNT and iSCNT embryos. While maternal RNA degradation occurred in both SCNT and iSCNT embryos, we saw more limited overall degradation of maternal RNA in iSCNT embryos than in SCNT embryos. Several important maternal RNAs, like GPF9, were not properly processed in SCNT embryos. Conclusions/Significance Our data suggested that iSCNT embryos are capable of triggering EGA, while a portion of somatic cell-associated genes maintain their expression. Maternal RNA degradation seems to be impaired in iSCNT embryos. Further understanding of the biological roles of these genes, networks, and pathways revealed by iSCNT may expand our knowledge about cell reprogramming, pluripotency, and differentiation.

BackgroundHuman embryonic stem cells (hESCs) are an attractive resource for new therapeutic approaches that involve tissue regeneration. hESCs have exhibited low immunogenicity due to low levels of Mayor Histocompatibility Complex (MHC) class-I and absence of MHC class-II expression. Nevertheless, the mechanisms regulating MHC expression in hESCs had not been explored.Methodology/principal findingsWe analyzed the expression levels of classical and non-classical MHC class-I, MHC class-II molecules, antigen-processing machinery (APM) components and NKG2D ligands (NKG2D-L) in hESCs, induced pluripotent stem cells (iPSCs) and NTera2 (NT2) teratocarcinoma cell line. Epigenetic mechanisms involved in the regulation of these genes were investigated by bisulfite sequencing and chromatin immunoprecipitation (ChIP) assays. We showed that low levels of MHC class-I molecules were associated with absent or reduced expression of the transporter associated with antigen processing 1 (TAP-1) and tapasin (TPN) components in hESCs and iPSCs, which are involved in the transport and load of peptides. Furthermore, lack of beta2-microglobulin (beta2m) light chain in these cells limited the expression of MHC class I trimeric molecule on the cell surface. NKG2D ligands (MICA, MICB) were observed in all pluripotent stem cells lines. Epigenetic analysis showed that H3K9me3 repressed the TPN gene in undifferentiated cells whilst HLA-B and beta2m acquired the H3K4me3 modification during the differentiation to embryoid bodies (EBs). Absence of HLA-DR and HLA-G expression was regulated by DNA methylation.Conclusions/significanceOur data provide fundamental evidence for the epigenetic control of MHC in hESCs and iPSCs. Reduced MHC class I and class II expression in hESCs and iPSCs can limit their recognition by the immune response against these cells. The knowledge of these mechanisms will further allow the development of strategies to induce tolerance and improve stem cell allograft acceptance.

The first two authors, Steven T. Suhr and Eun Ah Chang, should be noted as having contributed equally to this work.

BACKGROUND Human induced pluripotent stem cells (IPSCs) have enormous potential in the development of cellular models of human disease and represent a potential source of autologous cells and tissues for therapeutic use. A question remains as to the biological age of IPSCs, in particular when isolated from older subjects. Studies of cloned animals indicate that somatic cells reprogrammed to pluripotency variably display telomere elongation, a common indicator of cell "rejuvenation." METHODOLOGY/PRINCIPAL FINDINGS We examined telomere lengths in human skin fibroblasts isolated from younger and older subjects, fibroblasts converted to IPSCs, and IPSCs redifferentiated through teratoma formation and explant culture. In IPSCs analyzed at passage five (P5), telomeres were significantly elongated in 6/7 lines by >40% and approximated telomere lengths in human embryonic stem cells (hESCs). In cell lines derived from three IPSC-teratoma explants cultured to P5, two displayed telomeres shortened to lengths similar to input fibroblasts while the third line retained elongated telomeres. CONCLUSIONS/SIGNIFICANCE While these results reveal some heterogeneity in the reprogramming process with respect to telomere length, human somatic cells reprogrammed to pluripotency generally displayed elongated telomeres that suggest that they will not age prematurely when isolated from subjects of essentially any age.

Egg activation, which is the first step in the initiation of embryo development, involves both completion of meiosis and progression into mitotic cycles. In mammals, the fertilizing sperm delivers the activating signal, which consists of oscillations in free cytosolic Ca(2+) concentration ([Ca(2+)](i)). Intracytoplasmic sperm injection (ICSI) is a technique that in vitro fertilization clinics use to treat a myriad of male factor infertility cases. Importantly, some patients who repeatedly fail ICSI also fail to induce egg activation and are, therefore, sterile. Here, we have found that sperm from patients who repeatedly failed ICSI were unable to induce [Ca(2+)](i) oscillations in mouse eggs. We have also shown that PLC, zeta 1 (PLCZ1), the sperm protein thought to induce [Ca(2+)](i) oscillations, was localized to the equatorial region of wild-type sperm heads but was undetectable in sperm from patients who had failed ICSI. The absence of PLCZ1 in these patients was further confirmed by Western blot, although genomic sequencing failed to reveal conclusive PLCZ1 mutations. Using mouse eggs, we reproduced the failure of sperm from these patients to induce egg activation and rescued it by injection of mouse Plcz1 mRNA. Together, our results indicate that the inability of human sperm to initiate [Ca(2+)](i) oscillations leads to failure of egg activation and sterility and that abnormal PLCZ1 expression underlies this functional defect.

Each year many scientific meetings are held on stem cells to appraise the state of knowledge on their potency, differentiation and applications. So why did we hold another meeting? Because we thought one aspect was not adequately addressed in the others. When thinking of how our body is derived from a single fertilized egg, it is self-evident that the embryo is the mother of all stem cells. This fact is probably overlooked because it is so remote (decades back in our lives!) and because embryonic stem cells do not exist as such in the embryo. However, this also tends to be ignored on purpose in many stem cell meetings because working on (human) embryos brings up substantial ethical concerns that bear on the scientific undertaking like nothing else. The origin of stem cells has become even more of a sensitive issue since the discovery in 2006 that embryonic stem (ES) cell-like cells can be generated in a petri dish straight from somatic cells by retrovirus-mediated transfer of selected genes. These new cells have been named induced pluripotent stem (iPS) cells and have been obtained without any egg or embryo consumption (Takahashi and Yamanaka, 2006). This leads to the first topic of our meeting: natural and induced pluripotency... ispartof: The International Journal of Developmental Biology vol:52 issue:7 pages:801-809 ispartof: location:Spain status: published

Parthenogenesis is the biological phenomenon by which embryonic development is initiated without male contribution. Whereas parthenogenesis is a common mode of reproduction in lower organisms, the mammalian parthenote fails to produce a successful pregnancy. We herein describein vitroparthenogenetic development of monkey (Macaca fascicularis) eggs to the blastocyst stage, and their use to create a pluripotent line of stem cells. These monkey stem cells (Cyno-1 cells) are positive for telomerase activity and are immunoreactive for alkaline phosphatase, octamer-binding transcription factor 4 (Oct-4), stage-specific embryonic antigen 4 (SSEA-4), tumor rejection antigen 1-60 (TRA 1-60), and tumor rejection antigen 1-81 (TRA 1-81) (traditional markers of human embryonic stem cells). They have a normal chromosome karyotype (40 + 2) and can be maintainedin vitroin an undifferentiated state for extended periods of time. Cyno-1 cells can be differentiatedin vitrointo dopaminergic and serotonergic neurons, contractile cardiomyocyte-like cells, smooth muscle, ciliated epithelia, and adipocytes. When Cyno-1 cells were injected into severe combined immunodeficient mice, teratomas with derivatives from all three embryonic germ layers were obtained. When grown on fibronectin/laminin-coated plates and in neural progenitor medium, Cyno-1 cells assume a neural precursor phenotype (immunoreactive for nestin). However, these cells remain proliferative and express no functional ion channels. When transferred to differentiation conditions, the nestin-positive precursors assume neuronal and epithelial morphologies. Over time, these cells acquire electrophysiological characteristics of functional neurons (appearance of tetrodotoxin-sensitive, voltage-dependent sodium channels). These results suggest that stem cells derived from the parthenogenetically activated nonhuman primate egg provide a potential source for autologous cell therapy in the female and bypass the need for creating a competent embryo.

The field of regenerative medicine is moving toward translation to clinical practice. However, there are still knowledge gaps and safety concerns regarding stem cell-based therapies. Improving large animal models and methods for transplantation, engraftment, and imaging should help address these issues, facilitating eventual use of stem cells in the clinic.

While somatic cell nuclear transfer (SCNT) techniques have been successfully implemented in several species to produce cloned embryos and offspring, the efficiencies of the procedures are extremely low, possibly due to insufficient reprogramming of somatic nuclei. Employing GeneChip microarrays, we describe global gene expression analysis of bovine in vitro fertilized (IVF) and SCNT blastocysts as well as respective donor cell lines to characterize differences in their transcription profiles. Gene expression profiles of our donor cell lines were significantly different from each other; however, the SCNT and IVF blastocysts displayed surprisingly similar gene expression profiles, suggesting that a major reprogramming activity had been exerted on the somatic nuclei. Despite this remarkable phenomenon, a small set of genes appears to be aberrantly expressed and may affect critical developmental processes responsible for the failures observed in SCNT embryos. Our data provide the most comprehensive transcriptome database of bovine IVF and SCNT blastocysts to date.