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PubMed2005

Liver stem cell-derived beta-cell surrogates for treatment of type 1 diabetes.

Consistent with the common embryonic origin of liver and pancreas as well the similar glucose-sensing systems in hepatocytes and pancreatic beta-cells, it should not be surprising that liver stem cells/hepatocytes can transdifferentiate into insulin-producing cells under high-glucose culture conditions or by genetic reprogramming. Persistent expression of the pancreatic duodenal homeobox-1 (Pdx1) transcription factor or its super-active form Pdx1-VP16 fusion protein in hepatic cells reprograms these cells into pancreatic beta-cell precursors. In vitro culture at elevated glucose concentrations or in vivo exposure to a hyperglycemia are required for further differentiation and maturation of liver-derived pancreatic beta-cell precursor into functional insulin-producing pancreatic beta-like cells. Under appropriate conditions, multiple pancreatic transcription factors can work in concert to reprogram liver stem/adult liver cells into functional insulin-producing cells. If such autologous liver-derived insulin-producing cells can be made to escape the type 1 diabetes-associated autoimmunity, they may serve as a valuable cell source for future cell replacement therapy without the need for life-long immunosuppression.

Animals↗

PubMed2005

Oxidative-induced calcium mobilization is dependent on annexin VI release from lipid rafts.

BACKGROUND: Oxidative stress results in macrophage reprogramming through the formation of focal adhesion-like complexes on lipid rafts. Although the cellular mechanisms responsible for this reprogramming remain unknown, oxidative stress is known to result in a transient increase in intracellular calcium. This transient flux is thought to occur through the membrane dissociation of the calcium-bound protein annexin VI. The purpose of this study is to clarify the source of the calcium, and determine if it is responsible for the formation of focal adhesion-like complexes during oxidative stress through the activation of calcium/calmodulin dependent protein kinase II (CaMK II). METHODS: THP-1 cells were stimulated with hydrogen peroxide. Selected cells were pretreated with methyl beta-cyclodextrin (MbetaCD), a cholesterol-depleting agent; 1,2-bis aminophenoxy ethane-N,N,N',N'-tetraacetic acid, an intracellular calcium chelator; or autocamtide 2-related inhibitory peptide, a CaMK II inhibitor. Intracellular calcium flux was determined by a Fluo-3 technique. Lipid raft and cellular protein were extracted and analyzed for active CaMK II, annexin VI, and components of focal adhesion-like complexes. RESULTS: Hydrogen peroxide exposure led to mobilization of annexin VI from lipid rafts to the cytosol, which was followed by an increase in cytosolic calcium, phosphorylation of CaMK II, and formation of focal adhesion-like complexes. Cholesterol depletion from lipid rafts attenuated all of these effects. 1,2-bis Aminophenoxy ethane-N,N,N',N'-tetraacetic acid and autocamtide 2-related inhibitory peptide pretreatment attenuated CaMK II phopshorylation and formation of focal ahdesionlike complexes. CONCLUSIONS: Macrophage reprogramming during oxidative stress occurs through the cytosolic mobilization of annexin VI from lipid rafts. As a result, bound calcium dissociates, resulting in the activation of CaMK II and the formation of focal adhesion-like complexes.

Annexin A6↗

PubMed2026

Epigenetic reduction OF H3K9me3 and H3K27me3 by RK-701 and GSK 126 improves the developmental competence of bovine SCNT embryos.

Somatic cell nuclear transfer (SCNT) failure has largely been attributed to incomplete epigenetic reprogramming, particularly the dysregulation of repressive histone modifications such as H3K9me3 and H3K27me3. Reducing these repressive marks has been shown to improve reprogramming efficiency in SCNT embryos. Although histone demethylase mRNA injection has been used for this purpose, it is labor-intensive, technically demanding, and time-consuming. In this study, we investigated a simplified approach that combined RK-701 and GSK-126 to reduce H3K9me3 and H3K27me3 levels, respectively, in bovine SCNT embryos. Three experimental groups were established: IVF embryos (control), SCNT-control (SCNT-C) embryos, and inhibitor-treated SCNT embryos (SCNT-T). The IVF group was used as a reference standard. Fused one-cell SCNT embryos were treated with 2 μM RK-701 and 0.2 μM GSK-126 from the one-cell stage to the 16-cell stage. Gene expression analysis at the 16-cell stage revealed a significant reduction in histone methyltransferase (HMT) expression (p < 0.05), and immunofluorescence analysis confirmed marked decreases in H3K9me3 and H3K27me3 levels. In addition, the expression of genes associated with zygotic genome activation (ZGA) and pluripotency was significantly higher in SCNT-T embryos than in SCNT-C embryos. Assessment of blastocyst quality revealed reduced reactive oxygen species (ROS) levels, decreased expression of apoptosis-related genes, and improved mitochondrial membrane potential in the treated group, as indicated by JC1 staining. Overall, this approach effectively reduced repressive histone marks, enhanced epigenetic reprogramming, and improved ZGA, thereby increasing the developmental rate and adhesion potential of bovine SCNT embryos. These findings suggest that combined treatment with RK-701 and GSK-126 may provide a simple and practical strategy for improving the efficiency of bovine cloning.

Bovine embryos↗

PubMed2005

Differential nuclear remodeling of mammalian somatic cells by Xenopus laevis oocyte and egg cytoplasm.

The mechanisms governing nuclear reprogramming have not been fully elucidated yet; however, recent studies show a universally conserved ability of both oocyte and egg components to reprogram gene expression in somatic cells. The activation of genes associated with pluripotency by oocyte/egg components may require the remodeling of nuclear structures, such that they can acquire the features of early embryos and pluripotent cells. Here, we report on the remodeling of the nuclear lamina of mammalian cells by Xenopus oocyte and egg extracts. Lamin A/C is removed from somatic cells incubated in oocyte and egg extracts in an active process that requires permeable nuclear pores. Removal of lamin A/C is specific, since B-type lamins are not changed, and it is not dependent on the incorporation Xenopus egg specific lamin III. Moreover, transcriptional activity is differentially regulated in somatic cells incubated in the extracts. Pol I and II transcriptions are maintained in cells in oocyte extracts; however, both activities are abolished in egg extracts. Our study shows that components of oocyte and egg extracts can modify the nuclear lamina of somatic cells and that this nuclear remodeling induces a structural change in the nucleus which may have implications for transcriptional activity. These experiments suggest that modifications in the nuclear lamina structure by the removal of somatic proteins and the incorporation of oocyte/egg components may contribute to the reprogramming of somatic cell nuclei and may define a characteristic configuration of pluripotent cells.

Animals↗

PubMed1999

Survival of DDD pacing mode after atrioventricular junction ablation and pacing for refractory atrial fibrillation.

BACKGROUND: Patients with recurrent forms of atrial fibrillation may receive dual-chamber pacemakers after atrioventricular junction ablation for refractory symptoms. These patients are at risk for chronic atrial fibrillation, which would negate the benefits of dual-chamber pacing. The purpose of this study was to examine the survival of dual-chamber pacing modes in patients undergoing ablate and pace therapy. METHODS AND RESULTS: One hundred fifty-six patients underwent ablate and pace therapy for medically refractory chronic (70 patients) or recurrent (86 patients) atrial fibrillation. Seventy-eight percent of patients had structural heart disease. The mean age was 66 +/- 11 years, with an average ejection fraction of 48% +/- 18%. The choice of pacing mode and programming were at the discretion of the investigators. At implantation, 91 patients (58%) were programmed to VVI mode, 47 (30%) were programmed to DDD mode, and 18 (12%) were programmed to DDI mode. After 1 year of follow-up, 10 DDD patients were reprogrammed to VVI mode (7 patients) or DDI mode (3 patients), most frequently for chronic atrial fibrillation (7 patients). Two patients with DDI mode were reprogrammed to VVI and DDD modes (1 patient each). Survival of the DDD mode was 76% at 1 year by Kaplan-Meier analysis. Reprogramming from DDD mode was not associated with patient age, left ventricular ejection fraction, discontinuation of antiarrhythmic drugs, or the duration of atrial fibrillation symptoms before ablation. CONCLUSIONS: Seventy-six percent of patients with recurrent atrial fibrillation who are initially programmed to DDD mode remain in DDD mode 1 year after ablation and pacing therapy. The modest rate of progression to chronic atrial fibrillation supports the use of dual-chamber pacing in this setting.

Aged↗

PubMed1998

Cloning: new breakthroughs leading to commercial opportunities.

Research on cloning animals, again, came to the forefront of public attention in 1997. Most scientists involved in biomedical and agricultural research have emphasized the benefits, of which there are many, of cloning to the public. Basic studies on nuclear transfer have and will continue to contribute to our understanding of how genomic activation and cell cycle synchrony affect nuclear reprogramming and cloning efficiencies, specifically. Also, more basic information on actual mechanisms and specific factors in the oocyte causing nuclear reprogramming is forthcoming. As new molecular approaches in functional genomics are combined with nuclear transfer experiments, new genes involved in nuclear reprogramming will be found. The commercial potentials of products stemming from discoveries in cloning are vast. Cloning will be a more efficient, faster and more useful way of making transgenic fetuses for cell therapies, adult animals for protein production and organs for xenotransplantation. Clearly there are new opportunities in animal cloning technology that will produce many benefits to society.

Animals↗

PubMed2003

Teaching cells new tricks.

The direct conversion of one differentiated cell type into another--a process referred to as transdifferentiation--would be beneficial for producing isogenic (patient's own) cells to replace sick or damaged cells or tissue. Adult stem cells display a broader differentiation potential than anticipated and might contribute to tissues other than those in which they reside. As such, they could be worthy therapeutic agents. Recent advances in transdifferentiation involve nuclear transplantation, manipulation of cell culture conditions, induction of ectopic gene expression and uptake of molecules from cellular extracts. These approaches open the doors to new avenues for engineering isogenic replacement cells. To avoid unpredictable tissue transformation, nuclear reprogramming requires controlled and heritable epigenetic modifications. Considerable efforts remain to unravel the molecular processes underlying nuclear reprogramming and evaluate stable of the changes in reprogrammed cells.

Adipocytes↗

PubMed2000

Active demethylation of the paternal genome in the mouse zygote.

DNA methylation is essential for the control of a number of biological mechanisms in mammals [1]. Mammalian development is accompanied by two major waves of genome-wide demethylation and remethylation: one during germ-cell development and the other after fertilisation [2] [3] [4] [5] [6] [7]. Most previous studies have suggested that the genome-wide demethylation observed after fertilisation occurs passively, that is, by the lack of maintenance methylation following DNA replication and cell division [6] [7], although one other study has reported that replication-independent demethylation may also occur during early embryogenesis [8]. Here, we report that genes that are highly methylated in sperm are rapidly demethylated in the zygote only hours after fertilisation, before the first round of DNA replication commences. By contrast, the oocyte-derived maternal alleles are unaffected by this reprogramming. They either remain methylated after fertilisation or become further methylated de novo. These results provide the first direct evidence for active demethylation of single-copy genes in the mammalian zygote and, moreover, reveal a striking asymmetry in epigenetic methylation reprogramming. Whereas paternally (sperm)-derived sequences are exposed to putative active demethylases in the oocyte cytoplasm, maternally (oocyte)-derived sequences are protected from this reaction. These results, whose generality is supported by findings of Mayer et al. [9], have important implications for the establishment of biparental genetic totipotency after fertilisation, the establishment and maintenance of genomic imprinting, and the reprogramming of somatic cells during cloning.

Actins↗

PubMed2001

Aberrant methylation of donor genome in cloned bovine embryos.

Despite recent successes in cloning various animal species, the use of somatic cells as the source of donor nuclei has raised many practically relevant questions such as increased abortion rates, high birth weight and perinatal death. These anomalies may be caused by incomplete epigenetic reprogramming of donor DNA. Genome-wide demethylation occurs during early development, 'erasing' gamete-specific methylation patterns inherited from the parents. This process may be a prerequisite for the formation of pluripotent stem cells that are important for the later development. Here, we provide evidence that cloned bovine embryos may have impaired epigenetic reprogramming capabilities. We found highly aberrant methylation patterns in various genomic regions of cloned embryos. Cloned blastocysts closely resembled donor cells in their overall genomic methylation status, which was very different from that of normal blastocysts produced in vitro or in vivo. We found demethylation of the Bov-B long interspersed nuclear element sequence in normal embryos, but not in cloned embryos, in which the donor-type methylation was simply maintained during preimplantation development. There were also significant variations in the degree of methylation among individual cloned blastocysts. Our findings indicate that the developmental anomalies of cloned embryos could be due to incomplete epigenetic reprogramming of donor genomic DNA.

Animals↗

PubMed1999

The maternal, fetal and postnatal somatotrophic axes in intrauterine growth retardation.

Both the maternal and fetal somatotrophic axes are closely linked to fetal substrate supply. Nutritional insults at critical stages of fetal development may lead to permanent reprogramming of the relationships between these factors. The consequences of reprogramming during fetal life may be harmful to metabolic, endocrine and cardiovascular homoeostatic mechanisms in postnatal life. The exact mechanisms that lead to reprogramming during fetal life need thorough investigation before effective strategies to deal with this problem can be devised.

Amniotic Fluid↗

PubMed2000

The paternal methylation imprint of the mouse H19 locus is acquired in the gonocyte stage during foetal testis development.

BACKGROUND: Germline-specific differential DNA methylation that persists through fertilization and embryonic development is thought to be the 'imprint' distinguishing the parental alleles of imprinted genes. If such methylation is to work as the imprinting mechanism, however, it has to be reprogrammed following each passage through the germline. Previous studies on maternally methylated genes have shown that their methylation imprints are first erased in primordial germ cells (PGCs) and then re-established during oocyte growth. RESULTS: We have examined the timing of the reprogramming of the paternal methylation imprint of the mouse H19 gene during germ cell development. In both male and female PGCs, the paternal allele is partially methylated whereas the maternal allele is unmethylated. This partial methylation is completely erased in the female germline by entry into meiosis, establishing the oocyte methylation pattern. In the male germline, both alleles become methylated, mainly during the gonocyte stage, establishing the sperm methylation pattern. CONCLUSION: The paternal methylation imprint of H19 is established in the male germline and erased in the female germline at specific developmental stages. The identification of the timings of the methylation and demethylation should help to identify and characterize the biochemical basis of the reprogramming of imprinting.

Animals↗

PubMed2003

Automatic sensor algorithms expedite pacemaker follow-ups.

OBJECTIVE: Automatic algorithms can be used to optimize settings and reduce the duration of pacemaker (PM) clinical follow-up. METHODS: This study prospectively evaluated 87 patients (74.2 +/- 10.7 years old, 52% men) who received PM with the Autoslope algorithm. Patients randomized to the manual group (group M, n = 43) performed a walk test and used sensor-indicated rate histograms to adjust the sensor, while in the automatic group (group A, n = 44) the sensor was automatically adjusted by the Autoslope. The patients were followed for 6 months. Follow-up time required for device interrogation and optimal sensor set-up, and the number of sensor parameters reprogramming were recorded. Changes in the patients' activity level were also evaluated. RESULTS: Group A required significantly less follow-up time than group M (9.4 +/- 5.7 min vs 13.5 +/- 8.5 min, P = 0.0002). The average number of sensor parameters reprogrammed during visits was significantly lower in group A than M (0.6 +/- 0.9 vs 0.9 +/- 1.3, P = 0.048). Threshold was adjusted 34.4% of the time in the sensor evaluations in group M versus 12.9% in group A (P = 0.0004). Although more patients in group A reported being more active, the changes in patients' activity level did not lead to increasing sensor setup time or number of parameter reprogramming in either group. CONCLUSIONS: Auto sensor adjustment required less time during routine PM clinical follow-up by reducing steps needed for manual sensor threshold adjustment.

Aged↗

PubMed2004

Prospective randomized study of mode switching in a clinical trial of pacemaker therapy for sinus node dysfunction.

INTRODUCTION: Atrial fibrillation (AF) is common in pacemaker patients with sinus node dysfunction (SND) and may result in rapid ventricular pacing (RVP) in the DDDR mode. Mode switching (MS) reduces RVP, but its clinical benefit in patients with SND is unknown. METHODS AND RESULTS: Two hundred two patients in the Mode Selection Trial (MOST; 2,010-patient, 6-year trial of DDDR vs VVIR pacing in SND) randomized to DDDR pacemakers with atrial high-rate episode (AHRE) storage capabilities were subrandomized to MS ON (N = 96) or MS OFF (N = 106). Cardiovascular symptoms, quality of life (QOL), reprogramming due to RVP, death, stroke, and heart failure hospitalization (HFH) were compared between groups. The treatment groups were similar with regard to AF history (59% MS ON vs 57% MS OFF). AHREs occurred in 49% patients during median follow-up of 2.2 years. Median AHRE duration (in min; MS ON 116 vs MS OFF 58, P = 0.29), frequency AHREs/week (MS ON 3.5 vs MS OFF 6.4, P = 0.23), and time spent in AHRE (min/week) (MS ON 450, MS OFF 268) were similar. Reprogramming due to any RVP during AHREs occurred more in MS OFF vs MS ON (13.2% vs 3.1%, P = 0.011) and marginally more for symptomatic RVP (8.5% vs 2.1%, P = 0.062). Cardiovascular symptoms occurred in 93.6% MS ON vs 90.2% MS OFF (P = 0.38). Median number of symptoms reported per visit was similar (MS ON 1.3 vs MS OFF 1.5, P = 0.62). Median symptom frequency/severity, summed and averaged over visits, was similar (MS ON 4.3 vs MS OFF 4.5, P = 0.74). QOL was not different between groups. Death, stroke, and HFH were not different between groups. CONCLUSION: MS reduces pacemaker reprogramming due to RVP during AHREs in a small number of patients but does not improve QOL or cardiovascular symptoms overall among patients with SND.

Aged↗

PubMed2005

Utility of routine follow-up defibrillator threshold testing in congenital heart disease and pediatric populations.

INTRODUCTION: Recent studies have suggested that routine defibrillation threshold (DFT) testing of implantable cardioverter defibrillators (ICDs) in adults may not be necessary. The congenital heart disease and pediatric populations are a unique group of ICD recipients having a higher incidence of lead failure. We investigated the utility of follow-up DFT testing in this population. METHODS AND RESULTS: The records of 155 ICD recipients at one center were retrospectively reviewed, and patients having one or more follow-up DFT tests were analyzed. The patients were divided into two groups. The "routine" group consisted of 58 follow-up DFT procedures in 46 patients, without known changes in ICD parameters. The "prompted" group consisted of 21 follow-up DFT procedures in 18 patients, motivated by clinical concerns about changes in ICD lead status. Of 58 "routine" DFTs performed at a mean postimplant duration of 32 +/- 23 months (range 2-78), 7 (12%) had reprogramming, and 1 required a hardware change as a result of the testing. Of the 21 "prompted" DFTs performed, 7 required device reprogramming, and 3 required hardware upgrade. Overall, 19 (24%) of 79 procedures detected clinically significant changes, requiring reprogramming or ICD system revision. No complications were seen from follow-up DFT testing. CONCLUSION: A high rate of abnormalities was found at follow-up DFT testing in this population, especially in the group of patients with clinically prompted testing. Clinically indicated DFT testing, as expected, has a high yield of important information on device function in congenital heart disease and pediatric populations.

Adolescent↗

PubMed1997

Effect of enucleation on protein synthesis during maturation of bovine oocytes in vitro.

The role of the nucleus in protein synthesis reprogramming during oocyte maturation was examined in immature or mature bovine oocytes, enucleated at the germinal vesicle (GV) stage or the metaphase II (MII) stage. Cumulus-oocyte complexes (COCs) were denuded before or after maturation in vitro. Denuded oocytes were (i) enucleated at the GV or MII stage (after DNA staining and ultraviolet (UV) exposure), (ii) stained and exposed to UV but not enucleated, or (iii) used as controls. After treatment, oocytes were labelled for 4 h with 35S-methionine or were matured for 24 h before labelling. GV- or MII- karyoplasts and small portions of cytoplasm (cytoplasts), removed during enucleation, were also labelled. Labelled oocytes, karyoplasts or cytoplasts were prepared for one-dimensional polyacrylamide gel electrophoresis. Incorporation of labelled methionine into oocyte protein was measured. Enucleation did not affect protein synthesis reprogramming, but incorporation of 35S-methionine in immature UV-stained oocytes was high--possibly due to nuclear repair mechanisms. Protein profiles of GV- and MII- karyoplasts differed from those of immature and mature oocytes. In conclusion, normal protein synthesis reprogramming in the cytoplasm can occur in the absence of the nucleus, and specific proteins are synthesized in the nuclear region.

Animals↗

PubMed2005

Tetraploid embryonic stem cells contribute to the inner cell mass of mouse blastocysts.

The demonstration that mouse somatic cells can be reprogrammed following fusion with embryonic stem (ES) cells may provide an alternative to somatic cell nuclear transfer (therapeutic cloning) to generate autologous stem cells. In an attempt to produce cells with an increased pool of reprogramming factors, tetraploid ES cells were produced by polyethylene glycol mediated fusion of two ES cell lines transfected with plasmids carrying puromycin or neomycin resistance cassettes, respectively, followed by double antibiotic selection. Tetraploid ES cells retain properties characteristic of diploid ES cells, including the expression of pluripotent gene markers Oct4 and Rex1. On injection into the testis capsule of severe combined immunodeficient (SCID) mice, tetraploid ES cells are able to form teratomas containing cells representative of all three germ layers. Further, these cells demonstrated the ability to integrate into the inner cell mass of blastocysts. This study indicates that tetraploid ES cells are promising candidates as cytoplasm donors for reprogramming studies.

Animals↗

PubMed2006

Recombinant human albumin supports development of somatic cell nuclear transfer embryos in mice: toward the establishment of a chemically defined cloning protocol.

Culturing embryos in different media is a useful approach to characterize their nature in regard to "memory" of the donor nucleus and its "reprogramming" after somatic cell nuclear transfer (SCNT). However, efforts to elucidate the mechanisms of reprogramming are seriously undermined when embryo culture conditions are not completely defined. Using recombinant human albumin (rHA) is a step toward establishing defined culture conditions for mouse cloning. Recombinant HA supports blastocyst formation of cumulus cell-derived clones at a rate comparable with two types of bovine serum albumin (BSA); following transfer of blastocysts to the genital tract, rates of development to midgestation (10.5 dpc) were indistinguishable. rHA also supports the derivation of germline competent embryonic stem (ES) cells from SCNT blastocysts at a substantial rate compared with BSA counterparts and with zygotic blastocysts. Unlike the developmental parameters, the gene expression patterns of clones cultured in rHA or BSA were not superimposed; identical patterns were observed for zygotic blastocysts in the two albumins. In summary, the present study demonstrates that (1) rHA can replace BSA, proving a defined protein source for SCNT in mice; (2) although using rHA is similar to BSA, it is not equal (rHA leaves a mark on gene expression of clones but not zygotes). Future studies that investigate reprogramming after SCNT will need to consider not only the implications of culture media for cloning but also the supplement choice.

Animals↗

PubMedDate not supplied

The molecular foundations of the maternal to zygotic transition in the preimplantation embryo.

The maternal to zygotic transition is the first major transition that occurs following fertilization, and entails a dramatic reprogramming of gene expression that is essential for continued development. Although the major reprogramming of gene expression occurs during the 2-cell stage, transcription is evident in the 1-cell embryo, with the male pronucleus supporting a significantly higher level of transcription than the female pronucleus. This difference is likely due to differences in chromatin structure as a consequence of the protamine-histone exchange. Although the 1-cell embryo is transcriptionally competent, transcription and translation appear uncoupled. This transcription, however, may mark promoters for efficient utilization in the 2-cell embryo. Genome activation in the 2-cell embryo is accompanied by a requirement for an enhancer for efficient transcription and the more efficient utilization of TATA-less promoters. These changes in promoter utilization could contribute substantially to the reprogramming of gene expression. Superimposed on genome activation is the development of a chromatin-mediated transcriptionally repressive state that is relieved by either inducing histone hyperacetylation or inhibiting the second round of DNA replication. Since genome activation appears to be a relatively opportunistic process, the development of the transcriptionally repressive state may be a major determinant in establishing the appropriate gene expression profile that is essential for continued development.

Animals↗