Biomedical subjects

Cinzia Allegrucci

Publications and source records attributed to Cinzia Allegrucci.

7 recordsLinked to original sources

PubMed2006

Improved human embryonic stem cell embryoid body homogeneity and cardiomyocyte differentiation from a novel V-96 plate aggregation system highlights interline variability.

Although all human ESC (hESC) lines have similar morphology, express key pluripotency markers, and can differentiate toward primitive germ layers in vitro, the lineage-specific developmental potential may vary between individual lines. In the current study, four hESC lines were cultured in the same feeder-free conditions to provide a standardized platform for interline analysis. A high-throughput, forced-aggregation system involving centrifugation of defined numbers of hESCs in V-96 plates (V-96FA) was developed to examine formation, growth, and subsequent cardiomyocyte differentiation from >22,000 EBs. Homogeneity of EBs formed by V-96FA in mouse embryo fibroblast-conditioned medium was significantly improved compared with formation in mass culture (p < .02; Levene's test). V-96FA EB formation was successful in all four lines, although significant differences in EB growth were observed during the first 6 days of differentiation (p = .044 to .001; one-way analysis of variance [ANOVA]). Cardiomyocyte differentiation potential also varied; 9.5% +/- 0.9%, 6.6% +/- 2.4%, 5.2% +/- 3.1%, and 1.6% +/- 1.0% beating EBs were identified for HUES-7, NOTT2, NOTT1, and BG01, respectively (p = .008; one-way ANOVA). Formation of HUES-7 V-96FA EBs in defined medium containing activin A and basic fibroblast growth factor resulted in 23.6% +/- 3.6% beating EBs, representing a 13.1-fold increase relative to mass culture (1.8% +/- 0.7%), consistent with an observed 14.8-fold increase in MYH6 (alphaMHC) expression by real-time polymerase chain reaction. In contrast, no beating areas were derived from NOTT1-EBs and BG01-EBs formed in defined medium. Thus, the V-96FA system highlighted interline variability in EB growth and cardiomyocyte differentiation but, under the test conditions described, identified HUES-7 as a line that can respond to cardiomyogenic stimulation.

Animals↗

PubMed2006

Common culture conditions for maintenance and cardiomyocyte differentiation of the human embryonic stem cell lines, BG01 and HUES-7.

Development of generic differentiation protocols that function in a range of independently-derived human embryonic stem cell (hESC) lines remains challenging due to considerable diversity in culture methods practiced between lines. Maintenance of BG01 and HUES-7 has routinely been on mouse embryonic fibroblast (MEF) feeder layers using manual- and trypsin-passaging, respectively. We adapted both lines to trypsin-passaging on feeders or on Matrigel in feeder-free conditions and assessed proliferation and cardiac differentiation. On feeders, undifferentiated proliferation of BG01 and HUES-7 was supported by all three media tested (BG-SK, HUES-C and HUES-nL), although incidence of karyotypic instability increased in both lines in BG-SK. On Matrigel, KSR-containing conditioned medium (CM) promoted undifferentiated cell proliferation, while differentiation occurred in CM containing Plasmanate or ES-screened Fetal Bovine Serum (FBS) and in unconditioned medium containing 100 ng/ml bFGF. Matrigel cultures were advantageous for transfection but detrimental to embryoid body (EB) formation. However, transfer of hESCs from Matrigel back to feeders and culturing to confluence was found to rescue EB formation. EBs formed efficiently when hESCs on feeders were treated with collagenase, harvested by scraping and then cultured in suspension in CM. Subsequent culture in FBS-containing medium produced spontaneously contracting EBs, for which the mean beat rate was 37.2 +/- 2.3 and 41.1 +/- 3.1 beats/min for BG01-EBs and HUES-7-EBs, respectively. Derived cardiomyocytes expressed cardiac genes and responded to pharmacological stimulation. Therefore the same culture and differentiation conditions functioned in two independently-derived hESC lines. Similar studies in other lines may facilitate development of universal protocols.

Biomarkers↗

PubMed2005

Human embryonic stem cell methyl cycle enzyme expression: modelling epigenetic programming in assisted reproduction?

To investigate a possible mechanism for inducing epigenetic defects in the preimplantation embryo, a human embryonic stem cell model was developed, and gene expression of the key methyl cycle enzymes, MAT2A, MAT2B, GNMT, SAHH, CBS, CGL, MTR, MTRR, BHMT, BHMT2, mSHMT, cSHMT and MTHFR was demonstrated, while MAT1 was barely detectable. Several potential acceptors of cycle-generated methyl groups, the DNA methyltransferases (DNMT1, DNMT3A, DNMT3B and DNMT3L), glycine methyltransferase and the polyamine biosynthetic enzymes, SAM decarboxylase and ornithine decarboxylase, were also expressed. Expression of folate receptor alpha suggests a propensity for folate metabolism. Methotrexate-induced depletion of folate resulted in elevated intracellular homocysteine concentration after 7 days in culture and a concomitant increase in cysteine and glutathione, indicating clearance of homocysteine through the transulphuration pathway. These studies indicate that altered methyl group metabolism provides a potential mechanism for inducing epigenetic changes in the preimplantation embryo.

Cell Proliferation↗

PubMed2005

Epigenetics and the germline.

Epigenetic processes affect three stages of germline development, namely (1) specification and formation of primordial germ cells and their germline derivatives through lineage-specific epigenetic modifications, in the same manner as other embryonic lineages are formed, (2) a largely genome-wide erasure and re-establishment of germline-specific epigenetic modifications that only occurs in the embryonic primordial germ cell lineage, followed by re-establishment of sex-specific patterns during gametogenesis, and (3) differential epigenetic modifications to the mature male and female gamete genomes shortly after fertilisation. This review will detail current knowledge of these three processes both at the genome-wide level and at specific imprinted loci. The consequences of epigenetic perturbation are discussed and new in vitro models which may allow further understanding of a difficult developmental period to study, especially in the human, are highlighted.

Animals↗

PubMed2002

Ecto-diadenosine polyphosphates hydrolase activity on human prostasomes.

BACKGROUND: Ecto-diadenosine polyphosphates are ubiquitous compounds with several physiological roles. Ecto-diadenosine polyphosphates hydrolase control their actions by degrading and terminating their signaling. The present work deals with the identification and partial characterization of ecto-diadenosine polyphosphates hydrolase on human prostasomes. METHODS: Reverse-phase and paired-ion HPLC techniques have been used. RESULTS: Prostasomes have an ecto-diadenosine polyphosphates hydrolase that leads to the degradation of several diadenosine compounds. Kinetic parameters of the enzyme show that diadenosine tetraphosphate is the preferred substrate that is further metabolized by the prostasome-ecto-nucleotidases to adenosine. The ecto-enzyme is bound to the prostasome-membranes through a GPI-anchor and is activated by physiological concentration of Ca+2, Mg+2, and Mn+2. Its optimum pH is also in the slightly alkaline physiological range. Human spermatozoa do not possess this hydrolytic activity, but they can acquire it after fusion with prostasomes. CONCLUSIONS: The existence of an enzyme capable of degrading diadenosine compounds and can be transferred to human spermatozoa suggests new physiological implications for the role of prostasomes in fertilization.

Acid Anhydride Hydrolases↗

PubMedDate not supplied

Human embryonic stem cells as a model for nutritional programming: an evaluation.

Our laboratory is evaluating whether an epigenetic mechanism involving alterations in DNA methylation can alter the trajectory of embryonic/fetal development in response to maternal nutrients. A similar mechanism may operate in embryo culture environments commonly used in human assisted conception. Since developmental studies on early human embryos in utero are obviously not possible, we have begun to investigate the utility of human embryonic stem cells (hESC) to uncover potential programming mechanisms. This review highlights some of the advantages and problems associated with such a model and suggests that these issues are also broadly applicable to utility of hESC for more general toxicology and drug screening applications.

Adult↗

PubMedDate not supplied

Stem-cell consequences of embryo epigenetic defects.

CONTEXT: The genetic code in the DNA of virtually every somatic cell can produce the entire complement of encoded proteins. Acetylation of histones and methylation of histones and DNA cytosine residues are part of the complex epigenetic regulatory process determining lineage-specific gene expression by altering the local structure of chromatin. After fertilisation, sperm DNA exchanges protamines for histones recruited from oocyte cytoplasm, reconfiguring both parental genomes into an epigenetic state conducive to activating the embryonic developmental programme. The identification of epigenetic reprogramming mechanisms is a major interest, rekindled by the ability of at least some somatic cells to acquire totipotency after somatic-cell nuclear transfer. STARTING POINT: Recently, Woo SukHwang and colleagues (Science 2004; 303: 1669-74) derived a human embryonic stem-cell line from embryo therapeutic cloning. Chad Cowan and colleagues (N Engl JMed 2004; 350: 1353-56) produced 17 new lines from embryos supernumerary to infertility treatments. However, increasing evidence from a range of mammals shows a propensity for epigenetic errors with embryo technologies. If paralleled in human embryos, the effect on tumorigenic and differentiation properties of embryonic stem cells needs to be established. WHERE NEXT? Identifying the mechanisms in the oocyte that reprogramme a somatic cell to the embryonic state might allow somatic cells to be reprogrammed ex ovo by in-vitro manipulation of the epigenome. Because the oocyte is designed to reprogramme the sperm genome, which is in a different chromatin state from a somatic cell, perhaps many of the epigenetic errors induced by somatic-cell nuclear transfer could be avoided by a more targeted approach.

Animals↗