Helen Priddle

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; Levenes 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 (αMHC) 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.

SonoAVC: a novel method of automatic volume calculation.

To assess the ability of the new software SonoAVC to measure follicular volume and to compare these volume calculations with those made by conventional methods.

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.

Hematopoiesis from Human Embryonic Stem Cells: Overcoming the Immune Barrier in Stem Cell Therapies

The multipotency and proliferative capacity of human embryonic stem cells (hESCs) make them a promising source of stem cells for transplant therapies and of vital importance given the shortage in organ donation. Recent studies suggest some immune privilege associated with hESC‐derived tissues. However, the adaptability of the immune system makes it unlikely that fully differentiated tissues will permanently evade immune rejection. One promising solution is to induce a state of immune tolerance to a hESC line using tolerogenic hematopoietic cells derived from it. This could provide acceptance of other differentiated tissues from the same line. However, this approach will require efficient multilineage hematopoiesis from hESCs. This review proposes that more efficient differentiation of hESCs to the tolerogenic cell types required is most likely to occur through applying knowledge gained of the ontogeny of complex regulatory signals used by the embryo for definitive hematopoietic development in vivo. Stepwise formation of mesoderm, induction of definitive hematopoietic stem cells, and the application of factors key to their self‐renewal may improve in vitro production both quantitatively and qualitatively.

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.

Automated measurements of follicle diameter: a chance to standardize?

This study describes the use of an automated measurement of follicular diameter using the new Sono-Automatic Volume Calculation (Sono-AVC) software and compares the accuracy of automated measures and the time taken for such measures to those made manually from two-dimensional and three-dimensional ultrasound. Sono-AVC provides measurements of follicular diameter that are more accurate than the manual measures and has the potential to improve the clinical work flow because the time taken for the measurements is significantly shorter.

Human Embryonic Stem Cells Passaged Using Enzymatic Methods Retain a Normal Karyotype and Express CD30

Human embryonic stem cells (hESCs) are thought to be susceptible to chromosomal rearrangements as a consequence of single cell dissociation. Compared in this study are two methods of dissociation that do not generate single cell suspensions (collagenase and EDTA) with an enzymatic procedure using trypsin combined with the calcium-specific chelator EGTA (TEG), that does generate a single cell suspension, over 10 passages. Cells passaged by single cell dissociation using TEG retained a normal karyotype. However, cells passaged using EDTA, without trypsin, acquired an isochromosome p7 in three replicates of one experiment. In all of the TEG, collagenase and EDTA-treated cultures, cells retained consistent telomere length and potentiality, demonstrating that single cell dissociation can be used to maintain karyotypically and phenotypically normal hESCs. However, competitive genomic hybridization revealed that subkaryotypic deletions and amplifications could accumulate over time, reinforcing that present culture regimes remain suboptimal. In all cultures the cell surface marker CD30, reportedly expressed on embryonal carcinoma but not karyoptically normal ESCs, was expressed on hESCs with both normal and abnormal karyotype, but was upregulated on the latter.

Ablation of Undifferentiated Human Embryonic Stem Cells: Exploiting Innate Immunity Against the Gal α1‐3Galβ1‐4GlcNAc‐R (α‐Gal) Epitope

Although undifferentiated human embryonic stem cells (hESCs) are tumorigenic, this capacity is lost after differentiation, and hESCs are being widely investigated for applications in regenerative medicine. To engineer protection against the unintentional transplantation of undifferentiated cells, we generated hESCs carrying a construct in which the α1,3‐galactosyltransferase (GalT) open reading frame was transcribed from the hTERT promoter (pmGT). Because the endogenous GalT gene is inactive, GalT expression was limited to undifferentiated cells. A second chimeric construct (pmfGT) differed by replacement of the GalT leader sequence for that of the fucosyltransferase gene. Two subclones containing stable integrations of pmGT and pmfGT (M2 and F11, respectively) were assessed for their response to human serum containing antibodies to the Galα1‐3Galβ1‐4GlcNAc‐R (α‐gal) epitope. The low‐variegation line, M2, and to a lesser extent the more variegated line F11, were sensitive to human serum when exposed in the undifferentiated state. However, M2 cells were largely insensitive after differentiation and retained both a normal karyotype and the ability to differentiate into derivatives of the three germ layers in severe combined immunodeficient mice. These data exemplify a method of protection against residual, undifferentiated hESCs prior to engraftment and may provide ongoing immune surveillance after engraftment against dedifferentiation or against de novo tumorigenesis involving hTERT reactivation. Untransfected H9 cells were not sensitive to the human serum used in this study. Hence, in our system, interactions of hESCs with other circulating antibodies, such as anti‐Neu5Gc, were not observed.

Elevated expression of exogenous Rad51 leads to identical increases in gene-targeting frequency in murine embryonic stem (ES) cells with both functional and dysfunctional p53 genes

The Rad51 gene is the mammalian homologue of the bacterial RecA gene and catalyses homologous recombination in mammalian cells. In some cell types Rad51 has been shown to interact with p53, leading to inhibition of Rad51 activity. Here, we show a two- to four-fold increase in gene-targeting frequency at the HPRT locus using murine ES clones preengineered to overexpress Rad51, and a twofold increase in targeting frequency when a Rad51 expression cassette was cointroduced to wild-type ES cells with the targeting construct. In addition to its effect on homologous recombination, we show that Rad51 may down-regulate illegitimate recombination. We investigated the dependence of these phenomena upon p53 and found no evidence that the Rad 51-mediated increase is affected by the functional status of p53, a conclusion supported by the observed cytoplasmic localisation of p53 in ES cells following electroporation. Furthermore, in the absence of additional Rad51, p53-deficient ES cells do not have elevated rates of homologous recombination with extrachromosomal DNA. These findings demonstrate that Rad51 levels modify both homologous and illegitimate recombination, but that these phenomena are independent of p53 status.

Bioluminescence imaging of human embryonic stem cells transplanted in vivo in murine and chick models.

Research into the behavior, efficacy, and biosafety of stem cells with a view to clinical transplantation requires the development of noninvasive methods for in vivo imaging of cells transplanted into animal models. This is particularly relevant for human embryonic stem cells (hESCs), because transplantation of undifferentiated hESCs leads to tumor formation. The present study aimed to monitor hESCs in real time when injected in vivo. hESCs were stably transfected to express luciferase, and luciferase expression was clearly detected in the undifferentiated and differentiated state. When transfected hESCs were injected into chick embryos, bioluminescence could be detected both ex and in ovo. In the SCID mouse model, undifferentiated hESCs were detectable after injection either into the muscle layer of the peritoneum or the kidney capsule. Tumors became detectable between days 10-30, with approximately a 3 log increase in the luminescence signal by day 75. The growth phase occurred earlier in the kidney capsule and then reached a plateau, whilst tumors in the peritoneal wall grew steadily throughout the period analysed. These results show the widespread utility of bioluminescent for in vivo imaging of hESCs in a variety of model systems for preclinical research into regenerative medicine and cancer biology.