Jonathan S. Draper

Recurrent gain of chromosomes 17q and 12 in cultured human embryonic stem cells

We have observed karyotypic changes involving the gain of chromosome 17q in three independent human embryonic stem (hES) cell lines on five independent occasions. A gain of chromosome 12 was seen occasionally. This implies that increased dosage of chromosome 17q and 12 gene(s) provides a selective advantage for the propagation of undifferentiated hES cells. These observations are instructive for the future application of hES cells in transplantation therapies in which the use of aneuploid cells could be detrimental.

Gene expression patterns in human embryonic stem cells and human pluripotent germ cell tumors

Remarkably little is known about the transcriptional profiles of human embryonic stem (ES) cells or the molecular mechanisms that underlie their pluripotency. To identify commonalties among the transcriptional profiles of different human pluripotent cells and to search for clues into the genesis of human germ cell tumors, we compared the expression profiles of human ES cell lines, human germ cell tumor cell lines and tumor samples, somatic cell lines, and testicular tissue samples by using cDNA microarray analysis. Hierarchical cluster analysis of gene expression profiles showed that the five independent human ES cell lines clustered tightly together, reflecting highly similar expression profiles. The gene expression patterns of human ES cell lines showed many similarities with the human embryonal carcinoma cell samples and more distantly with the seminoma samples. We identified 895 genes that were expressed at significantly greater levels in human ES and embryonal carcinoma cell lines than in control samples. These genes are candidates for involvement in the maintenance of a pluripotent, undifferentiated phenotype.

Surface antigens of human embryonic stem cells: changes upon differentiation in culture

We have analysed the surface antigen phenotype of a human embryonic stem (hES) cell line (H7) and the changes that occur upon differentiation induced by retinoic acid, hexamethylene bisacetamide and dimethylsulphoxide. The undifferentiated stem cells expressed Stage Specific Embryonic Antigen‐3 (SSEA3), SSEA4, TRA‐1‐60, and TRA‐1‐8 but not SSEA1. In these characteristics they closely resemble human embryonal carcinoma (EC) cells derived from testicular teratocarcinomas, and are distinct from murine EC and ES cells. The undifferentiated cells also expressed the liver/bone/kidney isozyme of alkaline phosphatase detected by antibody TRA‐2‐54, the class 1 major histocompatability antigens, HLA‐ABC, and the human Thy1 antigen. Differentiation of hES cells was induced by retinoic acid, HMBA and DMSO with the appearance of various cell types including neurons and muscle cells. The surface antigens characteristically expressed by hES cells were down‐regulated following induction of differentiation and other antigens appeared, notably several ganglioside glycolipids detected by antibodies VIN‐IS‐56 (GD3 and GD2), VIN‐2PB‐22 (GD2), A2B5 (GT3) and ME311 (9‐O‐acetyl‐GD3). Whereas the expression of HLA was slightly down‐regulated upon differentiation, its expression was strongly induced by interferon‐γ in both the undifferentiated and the differentiated cells, although the induction in the differentiated cultures was considerably stronger than in the stem cells. In all of these features the human ES cells, and their pattern of differentiation, resembled the pluripotent human EC cell line NTERA‐2 although clearly the range of cells generated by the hES cells was considerably greater.

Preimplantation Human Embryos and Embryonic Stem Cells Show Comparable Expression of Stage‐Specific Embryonic Antigens

Cell‐surface antigens provide invaluable tools for the identification of cells and for the analysis of cell differentiation. In particular, stage‐specific embryonic antigens that are developmentally regulated during early embryogenesis are widely used as markers to monitor the differentiation of both mouse and human embryonic stem (ES) cells and their malignant counterparts, embryonic carcinoma (EC) cells. However, there are notable differences in the expression patterns of some such markers between human and mouse ES/EC cells, and hitherto it has been unclear whether this indicates significant differences between human and mouse embryos, or whether ES/EC cells correspond to distinct cell types within the early embryos of each species. We now show that human ES cells are characterized by the expression of the cell‐surface antigens, SSEA3, SSEA4, TRA‐1‐60, and TRA‐1‐81, and by the lack of SSEA1, and that inner cell mass cells of the human blastocyst express a similar antigen profile, in contrast to the corresponding cells of the mouse embryo.

Specific Knockdown of Oct4 and β2‐microglobulin Expression by RNA Interference in Human Embryonic Stem Cells and Embryonic Carcinoma Cells

We have used RNA interference (RNAi) to downregulate β2‐microglobulin and Oct4 in human embryonal carcinoma (hEC) cells and embryonic stem (hES) cells, demonstrating that RNAi is an effective tool for regulating specific gene activity in these human stem cells. The knockdown of Oct4 but not β2‐microglobulin expression in both EC and ES cells resulted in their differentiation, as indicated by a marked change in morphology, growth rate, and surface antigen phenotype, with respect to SSEA1, SSEA3, and TRA‐1‐60 expression. Expression of hCG and Gcm1 was also induced following knockdown of Oct4 expression, in both 2102Ep hEC cells and in H7 and H14 hES cells, consistent with the conclusion that, as in the mouse, Oct4 is required to maintain the undifferentiated stem cell state, and that differentiation to trophectoderm occurs in its absence. NTERA2 hEC cells also differentiated, but not to trophectoderm, suggesting their equivalence to a later stage of embryogenesis than other hEC and hES cells.

Isolation of human iPS cells using EOS lentiviral vectors to select for pluripotency

Induced pluripotent stem (iPS) cells may be of use in regenerative medicine. However, the low efficiency of reprogramming is a major impediment to the generation of patient-specific iPS cell lines. Here we report the first selection system for the isolation of human iPS cells. We developed the EOS (Early Transposon promoter and Oct-4 (Pou5f1) and Sox2 enhancers) lentiviral vector to specifically express in mouse and human embryonic stem cells but not in primary fibroblasts. The bicistronic EOS vector marked emerging mouse and human iPS cell colonies with EGFP, and we used puromycin selection to aid the isolation of iPS cell lines that expressed endogenous pluripotency markers. These lines differentiated into cell types from all three germ layers. Reporter expression was extinguished upon differentiation and therefore monitored the residual pluripotent cells that form teratomas. Finally, we used EOS selection to establish Rett syndrome–specific mouse and human iPS cell lines with known mutations in MECP2.

Establishment of Endoderm Progenitors by SOX Transcription Factor Expression in Human Embryonic Stem Cells

In this study, we explore endoderm cell fate regulation through the expression of lineage-determining transcription factors. We demonstrate that stable endoderm progenitors can be established from human ES cells by constitutive expression of SOX7 or SOX17, producing extraembryonic endoderm and definitive endoderm progenitors, respectively. In teratoma assays and growth factor-mediated differentiation, SOX7 cells appear restricted to the extraembryonic endoderm, and SOX17 cells demonstrate a mesendodermal phenotype in teratomas and the ability to undergo endoderm maturation in vitro in the absence of cytokine-mediated endoderm induction. These endoderm progenitor cells maintain a stable phenotype through many passages in culture, thereby providing new tools to explore the pathways of endoderm differentiation.

Transient and Stable Transgene Expression in Human Embryonic Stem Cells

Plasmid vectors remain a valuable yet capricious tool for the genetic manipulation of human embryonic stem (hES) cells. We have compared the efficacy of four promoters to mediate transient and stable transfection in hES and human embryonal carcinoma cell lines with the reporter enhanced green fluorescent protein (eGFP). In transient assays, the two mammalian promoters, UbiquitinC and Rosa26 (pUbiC and pR26), the human cytomegalovirus major immediate early promoter (HCMV‐MIE; pCMV), and the HCMV‐MIE/chicken β‐actin/rabbit β‐globin hybrid promoter (pCAGG) gave variable results that depended upon the cell line transfected but in an unpredictable way: each promoter supported strong transient expression in at least one cell line. The results for stable transfection were generally at variance with the transient assays. In each case, transgene silencing was quite marked, most notably with the pCMV, with which no eGFP‐positive clones were obtained. An exception was the pCAG vector, in which the CAGG composite promoter is linked to the polyoma virus mutant enhancer PyF101; stable eGFP‐positive transfectants were obtained, and these clones retained eGFP expression for over 120 passages, even in the absence of selection. However, if the PyF101 elements were removed, the resulting transfectants were also subjected to progressive gene silencing. Thus, the choice of promoter is critical for determining the desired effect of transgene expression in hES cells. Our data also demonstrate that pUbiC, pR26, pCAGG, and pCAG are more superior to the pCMV for generation of stable transfectants in hES cells.

Lengthened G1 Phase Indicates Differentiation Status in Human Embryonic Stem Cells

The cell cycle in pluripotent stem cells is notable for the brevity of the G1 phase, permitting rapid proliferation and reducing the duration of differentiation signal sensitivity associated with the G1 phase. Changes in the length of G1 phase are understood to accompany the differentiation of human embryonic stem cells (hESCs), but the timing and extent of such changes are poorly defined. Understanding the early steps governing the differentiation of hESCs will facilitate better control over differentiation for regenerative medicine and drug discovery applications. Here we report the first use of real-time cell cycle reporters in hESCs. We coexpressed the chromatin-decorating H2B-GFP fusion protein and the fluorescence ubiquitination cell cycle indicator (FUCCI)-G1 fusion protein, a G1 phase-specific reporter, in hESCs to measure the cell cycle status in live cells. We found that FUCCI-G1 expression is weakly detected in undifferentiated hESCs, but rapidly increases upon differentiation. hESCs in the G1 phase display a reduction in undifferentiated colony-initiating cell function, underscoring the relationship between G1 phase residence and differentiation. Importantly, we demonstrate inter- and intracolony variation in response to chemicals that induce differentiation, implying extensive cell-cell variation in the threshold necessary to alter the G1 phase length. Finally, gain of differentiation markers appears to be coincident with G1 phase lengthening, with distinct G1 phase profiles associated with different markers of early hESC differentiation. Our data demonstrate the tight coupling of cell cycle changes to hESC differentiation, and highlight the cell cycle reporter system and assays we have implemented as a novel avenue for investigating pluripotency and differentiation.

Embryonic stem cells: advances toward potential therapeutic use.

Established lines of human pluripotent stem cells provide a convenient tool for investigating cell differentiation in a way that is pertinent to human embryonic development, providing insights into the causes of birth defects and diseases such as cancer that involve aberrant cell proliferation and differentiation. In principle, human pluripotent stem cells, including embryonic stem and embryonic germ cells, are capable of differentiating into all of the cell types that are present in the adult human. They therefore have the potential to provide a source of tissues for replacement in diseases in which native cell types are inactivated or destroyed. Intense media and public interest has surrounded the announcement of human pluripotent stem cells derivation, focusing on the ethical implications of embryo-related work and on the prospects of an unlimited source of tissues for transplantation-based treatments. Recent studies have focused on identifying method for culture of these cells and inducing their differentiation into specific cell types.