Susan M. Hawes

NKX2-5eGFP/w hESCs for isolation of human cardiac progenitors and cardiomyocytes

NKX2-5 is expressed in the heart throughout life. We targeted eGFP sequences to the NKX2-5 locus of human embryonic stem cells (hESCs); NKX2-5eGFP/w hESCs facilitate quantification of cardiac differentiation, purification of hESC-derived committed cardiac progenitor cells (hESC-CPCs) and cardiomyocytes (hESC-CMs) and the standardization of differentiation protocols. We used NKX2-5 eGFP+ cells to identify VCAM1 and SIRPA as cell-surface markers expressed in cardiac lineages.

Transcriptional analysis of early lineage commitment in human embryonic stem cells.

BackgroundThe mechanisms responsible for the maintenance of pluripotency in human embryonic stem cells, and those that drive their commitment into particular differentiation lineages, are poorly understood. In fact, even our knowledge of the phenotype of hESC is limited, because the immunological and molecular criteria presently used to define this phenotype describe the properties of a heterogeneous population of cells.ResultsWe used a novel approach combining immunological and transcriptional analysis (immunotranscriptional profiling) to compare gene expression in hESC populations at very early stages of differentiation. Immunotranscriptional profiling enabled us to identify novel markers of stem cells and their differentiated progeny, as well as novel potential regulators of hESC commitment and differentiation. The data show clearly that genes associated with the pluripotent state are downregulated in a coordinated fashion, and that they are co-expressed with lineage specific transcription factors in a continuum during the early stages of stem cell differentiation.ConclusionThese findings, that show that maintenance of pluripotency and lineage commitment are dynamic, interactive processes in hESC cultures, have important practical implications for propagation and directed differentiation of these cells, and for the interpretation of mechanistic studies of hESC renewal and commitment. Since embryonic stem cells at defined stages of commitment can be isolated in large numbers by immunological means, they provide a powerful model for studying molecular genetics of stem cell commitment in the embryo.

Isolation, characterization, and differentiation of human embryonic stem cells

Publisher Summary The first derivation of human embryonic stem (ES) cells was reported in 1998, and although there have been a number of anecdotal reports of isolation of new ES cells since then, published data are based only on a few cell lines. Timely progress in this field depends upon the derivation of new ES cell lines, their proper characterization and comparison with existing isolates, and in-depth analysis of their differentiation under different conditions. Previous reviews have compared the properties of published human ES cells with those of pluripotent cell lines derived from nonhuman primates or with human embryonal carcinoma cell lines, and with the counterparts of both cell types in the mouse. These comparisons have given rise to some consensus regarding the canonical primate pluripotent cell phenotype; the data are limited, and the cultures are probably more heterogeneous than the published descriptions imply. The purpose of this chapter is to help provide some guidelines for human ES cell derivation, and a methodological basis for the characterization and comparison of stem cells and their differentiation in different laboratories. The chapter deals with the isolation, characterization, and differentiation of human embryonic stem (ES) cell lines from preimplantation blastocysts.

CD133 Expression by Neural Progenitors Derived from Human Embryonic Stem Cells and Its Use for Their Prospective Isolation

The ability to generate purified neural progenitors is critical to the development of embryonic stem cell-based therapies to alleviate human neurological disorders. While many cell culture protocols for directed differentiation of human embryonic stem (hES) cells into neural cells have been described, most yield mixed populations, some containing cells of different embryonic germ layer lineages, or even undifferentiated embryonic stem cells. In this study, we describe a method for single-cell dissociation, isolation by flow cytometry, and subsequent culture of neural progenitors from hES cells. As reported earlier, hES cells treated with the bone morphogenetic protein (BMP) antagonist noggin gave rise to neurospheres at a relatively high frequency. However, these noggin-treated embryonic stem cell cultures were heterogeneous, with cells expressing embryonic stem markers still detectable even following 14 days of differentiation. In order to isolate pure human neural progenitors, we fractionated noggin-treated ES cells on the basis of their expression of the putative neural stem cell marker, CD133, and the GCTM-2, and SSEA-1 antigens, which mark pluripotent stem cells and differentiated cells respectively from hES cell culture. CD133(+) cells formed larger spheres compared to CD133(-) cells. CD133(+)SSEA1(+) cells and CD133(+)SSEA-1(-) cells expressed similar levels of neural genes and formed neurospheres at similar frequencies. By contrast, CD133(+)GCTM-2(+) cells expressed high levels of OCT4 but not neural lineage genes and failed to form neurospheres. CD133(+)GCTM-2(-) cells formed neurospheres at the relative highest frequency. Thus, negative selection with GCTM-2 may be useful for the purification of specific cell types differentiated from hES cells.

Differential expression of the embryo/cancer gene ECSA(DPPA2), the cancer/testis gene BORIS and the pluripotency structural gene OCT4, in human preimplantation development

In this paper, we examine the expression profiles of two new putative pluripotent stem cell genes, the embryo/cancer sequence A gene (ECSA) and the cancer/testis gene Brother Of the Regulator of Imprinted Sites (BORIS), in human oocytes, preimplantation embryos, primordial germ cells (PGCs) and embryo stem (ES) cells. Their expression profiles are compared with that of the well-known pluripotency gene, OCT4, using a primer design that avoids amplification of the multiple OCT4 pseudogenes. As expected, OCT4 is high in human oocytes, down-regulated in early cleavage stages and then expressed de novo in human blastocysts and PGCs. BORIS and ECSA show distinct profiles of expression in that BORIS is predominantly expressed in the early stages of preimplantation development, in oocytes and 4-cell embryos, whereas ECSA is predominantly expressed in the later stages, blastocysts and PGCs. BORIS is not detected in blastocysts, PGCs or other fetal and adult somatic tissue tested. Thus, BORIS and ECSA may be involved in two different aspects of reprogramming in development, viz., in late gametogenesis, and at the time of formation of the ES cells (inner cell mass (ICM) and PGC), respectively. However, in human ES cells, where a deprogrammed stem cell state is stably established in culture, an immunofluoresence study shows that all three genes are co-expressed at the protein level. Thus, following their derivation from ICM cells, ES cells may undergo further transformation in culture to express a number of embryo and germ line stem cell functions, which, in normal development, show different temporal and spatial specificity of expression.

A Novel Cell‐Surface Marker Found on Human Embryonic Hepatoblasts and a Subpopulation of Hepatic Biliary Epithelial Cells

The nature of the cells that contribute to the repopulation of the liver after hepatic necrosis or cirrhosis remains uncertain, in part because we lack specific markers to facilitate identification and prospective isolation of progenitor cells. The monoclonal antibody GCTM‐5 reacts with a minority subpopulation of cells in spontaneously differentiating cultures of pluripotent human embryonal carcinoma or embryonic stem cells. The epitope recognized by GCTM‐5 is found on a 50‐kDa protein present on the surface of these cells. In tissue sections of first‐trimester human embryos, GCTM‐5 specifically stained hepatoblasts and no other cell type examined. In normal pediatric or adult liver, GCTM‐5 reacted with a minority population of luminal bile duct cells. In diseased livers, the numbers of GCTM‐5–positive cells were increased compared with normal liver; antibody staining was restricted to a subpopulation of ductular reactive cells, and among this subpopulation we observed GCTM‐5–positive cells that did not express cytokeratin 19 or N‐CAM, classical makers of ductular reactive cells. Live GCTM‐5–positive cells could be isolated from diseased livers by immunomagnetic sorting. These results suggest that GCTM‐5 will be a useful reagent for defining cell lineage relationships between putative progenitor populations in embryonic liver and in the biliary epithelium during tissue repair.

G-protein Coupled Receptors in Stem Cell Self-Renewal and Differentiation

Stem cells have great potential for understanding early development, treating human disease, tissue trauma and early phase drug discovery. The factors that control the regulation of stem cell survival, proliferation, migration and differentiation are still emerging. Some evidence now exists demonstrating the potent effects of various G-protein coupled receptor (GPCR) ligands on the biology of stem cells. This review aims to give an overview of the current knowledge of the regulation of embryonic and somatic stem cell maintenance and differentiation by GPCR ligands.

Characterisation of Histone Variant Distribution in Human Embryonic Stem Cells by Transfection of In Vitro Transcribed mRNA

Recent studies, primarily in mouse embryonic stem cells, have highlighted the unique chromatin state of pluripotent stem cells, including the incorporation of histone variants into specific genomic locations, and its role in facilitating faithful expression of genes during development. However, there is little information available on the expression and subcellular localisation of histone variants in human embryonic stem cells (hESCs). In this study, we confirmed the expression of a panel of histone variant genes in several hESC lines and demonstrated the utility of transfection of in vitro transcribed, epitope‐tagged mRNAs to characterise the subcellular localisation of these proteins. The subcellular localisations of variant histone H3 (CENP‐A, H3.3), H2A (MACROH2A, H2AX, H2AZ, H2ABBD) and H1 (H1A, HB, H1C, H1D) were examined, revealing distinct nuclear localisation profiles for each protein. These data highlight the differences between murine (m) ESCs and hESCs, including the presence of a MACROH2A‐enriched inactive X chromosome in undifferentiated XX hESC lines. We also provide the first evidence for MACROH2A accumulation on the Y‐chromosome in XY hESCs. Mol. Reprod. Dev. 76: 1128–1142, 2009.

The GCTM‐5 Epitope Associated with the Mucin‐Like Glycoprotein FCGBP Marks Progenitor Cells in Tissues of Endodermal Origin

Monoclonal antibodies against cell surface markers are powerful tools in the study of tissue regeneration, repair, and neoplasia, but there is a paucity of specific reagents to identify stem and progenitor cells in tissues of endodermal origin. The epitope defined by the GCTM‐5 monoclonal antibody is a putative marker of hepatic progenitors. We sought to analyze further the distribution of the GCTM‐5 antigen in normal tissues and disease states and to characterize the antigen biochemically. The GCTM‐5 epitope was specifically expressed on tissues derived from the definitive endoderm, in particular the fetal gut, liver, and pancreas. Antibody reactivity was detected in subpopulations of normal adult biliary and pancreatic duct cells, and GCTM‐5‐positive cells isolated from the nonparenchymal fraction of adult liver expressed markers of progenitor cells. The GCTM‐5‐positive cell populations in liver and pancreas expanded greatly in numbers in disease states such as biliary atresia, cirrhosis, and pancreatitis. Neoplasms arising in these tissues also expressed the GCTM‐5 antigen, with pancreatic adenocarcinoma in particular showing strong and consistent reactivity. The GCTM‐5 epitope was also strongly displayed on cells undergoing intestinal metaplasia in Barretts esophagus, a precursor to esophageal carcinoma. Biochemical, mass spectrometry, and immunochemical studies revealed that the GCTM‐5 epitope is associated with the mucin‐like glycoprotein FCGBP. The GCTM‐5 epitope on the mucin‐like glycoprotein FCGBP is a cell surface marker for the study of normal differentiation lineages, regeneration, and disease progression in tissues of endodermal origin. Stem Cells2012;30:1999–2009

Effective adenovirus-mediated gene transfer into neural stem cells derived from human embryonic stem cells

Human embryonic stem cell-derived neural stem cells (hESC-NSCs) are an attractive cell type for studying aspects of brain development and pathology. To develop the full potential of this model system, it is important to establish a reliable methodology for the manipulation of gene expression in hNSCs. To address this issue, we used an adenoviral vector with a CMV promoter-driven green fluorescent protein (GFP) reporter gene (Ad5-GFP). We optimized conditions for Ad5-GFP infection and assessed the efficiency of infection of whole and dissociated embryonic stem cell (ESC)-derived neurospheres as well as the effect of adenoviral vectors on cell surface marker expression, proliferation, and differentiation potential. Our results demonstrate that most neurosphere cells ( approximately 70%) express the coxsackie and adenovirus receptor and can be infected with Ad5. More specifically, the CD133+ hESC-NSC population could be infected more efficiently than the CD133 population and both populations expressed GFP at high levels. At low multiplicity of infection (MOI < 25), the virus had no significant effect on stem cell marker expression (CD133 and Nestin), cell survival, cell proliferation rate, or differentiation potential. This model system provides a practical new approach to study human NSC function in the context of neurodegenerative and neoplastic disorders.