enrichment-purging-human-embryonic-stem-cells-detection-cell-surface

Enrichment and Purging of Human Embryonic Stem Cells by Detection of Cell Surface Antigens Using the Monoclonal Antibodies TG30 and GCTM-2

Human embryonic stem cells (hESC) can self-renew indefinitely in vitro, and with the appropriate cues can be induced to differentiate into potentially all ...

CSIRO

Totipotency can be restored to the nuclei of somatic cells by reprogramming the nucleus via the technique of nuclear transfer. As genes expressed in somatic cells differ from those expressed in early embryos, a change in gene expression must accompany nuclear reprogramming. In this study, nuclear reprogramming of somatic cell nuclei following nuclear transfer (NT) was demonstrated by the reactivation of developmentally regulated lacZ reporter genes. NT embryos were generated by direct injection of adult cumulus cell nuclei into mature mouse oocytes from which maternal chromosomes were subsequently removed. Cumulus cells were collected from transgenic mice which show developmentally regulated lacZ reporter gene expression as a result of integration and functional coupling of reporter gene expression with the endogenous Oct4 or COB54 genes. As both genes are transcriptionally silent in somatic cells but are expressed during early embryonic development, reactivation of reporter gene expression in cumulus cell-derived NT embryos was assessed as a means of monitoring nuclear reprogramming. The pattern of X-gal staining observed in individual NT embryos derived from both transgenic lines revealed that coordination of reactivation appeared to be specific for each gene, and that the timing of expression was consistent with that seen in control non-manipulated transgenic embryos. However, the staining in some NT embryos appeared to be diminished or absent. This reduction in staining may indicate a failure to reprogram gene expression in these embryos. Similar transgenic strategies employing lacZ vital stains, or alter-native reporters such as GFP, may facilitate real-time monitoring of reprogramming and a potential selection strategy to increase cloning efficiency through the identification and selection of reprogrammed, preimplantation stage NT embryos prior to embryo transfer.

Transgenic strategy for demonstrating nuclear reprogramming in the mouse.

We have generated embryonic stem (ES) cells and transgenic mice with green fluorescent protein (GFP) inserted into the Pitx3 locus via homologous recombination. In the central nervous system, Pitx3-directed GFP was visualized in dopaminergic (DA) neurons in the substantia nigra and ventral tegmental area. Live primary DA neurons can be isolated by fluorescence-activated cell sorting from these transgenic mouse embryos. In culture, Pitx3-GFP is coexpressed in a proportion of ES-derived DA neurons. Furthermore, ES cell-derived Pitx3-GFP expressing DA neurons responded to neurotrophic factors and were sensitive to DA-specific neurotoxin N-4-methyl-1, 2, 3, 6-tetrahydropyridine. We anticipate that the Pitx3-GFP ES cells could be used as a powerful model system for functional identification of molecules governing mDA neuron differentiation and for preclinical research including pharmaceutical drug screening and transplantation. The Pitx3 knock-in mice, on the other hand, could be used for purifying primary neurons for molecular studies associated with the midbrain-specific DA phenotype at a level not previously feasible. These mice would also provide a useful tool to study DA fate determination from embryo- or adult-derived neural stem cells.

Generation of embryonic stem cells and transgenic mice expressing green fluorescence protein in midbrain dopaminergic neurons.

ES cells are extraordinary cells, capable of proliferating in a pluripotent state indefinitely and of differentiating spontaneously into all cell types in vivo and many in vitro. However, the manipulation and modification of ES cells by processes such as directed differentiation and genetic modification have placed ES cells at the forefront of many biological studies and could lead to their application in biopharmaceutical areas such as cellular therapy and drug screening. Here we describe some of the ES cell based technologies that have lead to this realisation of ES cell potential.

ES cell technology: an introduction to genetic manipulation, differentiation and therapeutic cloning.

The process of bringing a new pharmacologically active drug to market is laborious, time consuming, and costly. From drug discovery to safety assessment, new methods are constantly sought to develop faster and more efficient procedures to eliminate drugs from further investigation because of their limited effectiveness or high toxicity. Because in vitro cell assays are an important arm of this discovery process, it is therefore somewhat unsurprising that there is an emerging contribution of embryonic stem (ES) cell technology to this area. This technology utilizes the in vitro differentiation of ES cells into somatic cell target populations that, when coupled to the use of "lineage selection" protocols, allows for the production of infinite numbers of pure populations of the desired cells for both bioactivity and toxicological screens. Unlike the use of transformed cell lines, ES-derived cells remain karyotypically normal and therefore better reflect the potential responses of cells in vivo, and when selected are more homogeneous than those obtained using primary cultures. In this chapter we discuss the use of ES cell-derived somatic cells in pharmacological screens, with particular emphasis on neural cells, and describe the methods and protocols associated with the development of ES cell-derived neural cell assays.

Embryonic stem cells as a source of differentiated neural cells for pharmacological screens.

The International Stem Cell Initiative characterized 59 human embryonic stem cell lines from 17 laboratories worldwide. Despite diverse genotypes and different techniques used for derivation and maintenance, all lines exhibited similar expression patterns for several markers of human embryonic stem cells. They expressed the glycolipid antigens SSEA3 and SSEA4, the keratan sulfate antigens TRA-1-60, TRA-1-81, GCTM2 and GCT343, and the protein antigens CD9, Thy1 (also known as CD90), tissue-nonspecific alkaline phosphatase and class 1 HLA, as well as the strongly developmentally regulated genes NANOG, POU5F1 (formerly known as OCT4), TDGF1, DNMT3B, GABRB3 and GDF3. Nevertheless, the lines were not identical: differences in expression of several lineage markers were evident, and several imprinted genes showed generally similar allele-specific expression patterns, but some gene-dependent variation was observed. Also, some female lines expressed readily detectable levels of XIST whereas others did not. No significant contamination of the lines with mycoplasma, bacteria or cytopathic viruses was detected.

Characterization of human embryonic stem cell lines by the International Stem Cell Initiative.

Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), collectively termed human pluripotent stem cells (hPSCs), are typically derived and maintained in adherent and semi-defined culture conditions. Recently a number of groups, including Chen et al., 2012, have demonstrated that hESCs can now be expanded efficiently and maintain pluripotency over long-term passaging as aggregates in a serum-free defined suspension culture system, permitting the preparation of scalable cGMP derived hPSC cultures for cell banking, high throughput research programs and clinical applications. In this short commentary we describe the utility and potential future uses of suspension culture systems for hPSCs.

Suspended in culture--human pluripotent cells for scalable technologies.

Human induced pluripotent stem cells (hiPSC) have the potential to generate healthy cells and tissues for the study and medical treatment of a large number of diseases. The utility of putative hiPSC-based therapies is constrained by a lack of robust quality-control assays that address the stability of the cells or their capacity to form teratomas after differentiation. Here we report that virally derived hiPSC, but not human embryonic stem cells (hESC) or hiPSC derived using episomal nonintegrating vectors, exhibit a propensity to revert to a pluripotent phenotype following differentiation. This instability was revealed using our published method to identify pluripotent cells undergoing very early-stage differentiation in standard hESC cultures, by fluorescence activated cell sorting (FACS) based on expression of the cell surface markers TG30 (CD9) and GCTM-2. Differentiated cells cultured post-FACS fractionation from virally derived hiPSC lines reacquired immunoreactivity to TG30 (CD9) and GCTM-2, formed stem cell-like colonies, and re-expressed canonical pluripotency markers. Furthermore, differentiated cells from pluripotency-reverting hiPSC lines generated teratomas in immunocompromised mice, raising concerns about their safety in downstream applications. In contrast, differentiated cell populations from hESC and episomally derived hiPSC did not show any of these abnormalities. Our assays may be used to identify "unsafe" hiPSC cell lines and this information should be considered when selecting hiPSC lines for clinical use and indicate that experiments using these "unsafe" hiPSC lines should be interpreted carefully.

Article	Year
Transgenic strategy for demonstrating nuclear reprogramming in the mouse. Cloning and stem cells\| PubMed ID: 12180428	2002
Generation of embryonic stem cells and transgenic mice expressing green fluorescence protein in midbrain dopaminergic neurons. The European journal of neuroscience\| PubMed ID: 15016072	2004
ES cell technology: an introduction to genetic manipulation, differentiation and therapeutic cloning. Advanced drug delivery reviews\| PubMed ID: 16253387	2005
Embryonic stem cells as a source of differentiated neural cells for pharmacological screens. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 16846004	2006
Characterization of human embryonic stem cell lines by the International Stem Cell Initiative. Nature biotechnology\| PubMed ID: 17572666	2007
Suspended in culture--human pluripotent cells for scalable technologies. Stem cell research\| PubMed ID: 22771716	2012
Identification of unsafe human induced pluripotent stem cell lines using a robust surrogate assay for pluripotency. Stem cells (Dayton, Ohio)\| PubMed ID: 23728894	2013

Carmel O'Brien

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