Scientific Resources

Cancer stem cells are known for their inherent resistance to therapy. Here we investigated whether normal stem cells with acquired resistance to stress can be used to identify novel markers of cancer stem cells. For this,we generated a human embryonic stem cell line resistant to Trichostatin A and analyzed changes in its gene expression. The resistant cells over-expressed various genes associated with tumor aggressiveness,many of which are also expressed in the CD133+ glioma cancer stem cells. These findings suggest that stress-resistant stem cells generated in vitro may be useful for the discovery of novel markers of cancer stem cells. View Publication

Four commercially available serum-free and defined culture media tested on 2 human embryonic stem cell (hESC) lines were all found to support undifferentiated growth for textgreater10 continuous passages. For hESC cultured with defined StemPro and mTeSR1 media,the cells were maintained feeder-free on culture dishes coated with extracellular matrices (ECMs) with no requirement of feeder-conditioned media (CM). For xeno-free serum replacer (XSR),HEScGRO,and KnockOut media,mitotically inactivated human foreskin feeders (hFFs) were required for hESC growth. Under the different media conditions,cells continued to exhibit alkaline phosphatase activity and expressed undifferentiated hESC markers Oct-4,stage-specific embryonic antigens 4 (SSEA-4),and Tra-1-60. In addition,hESC maintained the expression of podocalyxin-like protein-1 (PODXL),an antigen recently reported in another study to be present in undifferentiated hESC. The cytotoxic antibody mAb 84 binds via PODXL expressed on hESC surface and kills textgreater90% of hESC within 45 min of incubation. When these cells were spontaneously differentiated to form embryoid bodies,derivatives representing the 3 germ layers were obtained. Injection of hESC into animal models resulted in teratomas and the formation of tissue types indicative of ectodermal,endodermal,and mesodermal lineages were observed. Our data also suggested that StemPro and mTeSR1 media were more optimal for hESC proliferation compared to cells grown on CM because the growth rate of hESC increased by 30%-40%,higher split ratio was thus required for weekly passaging. This is advantageous for the large-scale cultivation of hESC required in clinical applications. View Publication

Realizing the full potential of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) requires efficient methods for genetic modification. However,techniques to generate cell type-specific lineage reporters,as well as reliable tools to disrupt,repair or overexpress genes by gene targeting,are inefficient at best and thus are not routinely used. Here we report the highly efficient targeting of three genes in human pluripotent cells using zinc-finger nuclease (ZFN)-mediated genome editing. First,using ZFNs specific for the OCT4 (POU5F1) locus,we generated OCT4-eGFP reporter cells to monitor the pluripotent state of hESCs. Second,we inserted a transgene into the AAVS1 locus to generate a robust drug-inducible overexpression system in hESCs. Finally,we targeted the PITX3 gene,demonstrating that ZFNs can be used to generate reporter cells by targeting non-expressed genes in hESCs and hiPSCs. View Publication

The multidrug transporter ABCG2 in cell membranes enables various stem cells and cancer cells to efflux chemicals,including the fluorescent dye Hoechst 33342. The Hoechst(-) cells can be sorted out as a side population with stem cell properties. Abcg2 expression in mouse embryonic stem cells (ESCs) reduces accumulation of DNA-damaging metabolites in the cells,which helps prevent cell differentiation. Surprisingly,we found that human ESCs do not express ABCG2 and cannot efflux Hoechst. In contrast,trophoblasts and neural epithelial cells derived from human ESCs are ABCG2(+) and Hoechst(-). Human ESCs ectopically expressing ABCG2 become Hoechst(-),more tolerant of toxicity of mitoxantrone,a substrate of ABCG2,and more capable of self-renewal in basic fibroblast growth factor (bFGF)-free condition than control cells. However,Hoechst(low) cells sorted as a small subpopulation from human ESCs express lower levels of pluripotency markers than the Hoechst(high) cells. Similar results were observed with human induced pluripotent stem cells. Conversely,mouse ESCs are Abcg2(+) and mouse trophoblasts,Abcg2(-). Thus,absence of ABCG2 is a novel feature of human pluripotent stem cells,which distinguishes them from many other stem cells including mouse ESCs,and may be a reason why they are sensitive to suboptimal culture conditions. View Publication

The reprogramming of somatic cells into induced pluripotent stem (iPS) cells upon overexpression of the transcription factors Oct4,Sox2,Klf4 and cMyc is inefficient. It has been assumed that the somatic differentiation state provides a barrier for efficient reprogramming; however,direct evidence for this notion is lacking. Here,we tested the potential of mouse hematopoietic cells at different stages of differentiation to be reprogrammed into iPS cells. We show that hematopoietic stem and progenitor cells give rise to iPS cells up to 300 times more efficiently than terminally differentiated B and T cells do,yielding reprogramming efficiencies of up to 28%. Our data provide evidence that the differentiation stage of the starting cell has a critical influence on the efficiency of reprogramming into iPS cells. Moreover,we identify hematopoietic progenitors as an attractive cell type for applications of iPS cell technology in research and therapy. View Publication

Magnetic resonance imaging (MRI) may emerge as an ideal non-invasive imaging modality to monitor stem cell therapy in the failing heart. This imaging modality generates any arbitrary tomographic view at high spatial and temporal resolution with exquisite intrinsic tissue contrast. This capability enables robust evaluation of both the cardiac anatomy and function. Traditionally,superparamagnetic iron oxide nanoparticle (SPIO) has been widely used for cellular MRI due to SPIO's ability to enhance sensitivity of MRI by inducing remarkable hypointense,negative signal,blooming effect" on T2*-weighted MRI acquisition. Recently� View Publication

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Surface marker expression forms the basis for characterization and isolation of human embryonic stem cells (hESCs). Currently,there are few well-defined protein epitopes that definitively mark hESCs. Here we combine immunotranscriptional profiling of hESC lines with membrane-polysome translation state array analysis (TSAA) to determine the full set of genes encoding potential hESC surface marker proteins. Three independently isolated hESC lines (HES2,H9,and MEL1) grown under feeder and feeder-free conditions were sorted into subpopulations by fluorescence-activated cell sorting based on coimmunoreactivity to the hESC surface markers GCTM-2 and CD9. Colony-forming assays confirmed that cells displaying high coimmunoreactivity to GCTM-2 and CD9 constitute an enriched subpopulation displaying multiple stem cell properties. Following microarray profiling,820 genes were identified that were common to the GCTM-2(high)/CD9(high) stem cell-like subpopulation. Membrane-polysome TSAA analysis of hESCs identified 1,492 mRNAs encoding actively translated plasma membrane and secreted proteins. Combining these data sets,88 genes encode proteins that mark the pluripotent subpopulation,of which only four had been previously reported. Cell surface immunoreactivity was confirmed for two of these markers: TACSTD1/EPCAM and CDH3/P-Cadherin,with antibodies for EPCAM able to enrich for pluripotent hESCs. This comprehensive listing of both hESCs and spontaneous differentiation-associated transcripts and survey of translated membrane-bound and secreted proteins provides a valuable resource for future study into the role of the extracellular environment in both the maintenance of pluripotency and directed differentiation. View Publication

Development of non-invasive and accurate methods to track cell fate after delivery will greatly expedite transition of embryonic stem (ES) cell therapy to the clinic. In this protocol,we describe the in vivo monitoring of stem cell survival,proliferation and migration using reporter genes. We established stable ES cell lines constitutively expressing double fusion (DF; enhanced green fluorescent protein and firefly luciferase) or triple fusion (TF; monomeric red fluorescent protein,firefly luciferase and herpes simplex virus thymidine kinase (HSVtk)) reporter genes using lentiviral transduction. We used fluorescence-activated cell sorting to purify these populations in vitro,bioluminescence imaging and positron emission tomography (PET) imaging to track them in vivo and fluorescence immunostaining to confirm the results ex vivo. Unlike other methods of cell tracking,such as iron particle and radionuclide labeling,reporter genes are inherited genetically and can be used to monitor cell proliferation and survival for the lifetime of transplanted cells and their progeny. View Publication

Enumeration of human embryonic stem cell (hESC) numbers through single cell digestion can be time consuming especially in high-throughput or multi-factorial analysis containing 50+ samples. We have developed a reproducible,cost-effective method of counting hESCs in clumps circumventing the need to manually dissociate each sample to single cells. The method is based on the DNA binding capacity of propidium iodide (PI) and subsequent fluorescent signal detection. Standard curves generated for cell numbers versus PI fluorescence as single cells or clumps showed an almost identical relationship in the lines of best fit. The reproducibility of the assay was first demonstrated by seeding hESC clumps at specific cell densities ranging 0.05[x02013]2x105 cells/well and then secondly by using the assay to count cell numbers after different growth conditions. Validation tests showed that consistent seeding densities are important in maintaining undifferentiated hESC culture and that the assay can be used to estimate relative cell numbers and growth curves with high accuracy. View Publication

MLL rearrangements are hallmark genetic abnormalities in infant leukemia known to arise in utero. They can be induced during human prenatal development upon exposure to etoposide. We also hypothesize that chronic exposure to etoposide might render cells more susceptible to other genomic insults. Here,for the first time,human embryonic stem cells (hESCs) were used as a model to test the effects of etoposide on human early embryonic development. We addressed whether: (i) low doses of etoposide promote MLL rearrangements in hESCs and hESCs-derived hematopoietic cells; (ii) MLL rearrangements are sufficient to confer hESCs with a selective growth advantage and (iii) continuous exposure to low doses of etoposide induces hESCs to acquire other chromosomal abnormalities. In contrast to cord blood-derived CD34(+) and hESC-derived hematopoietic cells,exposure of undifferentiated hESCs to a single low dose of etoposide induced a pronounced cell death. Etoposide induced MLL rearrangements in hESCs and their hematopoietic derivatives. After long-term culture,the proportion of hESCs harboring MLL rearrangements diminished and neither cell cycle variations nor genomic abnormalities were observed in the etoposide-treated hESCs,suggesting that MLL rearrangements are insufficient to confer hESCs with a selective proliferation/survival advantage. However,continuous exposure to etoposide induced MLL breaks and primed hESCs to acquire other major karyotypic abnormalities. These data show that chronic exposure of developmentally early stem cells to etoposide induces MLL rearrangements and make hESCs more prone to acquire other chromosomal abnormalities than postnatal CD34(+) cells,linking embryonic genotoxic exposure to genomic instability. View Publication

BACKGROUND: The presence of chromosomal abnormalities could have a negative impact for human embryonic stem cell (hESC) applications both in regenerative medicine and in research. A biomarker that allows the identification of chromosomal abnormalities induced in hESC in culture before they take over the culture would represent an important tool for defining optimal culture conditions for hESC. Here we investigate the expression of CD30,reported to be a biomarker of hESCs with abnormal karyotype,in undifferentiated and spontaneously differentiated hESC.backslashnbackslashnMETHODS AND RESULTS: hESC were derived and cultured on mouse fibroblasts in KO-SR containing medium (serum free media) and passaged mechanically. Our results based on analysis at mRNA (RT-PCR) and protein (fluorescence-activated cell sorting and immunocytochemistry) level show that CD30 is expressed in undifferentiated hESC,even at very early passages,without any correlation with the presence of chromosomal anomalies. We also show that the expression of CD30 is rapidly lost during early spontaneous differentiation of hESC.backslashnbackslashnCONCLUSION: We conclude that CD30 expression in hESC cultures is probably a consequence of culture conditions,and that KO-SR may play a role. In addition,the expression of so-called 'stemness' markers does not change in undifferentiated hESC during long-term culture or when cells acquire chromosomal abnormalities. View Publication