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  • Human mesenchymal stem cell transformation is associated with a mesenchymal-epithelial transition. 18201695

    Carcinomas are widely thought to derive from epithelial cells with malignant progression often associated with an epithelial-mesenchymal transition (EMT). We have characterized tumors generated by spontaneously transformed human mesenchymal cells (TMC) previously obtained in our laboratory. Immunohistopathological analyses identified these tumors as poorly differentiated carcinomas, suggesting that a mesenchymal-epithelial transition (MET) was involved in the generation of TMC. This was corroborated by microarray and protein expression analysis that showed that almost all mesenchymal-related genes were severely repressed in these TMC. Interestingly, TMC also expressed embryonic antigens and were able to integrate into developing blastocysts with no signs of tumor formation, suggesting a dedifferentiation process was associated with the mesenchymal stem cell (MSC) transformation. These findings support the hypothesis that some carcinomas are derived from mesenchymal rather than from epithelial precursors.
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  • Isolation of human embryonal carcinoma stem cells by immunomagnetic sorting. 11713341

    Embryonal carcinoma cells are pluripotent stem cells derived from germ cell tumors and can be used to study cell differentiation in vitro. This report describes an approach designed to isolate pluripotent stem cells from primary/parent stock cultures of explanted tumor material. Cells expressing the pluripotent stem cell marker, SSEA-3, were isolated from heterogeneous stock cultures of the human teratoma line, TERA2, using immunomagnetic isolation. Single cell selection was performed on isolated SSEA-3+ cells and clonal lines were established. Each line was ultimately grown as a homogeneous monolayer, independent of feeder cells and expressed high levels of markers for pluripotent stem cells. In response to retinoic acid, clone TERA2.cl.SP-12 cells displayed enhanced neural differentiation compared to previously isolated TERA2 sublines and formed both neurons and glia. Deriving human pluripotent stem cell lines that differentiate into a range of cell types will provide useful tools to understand the molecular mechanisms controlling cell differentiation in a manner pertinent to human embryonic development.
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  • Establishment and characterization of embryonic stem-like cells from porcine somatic cell nuclear transfer blastocysts. 20307349

    This study was aimed to establish embryonic stem (ES)-like cells from blastocysts derived from somatic cell nuclear transfer (SCNT) in pig. Somatic cells isolated from both day-30 fetus and neonatal cloned piglet were used for donor cells. A total of 60 blastocysts (46 and 14 derived from fetal and neonatal fibroblast donor cells, respectively) were seeded onto a mitotically inactive mouse embryonic fibroblast (MEF) monolayer and two ES-like cell lines, one from each donor cell type, were established. They remained undifferentiated over more than 52 (fetal fibroblast-derived) and 48 (neonatal fibroblast-derived) passages, while retaining alkaline phosphatase activity and reactivity with ES specific markers Oct-4, stage-specific embryonic antigen-1 (SSEA-1), SSEA-4, TRA-1-60 and TRA-1-81. These ES-like cells maintained normal diploid karyotype throughout subculture and successfully differentiated into embryoid bodies that expressed three germ layer-specific genes (ectoderm: beta-III tubulin; endoderm: amylase; and mesoderm: enolase) after culture in leukemia inhibitory factor-free medium. Microsatellite analysis confirmed that they were genetically identical to its donor cells. Combined with gene targeting, our results may contribute to developing an efficient method for producing transgenic pigs for various purposes.
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  • Novel autogenic feeders derived from human embryonic stem cells (hESCs) support an undifferentiated status of hESCs in xeno-free culture conditions. 19202140

    BACKGROUND: Clinical-grade human embryonic stem cells (hESCs) ideally should be derived and maintained in xeno-free culture conditions using defined chemicals or materials of human origin. This will reduce the possibility of xeno-derived pathogenic infection and/or unfavorable immune reaction in clinical application. The present study therefore aimed to derive autogenic feeders from hESCs and evaluate their capability to support the pluripotency of hESCs in xeno-free culture conditions. METHODS AND RESULTS: H9 hESCs were cultured in media containing human serum (HS), serum replacement (SR) or KFM combination, to generate autogenic feeders (named HSdF, SRdF and KFMdF, respectively). Reverse transcription polymerase chain reaction, flow cytometry and immunofluorescence analysis using pluripotent stem cell markers, markers of early cell lineages and surface markers revealed that HSdF, SRdF and KFMdF likely belonged to different cellular subpopulations. The efficiency of the autogenic feeders in maintaining pluripotency of H9 hESCs using media containing SR, fetal bovine serum, HS or 1% HS plus various combinations of growth factors was evaluated by flow cytometric analysis of Oct4 expression. All three autogenic feeders were shown to be capable of maintaining the undifferentiated status of H9 hESCs in SR-containing media in long-term culture. When supplemented with bFGF, activin A and noggin, hESCs could also be maintained favorably on KFMdF in a medium containing 1% HS without losing their pluripotent potentials both in vitro and in vivo. CONCLUSIONS: Novel autogenic feeders can be derived from hESCs under xeno-free conditions and they can robustly maintain the pluripotent identity of hESCs in xeno-free media containing a low concentration of HS.
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  • Characterization of CD30 expression in human embryonic stem cell lines cultured in serum-free media and passaged mechanically. 19584135

    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.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.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.
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