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  • Somatic coding mutations in human induced pluripotent stem cells. 21368825

    Defined transcription factors can induce epigenetic reprogramming of adult mammalian cells into induced pluripotent stem cells. Although DNA factors are integrated during some reprogramming methods, it is unknown whether the genome remains unchanged at the single nucleotide level. Here we show that 22 human induced pluripotent stem (hiPS) cell lines reprogrammed using five different methods each contained an average of five protein-coding point mutations in the regions sampled (an estimated six protein-coding point mutations per exome). The majority of these mutations were non-synonymous, nonsense or splice variants, and were enriched in genes mutated or having causative effects in cancers. At least half of these reprogramming-associated mutations pre-existed in fibroblast progenitors at low frequencies, whereas the rest occurred during or after reprogramming. Thus, hiPS cells acquire genetic modifications in addition to epigenetic modifications. Extensive genetic screening should become a standard procedure to ensure hiPS cell safety before clinical use.
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  • Patient-specific stem cell lines derived from human parthenogenetic blastocysts. 17594198

    Parthenogenetic activation of human oocytes may be one way to produce histocompatible cells for cell-based therapy. We report the successful derivation of six pluripotent human embryonic stem cell (hESC) lines from blastocysts of parthenogenetic origin. The parthenogenetic human embryonic stem cells (phESC) demonstrate typical hESC morphology, express appropriate markers, and possess high levels of alkaline phosphatase and telomerase activity. The phESC lines have a normal 46, XX karyotype, except one cell line, and have been cultured from between 21 to 35 passages. The phESC lines form embryoid bodies in suspension culture and teratomas after injection to immunodeficient animals and give differentiated derivatives of all three embryonic germ layers. DNA profiling of all six phESC lines demonstrates that they are MHC matched with the oocyte donors. The study of imprinted genes demonstrated further evidence of the parthenogenetic origin of the phESC lines. Our research has resulted in a protocol for the production of human parthenogenetic embryos and the derivation of stem cell lines from them, which minimizes the presence of animal-derived components, making the derived phESC lines more suitable for potential clinical use.
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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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  • Reprogramming of human fibroblasts to induced pluripotent stem cells under xeno-free conditions. 19890879

    The availability of induced pluripotent stem cells (iPSCs) has created extraordinary opportunities for modeling and perhaps treating human disease. However, all reprogramming protocols used to date involve the use of products of animal origin. Here, we set out to develop a protocol to generate and maintain human iPSC that would be entirely devoid of xenobiotics. We first developed a xeno-free cell culture media that supported the long-term propagation of human embryonic stem cells (hESCs) to a similar extent as conventional media containing animal origin products or commercially available xeno-free medium. We also derived primary cultures of human dermal fibroblasts under strict xeno-free conditions (XF-HFF), and we show that they can be used as both the cell source for iPSC generation as well as autologous feeder cells to support their growth. We also replaced other reagents of animal origin (trypsin, gelatin, matrigel) with their recombinant equivalents. Finally, we used vesicular stomatitis virus G-pseudotyped retroviral particles expressing a polycistronic construct encoding Oct4, Sox2, Klf4, and GFP to reprogram XF-HFF cells under xeno-free conditions. A total of 10 xeno-free human iPSC lines were generated, which could be continuously passaged in xeno-free conditions and maintained characteristics indistinguishable from hESCs, including colony morphology and growth behavior, expression of pluripotency-associated markers, and pluripotent differentiation ability in vitro and in teratoma assays. Overall, the results presented here demonstrate that human iPSCs can be generated and maintained under strict xeno-free conditions and provide a path to good manufacturing practice (GMP) applicability that should facilitate the clinical translation of iPSC-based therapies.
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  • Comparative study of mouse and human feeder cells for human embryonic stem cells. 18415935

    Various types of feeder cells have been adopted for the culture of human embryonic stem cells (hESCs) to improve their attachment and provide them with stemness-supporting factors. However, feeder cells differ in their capacity to support the growth of undifferentiated hESCs. Here, we compared the expression and secretion of four well-established regulators of hESC pluripotency and/or differentiation among five lines of human foreskin fibroblasts and primary mouse embryonic fibroblasts throughout a standard hESC culture procedure. We found that human and mouse feeder cells secreted comparable levels of TGF beta 1. However, mouse feeder cells secreted larger quantities of activin A than human feeder cells. Conversely, FGF-2, which was produced by human feeder cells, could not be detected in culture media from mouse feeder cells. The quantity of BMP-4 was at about the level of detectability in media from all feeder cell types, although BMP-4 dimers were present in all feeder cells. Production of TGF beta 1, activin A, and FGF-2 varied considerably among the human-derived feeder cell lines. Low- and high-producing human feeder cells as well as mouse feeder cells were evaluated for their ability to support the undifferentiated growth of hESCs. We found that a significantly lower proportion of hESCs maintained on human feeder cell types expressed SSEA3, an undifferentiated cell marker. Moreover, SSEA3 expression and thus the pluripotent hESC compartment could be partially rescued by addition of activin A. Cumulatively, these results suggest that the ability of a feeder layer to promote the undifferentiated growth of hESCs is attributable to its characteristic growth factor production.
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  • Characterization of a novel embryonic stem cell line from an ICSI-derived blastocyst in the African green monkey. 19955206

    Several cell types from the African green monkey (Cercopithecus aethiops), such as red blood cells, primary culture cells from kidney, and the Vero cell line, are valuable sources for biomedical research and testing. Embryonic stem (ES) cells that are established from blastocysts have pluripotency to differentiate into these and other types of cells. We examined an in vitro culture system of zygotes produced by ICSI in African green monkeys and attempted to establish ES cells. Culturing with and without a mouse embryonic fibroblast (MEF) cell monolayer resulted in the development of ICSI-derived zygotes to the blastocyst stage, while culturing with a buffalo rat liver cell monolayer yielded no development (3/14, 21.4% and 6/31, 19.4% vs 0/23, 0% respectively; P0.05). One of the nine blastocysts, which had been one of the zygotes co-cultured with MEF cells, formed flat colonies consisting of cells with large nuclei, similar to other primate ES cell lines. The African green monkey ES (AgMES) cells expressed pluripotency markers, formed teratomas consisting of three embryonic germ layer tissues, and had a normal chromosome number. Furthermore, expression of the germ cell markers CD9 and DPPA3 (STELLA) was detected in the embryoid bodies, suggesting that AgMES cells might have the potential ability to differentiate into germ cells. The results suggested that MEF cells greatly affected the quality of the inner cell mass of the blastocysts. In addition, AgMES cells would be a precious resource for biomedical research such as other primate ES cell lines.
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  • Glycan stem-cell markers are specifically expressed by spermatogonia in the adult non-human primate testis. 18621756

    The glycan cell surface molecules, stage-specific embryonic antigen (SSEA)-1, -3 and -4 and tumor-rejection antigen (TRA)-1-60 and -1-81, are expressed in specific combinations by undifferentiated pluripotent cells, i.e. embryonic stem cells, induced pluripotent stem cells, embryonal carcinoma cells, primordial germ cells and embryonic germ cells. Upon differentiation of the cells, these markers vanish. Recently, it has been shown that also neonatal and adult mouse testes contain pluripotent cells. Here, we aimed at identifying in situ possibly pluripotent cells in the adult primate testis.Monoclonal antibodies raised against the glyco-epitopes SSEA-1, -3 and -4 and TRA-1-60 and -1-81, respectively, were tested to detect cells expressing the antigens, by immunohistochemistry on Bouin's-fixed and paraffin-embedded adult primate testes. Man, the new-world monkey, Callithrix jacchus (common marmoset), and the old-world monkey species, Macaca mulatta (Rhesus macaque) and Macaca silenus (Lion-tailed macaque), were included. The percentage of SSEA-4-positive cells in three adult marmoset testes was determined using flow cytometry.Spermatogonia in the testes of C. jacchus were labeled by SSEA-4, TRA-1-60 and -1-81-antibodies. In the macaques, spermatogonia were detected by SSEA-4 and TRA-1-81-antibodies. TRA-1-61 did not bind to macaque spermatogonia. Also, SSEA-1 and -3 did not bind to spermatogonia in any species. In human testes, we never obtained any clear staining. The total percentage of SSEA-4-positive cells in marmoset testes was 8.6 +/- 1.61%.SSEA-4 and TRA-1-81-antibodies may be very well suited for the identification and isolation of spermatogonia, and possibly also germline stem cells, in the non-human primate testis.
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  • Scalable passaging of adherent human pluripotent stem cells. 24498239

    Current laboratory methods used to passage adherent human pluripotent stem cells (hPSCs) are labor intensive, result in reduced cell viability and are incompatible with larger scale production necessary for many clinical applications. To meet the current demand for hPSCs, we have developed a new non-enzymatic passaging method using sodium citrate. Sodium citrate, formulated as a hypertonic solution, gently and efficiently detaches adherent cultures of hPSCs as small multicellular aggregates with minimal manual intervention. These multicellular aggregates are easily and reproducibly recovered in calcium-containing medium, retain a high post-detachment cell viability of 97%±1% and readily attach to fresh substrates. Together, this significantly reduces the time required to expand hPSCs as high quality adherent cultures. Cells subcultured for 25 passages using this novel sodium citrate passaging solution exhibit characteristic hPSC morphology, high levels (greater than 80%) of pluripotency markers OCT4, SSEA-4, TRA-1-60 andTRA-1-81, a normal G-banded karyotype and the ability to differentiate into cells representing all three germ layers, both in vivo and in vitro.
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