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  • Karyotyping ES Cells

    Document Type:
    Protocols
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  • Optimized mouse ES cell culture system by suspension growth in a fully defined medium. 18536648

    Mouse and human embryonic stem (mES and hES) cells have become one of the most intensively studied primary cell types in biomedical research. However, culturing ES cells is notoriously labor intensive. We have optimized current ES cell culture methods by growing mES cells in suspension in a defined medium. This protocol is unsurpassed in time efficiency and typically requires only 20 min of effective hands-on time per week. This protocol maintains a very high degree of pluripotent cells partly by mechanical separation of spontaneously differentiating cells. mES cells can be cultured for extended periods (>6 months) without the loss of pluripotency markers. High passage (>20) adherent mES cultures containing contaminating differentiated cells can be rescued and enriched in undifferentiated ES cells.
    Document Type:
    Reference
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  • Using small molecules to improve generation of induced pluripotent stem cells from somatic cells 20336525

    Induction of pluripotent stem cells from somatic cells by defined factors was shown to be possible only recently, but already several laboratories have made tremendous strive toward improving and understanding the process. Originally, Oct4, Sox2, Klf4, and cMyc were identified as being the combination of genes necessary to induce reprogramming. It was later shown that cMyc was dispensable; however, in its absence the process was less efficient and took a considerably longer period of time to occur. Furthermore, others have shown that the combination of Oct4, Sox2, Nanog, and Lin28 could also induce reprogramming. One major caveat associated with these techniques remains the need for overexpression of several genes using viral systems. Until very recently, most studies were done using integrating viruses such as retroviruses and lentiviruses. This method ensured that the protein of interested would be expressed at a high concentration and for an adequate period of time necessary to induce reprogramming. Up to date, others have now been able to use different nonintegrative method such as adenovirus and plasmid transfection to induce reprogramming. Furthermore, piggyBac transposition was successfully used to induce reprogramming of murine cells. Most importantly, it was recently published that reprogramming can be induced in the absence of virus, with proteins and small molecules. All of the later methods are appealing since they do not require the integration of the virus or plasmid to exert its effect. However, one avenue that would be all the more therapeutically appealing would be to induce reprogramming in the absence of gene overexpression systems, using small molecules to modulate signaling pathways in the somatic cells. A few molecules have already been identified with the ability to either improve the process or replace one or two of the genes deemed necessary for reprogramming. We have screened successfully for compounds that can replace some of these factors, and share the methods developed following these screens.
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    Reference
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  • Derivation of mouse embryonic stem cells 17487198

    Here we describe a simple and efficient protocol for derivation of germline chimera-competent mouse embryonic stem cells (mESCs) from embryonic day 3.5 (E3.5) blastocysts. The protocol involves the use of early-passage mouse embryonic fibroblast feeders (MEF) and the alternation of fetal bovine serum- and serum replacement (SR)-containing media. As compared to other available protocols for mESCs derivation, our protocol differs in the combination of commercial availability of all reagents, technical simplicity and high efficiency. mESC lines are derived with approximately 50% efficiency (50 independent mESC lines derived from 96 blastocysts). We believe that this protocol could be a good starting point for (i) setting up the derivation of mESC lines in a laboratory and (ii) incorporating further steps to improve efficiency or adapt the protocol to other applications. The whole process (from blastocyst extraction to the freezing of mESC line) usually takes between 15 and 20 d.
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    Reference
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  • Systematic delineation of optimal cytokine concentrations to expand hematopoietic stem/progenitor cells in co-culture with mesenchymal stem cells. 20424784

    The major obstacle to the widespread use of umbilical cord blood (UCB) in hematopoietic stem/progenitor (HSC) cell therapy is the low cell dose available. A cytokine cocktail for the ex vivo expansion of UCB HSC, in co-culture with a bone marrow (BM) mesenchymal stem cells (MSC)-derived stromal layer was optimized using an experimental design approach. Proliferation of total cells (TNC), stem/progenitor cells (CD34(+)) and colony-forming units (CFU) was assessed after 7 days in culture, while sole and interactive effects of each cytokine on HSC expansion were statistically determined using a two-level Face-Centered Cube Design. The optimal cytokine cocktail obtained for HSC-MSC co-cultures was composed by SCF, Flt-3L and TPO (60, 55 and 50 ng mL(-1), respectively), resulting in 33-fold expansion in TNC, 17-fold in CD34(+) cells, 3-fold in CD34(+)CD90(+) cells and 21-fold in CFU-MIX. More importantly, these short-term expanded cells preserved their telomere length and extensively generated cobblestone area-forming cells (CAFCs) in vitro. The statistical tools used herein contributed for the rational delineation of the cytokine concentration range, in a cost-effective way, while systematically addressing complex cytokine-to-cytokine interactions, for the efficient HSC expansion towards the generation of clinically significant cell numbers for transplantation.
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    Reference
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  • Proliferating versus differentiating stem and cancer cells exhibit distinct midbody-release behaviour. 22009035

    The central portion of the midbody, a cytoplasmic bridge between nascent daughter cells at the end of cell division, has generally been thought to be retained by one of the daughter cells, but has, recently, also been shown to be released into the extracellular space. The significance of midbody-retention versus -release is unknown. Here we show, by quantitatively analysing midbody-fate in various cell lines under different growth conditions, that the extent of midbody-release is significantly greater in stem cells than cancer-derived cells. Induction of cell differentiation is accompanied by an increase in midbody-release. Knockdown of the endosomal sorting complex required for transport family members, Alix and tumour-suppressor gene 101, or of their interaction partner, centrosomal protein 55, impairs midbody-release, suggesting mechanistic similarities to abscission. Cells with such impaired midbody-release exhibit enhanced responsiveness to a differentiation stimulus. Taken together, midbody-release emerges as a characteristic feature of cells capable of differentiation.
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    Reference
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