胚胎干细胞
Embryonic stem (ES) cells, which are derived from the inner cell mass of preimplantation embryos, have been recognized as the most pluripotent stem cell population. These cells are capable of unlimited, undifferentiated proliferation in vitro and still maintain the capacity for differentiation into a wide variety of somatic tissues. In this capacity ES cells have widespread clinical potential in the treatments of heart disease, diabetes, spinal cord injury, and a variety of neurodegenerative disorders.
R&D Systems offers a range of products designed to maintain and expand ES cells in culture, as well as monitor their differentiation status.
Feeder Cells
R&D Systems offers cryopreserved irradiated mouse embryonic fibroblasts (iMEFs). These cells are isolated from pathogen-free E13.5 CF1 embryos and irradiated at passage 3 to arrest mitosis. Each order includes 5 vials, enough to prepare ten 10 cm tissue culture plates. Every lot of iMEF feeders is tested for its ability to maintain the expression levels of pluripotency markers as evaluated by immunocytochemistry or flow cytometry (see figure below).
Please see pages 12-13 for a complete protocol using iMEF.
Human Embryonic Stem Cells (Cell Line BG01V) Cultured on iMEFs are Assessed Using Markers of Pluripotency. BG01V colonies (highlighted by arrows) growing in culture on iMEFs (A) were analyzed for markers of pluripotency after 3 passages. Cells were evaluated for SSEA-4 expression by flow cytometry (B) using PE-conjugated anti-human SSEA-4 (Catalog # FAB1435P; filled histogram) or isotype control antibody (open histogram). BG01V colonies were incubated with anti-human Oct-3/4 (C; Catalog # AF1759) or anti-human Nanog (D; Catalog # AF1997) followed by staining with NorthernLights™ 557-conjugated anti-goat secondary antibody (Catalog # NL001).
Feeder-Free Culture using Feeder Cell Conditioned Media
Human embryonic stem cells can be maintained and expanded using mouse or human feeder cell conditioned media. R&D Systems offers both Mouse Embryonic Fibroblast Conditioned Medium and Human Feeder Cell Conditioned Medium capable of supporting the growth of ES cells as assessed by the expression of pluripotency markers.
Please see page 14-15 for a complete protocol using conditioned media to grow the BG01V human ES cell line.
SSEA-4 Expression in Embryonic Stem Cells. Human embryonic stem cells were cultured with recombinant human FGF basic (Catalog # 233-FB) in the presence (A) or absence (B) of Mouse Embryonic Fibroblast (MEF)-conditioned medium (Catalog # AR005). SSEA-4 and Oct-3/4 were detected using anti-human SSEA-4 monoclonal antibody (Catalog # MAB1435) and anti-human Oct-3/4 polyclonal antibody (Catalog # AF1759). Cells were stained with Alexa Fluor® 568-conjugated anti-mouse secondary antibody (SSEA-4; red) and Alexa Fluor 488-conjugated anti-goat secondary antibody (Oct-3/4; green). Cells were counterstained with DAPI (blue). Image courtesy of Dr. Frank Soldner of the National Institutes of Health.
Embryonic Stem Cell Marker Panels
R&D Systems offers kits for assessing the differentiation status of human ES cells. Multiple kits are offered using either antibody or primer-based identification techniques. These kits offer an economical alternative to buying separate reagents.
An Embryoid Body Derived from Human Embryonic Stem Cells Stained for Pluripotency Markers. Cells were stained using antibodies from the Human Embryonic Stem Cell Marker Antibody Panel (Catalog # SC008) for Nanog (red; A) and the nuclei counterstained with DAPI (blue; A) or for Oct-3/4 (green; B). Merge of images in (A) and (B) shows the overlap of the 3 fluorochromes (white; C). Images courtesy of Dr. Ronald McKay, NINDS.
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