The best assay for confirmation of pluripotency for mouse embryonic stem cells (ESCs) is blastocyst chimerism and for human ESCs the production of teratomas in immunodeficient mice that contain lineages from all three primordial germ layers. However, the latter assay has been interpreted by some as one type of human-animal chimera and studies involving human-animal chimerism present a wide range of ethical issues with restrictive legislation in some countries. The need for teratoma assays is compelling because unproven differentiation may suggest a dangerously flawed cell resource for use in future therapies. We find that a three dimensional anchorage independent growth protocol for hESCs using methylcellulose and matrigel encourages prolonged growth of embryoid bodies (EBs) leading onto formation of teratomas in vitro. Our late EBs are a miniature form of teratomas containing lineages from all three germ layers, do not show embryonal carcinoma cells and allows integration of microinjected cancer cells (HepG2-GFP).
Additional data files are linked below as supplementary files: [1] Beadstudio_Controls_non-normalized.txt: Control Probe Profile intensity data exported from Beadstudio. [2] Beadstudio_background_removed_non-normalized.txt: Background corrected intensity data exported from Beadstudio using the default Beadstudio background correction setting. No normalization carried out on the data. [3] VST_quantile_filterp0.01_normalized.txt: Data after VST transformation, quantile normalized and filtered with detection p-value 0.01. This data set was used for analysis in the accompanying publication.
Overall design
Total RNA obtained from embryoid bodies grown in different culture conditions (EB: embryoid body medium, BCM: bulk conditioned medium, MAT: matrigel) and harvested at several time points (7day, 14 day, 30 day, 60 day, 90day).
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