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US 9,428,811 B2Granted
Epigenomic induced pluripotent stem cell signatures
AssigneeThe Salk Institute for Biological Studies
InventorsRonald M. Evans, Joseph R. Ecker, Ryan Lister
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Abstract
Provided herein are methods of characterizing the epigenetic signature of human induced pluripotent stem cells. The methods are useful in identifying human induced pluripotent stem cells (hiPSCs), diagnostic markers for incomplete hiPSCs reprogramming, and characterization of the efficacy of different reprogramming techniques.
Claims12
A method of identifying an incompletely reprogrammed human induced pluripotent stem cell, comprising:
- obtaining a human induced pluripotent stem cell produced using reprogramming factors;
- isolating genomic DNA from the human induced pluripotent stem cell;
- contacting the genomic DNA with bisulfate to detect methylation of the genomic DNA of the human induced pluripotent stem cell;
- evaluating the methylation of the genomic DNA of the human induced pluripotent cell to identify a methylation pattern of at least one CpG differentially methylated region (DMR) in the genomic DNA of the human induced pluripotent stem cell and at least one non-CpG DMR in the genomic DNA of the human induced pluripotent stem cell, wherein the at least one CpG DMR is:
- a) a CpG-DMR comprising position 125033730 to position 125034829 of chromosome 12 of the genomic DNA of the human induced pluripotent stem cell;
- b) a CpG-DMR comprising position 128952730 to position 128955529 of chromosome 12 of the genomic DNA of the human induced pluripotent stem cell;
- c) a CpG-DMR comprising position 129064330 to position 129065329 of chromosome 12 of the genomic DNA of the human induced pluripotent stem cell;
- d) a CpG-DMR comprising position 129092430 to position 129093429 of chromosome 12 of the genomic DNA of the human induced pluripotent stem cell;
- e) a CpG-DMR comprising position 129209230 to position 129215029 of chromosome 12 of the genomic DNA of the human induced pluripotent stem cell;
- f) a CpG-DMR comprising position 87905315 to position 87906414 of chromosome 2 of the genomic DNA of the human induced pluripotent stem cell;
- g) a CpG-DMR comprising position 132998500 to position 133001399 of chromosome 10 of the genomic DNA of the human induced pluripotent stem cell;
- h) a CpG-DMR comprising position 157173784 to position 157180183 of chromosome 7 of the genomic DNA of the human induced pluripotent stem cell;
- i) a CpG-DMR comprising position 47143596 to position 47145195 of chromosome 22 of the genomic DNA of the human induced pluripotent stem cell;
- j) a CpG-DMR comprising position 47263296 to position 47266095 of chromosome 22 of the genomic DNA of the human induced pluripotent stem cell;
- k) a CpG-DMR comprising position 47349296 to position 47351495 of chromosome 22 of the genomic DNA of the human induced pluripotent stem cell;
- and comparing the methylation pattern of the at least one CpG DMR in the genomic DNA of the human induced pluripotent stem cell and the at least one non-CpG DMR in the genomic DNA of the human induced pluripotent stem cell to the methylation pattern of the at least one CpG DMR in genomic DNA of an embryonic stem cell and the at least one non-CpG DMR in genomic DNA of the embryonic stem cell, wherein hypermethylation of the at least one CpG-DMRs as compared to corresponding CpG-DMRs in the DNA of the embryonic stem cell identifies the induced pluripotent stem cell as a reprogrammed human induced pluripotent stem cell;
- and wherein a difference in methylation of the at least one non-CpG DMR in the genomic DNA of the human induced pluripotent stem cell as compared to the methylation pattern of the at least one non-CpG DMR in the genomic DNA of the embryonic stem cell, identifies the human pluripotent stem cell as an incompletely reprogrammed human induced pluripotent stem cell.
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