Embryonic stem cell specific microRNAs promote induced pluripotency
The methods of the present application describe that introduction of physiologically relevant miRNAs can enhance or modulate somatic cell reprogramming, generating induced pluripotent stem cells (iPS cells). These miRNAs did not further enhance reprogramming in the presence of cMyc. Furthermore, unlike previously described methods of generating iPS cells, such as through the introduction of genetic elements using viruses, the methods of the present invention reduce the risk of activating oncogenes in the iPS cells. The methods of the invention generate iPS cells that can be free of genetic modifications and thus have greater potential for use as therapeutic agents than those generated by existing methods.
1. A method of inducing pluripotency in an isolated cell comprising:
(a) introducing a vector(s) encoding the reprogramming factors Oct-4, Sox2, KLF-4 (OSK) into the cell; and
(b) introducing a physiologically relevant miRNA into the cell, wherein the miRNA contains the seed sequence AAGUGCU (SEQ ID NO: 15) or AAAGUGC (SEQ ID NO:16) or AAGUGC; and
(c) culturing the cell from step (b) to produce a pluripotent cell, wherein expression of the reprogramming factors and the miRNA results in an enhanced production of the pluripotent cells when compared to introduction of the reprogramming factors in the absence of the miRNA.
2. The method of claim 1 , wherein the miRNA is a member of the embryonic stem cell cycle (ESCC) regulating miR-290 cluster.
3. The method of claim 1 , wherein the miRNA is a member of the embryonic stem cell cycle (ESCC) regulating 302 cluster, 17-92 cluster, 106a, and 370 family of human microRNAs.
4. The method of claim 1 , wherein the miRNA is a human or mouse miRNA.
5. The method of claim 4 wherein the cell is a human cell.
6. The method of claim 1 , wherein the miRNA is
(i) 80% or more identical to one of miR-291-3p (SEQ ID NO:1), miR-294 (SEQ ID NO:2), miR-295 (SEQ ID NO:3), miR-302d (SEQ ID NO:4), miR-292-3p (SEQ ID NO:5), hsa-miR-302a (SEQ ID NO:8), hsa-miR-302b (SEQ ID NO:9), hsa-miR-302c (SEQ ID NO:10), hsa-miR-302d (SEQ ID NO:11), hsa-miR-372 (SEQ ID NO:13), hsa-miR-373 (SEQ ID NO:14), hsa-miR-17 (SEQ ID NO:17), hsa-miR-20a (SEQ ID NO:18), hsa-miR-20b (SEQ ID NO:19), hsa-miR-93 (SEQ ID NO:20), hsa-miR-106a (SEQ ID NO:21), or hsa-miR-106b (SEQ ID NO:22); and
(ii) contains the seed sequence AAGUGCU (SEQ ID NO:15) or AAAGUGC (SEQ ID NO:16) or AAGUGC.
7. The method of claim 2 wherein the miR-290 cluster member is one of miR-291-3p (SEQ ID NO:1), miR-294 (SEQ ID NO:2) miR-295 (SEQ ID NO:3), miR-302d (SEQ ID NO:4), miR-292-3p (SEQ ID NO:5), hsa-miR-302a (SEQ ID NO:8), hsa-miR-302b (SEQ ID NO:9), hsa-miR-302c (SEQ ID NO:10), hsa-miR-302d (SEQ ID NO:11), hsa-miR371-5p (SEQ ID NO:12), hsa-miR-372 (SEQ ID NO:13), or hsa-miR-373 (SEQ ID NO:14).
8. The method of claim 1 , wherein the miRNA that contains the seed sequence AAGUGCU (SEQ ID NO: 15) or AAAGUGC (SEQ ID NO:16) or AAGUGC is an miRNA of Example 3 or Example 4 or their human orthologs.