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;
(b) introducing an inhibitor of a member of the let-7 family, wherein the member of the let-7 family has the seed sequence GAGGUAG and the inhibitor comprises a sequence complementary to the seed sequence GAGGUAG; and
(c) culturing the cell from step (b) to produce a pluripotent cell, wherein the inhibitor and the expression of the reprogramming factors results in an enhanced production of pluripotent cells when compared to introduction of the reprogramming factors in the absence of the inhibitor.
2. The method of claim 1 , wherein the inhibitor inhibits at least two let-7 family members simultaneously.
3. The method of claim 1 , further comprising introducing a physiologically relevant miRNA into the cell, wherein the relevant miRNA contains the seed sequence AAGUGCU (SEQ ID NO:15), AAAGUGC (SEQ ID NO:16) or AAGUGC.
4. The method of claim 3 , wherein the relevant miRNA is a member of the embryonic stem cell cycle (ESCC) regulating miR-290 cluster.
5. The method of claim 3 , wherein the relevant miRNA is a member of the embryonic stem cell cycle (ESCC) regulating 302 cluster, 17-92 cluster, 106a, and 370 family of human miRNAs.
6. The method of claim 3 , wherein the relevant miRNA is a human or mouse miRNA.
7. The method of claim 1 , wherein the cell is a human cell.
8. The method of claim 7 , wherein the human cell is a somatic cell.
9. The method of claim 3 , wherein the relevant 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), AAAGUGC (SEQ ID NO:16) or AAGUGC.
10. The method of claim 3 , 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).
11. The method of claim 3 , wherein the miRNA that contains the seed sequence AAGUGCU (SEQ ID NO:15), AAAGUGC (SEQ ID NO:16) or AAGUGC is a miRNA of Example 3 or Example 4 or a human ortholog thereof.