Induced pluripotent stem cells
Described herein is a major breakthrough in nuclear reprogramming and induced pluripotent stem cell (iPSC) technology. Fusion of the powerful transcription activation domain (TAD) of MyoD to the Oct4 protein makes iPSCs generation faster, more efficient, purer, safer and feeder-free. Also, disclosed herein is the first report of the use of a TAD fused to a transcription factor as a method for making iPSCs. By combining transcription factors and TADs, this approach to nuclear reprogramming can have a range of applications from inducing pluriopotency to inducing transdifferentiation without transitioning through iPSCs.
1. A method to increase efficiency of reprogramming cells to form induced pluripotent stem cells (iPSC) comprising a) introducing i) a nucleic acid encoding a fusion protein comprising a transactivation domain from MyoD fused to the N-terminus or C-terminus of Oct4 operably linked to a promoter, and ii) at least one nucleic acid encoding Sox2, Klf4, and optionally c-Myc operably linked to a promoter into a somatic cell, wherein the somatic cell expresses the nucleic acids of i) and ii); and
b) culturing said somatic cell for a time to reprogram into a pluripotent stein cell, wherein the fusion protein increases the efficiency of reprogramming the somatic cell into a iPSC.
2. The method of claim 1 , wherein the transactivation domain of MyoD comprises an N terminus region of MyoD.
3. The method of claim 1 , wherein the nucleic acid encoding the fusion protein comprises the sequence set forth in SEQ ID NO: 20 or a sequence that is at least 95% identical thereto.
4. The method of claim 1 , wherein the cell is mammalian.
5. The method of claim 4 , wherein the mammalian cell is human.
6. The method of claim 1 , wherein the somatic cells are placed in cell culture medium comprising a serum replacement.
7. An in vivo method comprising: (a) isolating a somatic cell from a subject; (b) reprogramming said somatic cell by the method of claim 1 to produce an iPSC; (c) differentiating the iPSC ex vivo into a differentiated cell; and (d) administering the differentiated cell to the subject.
8. The method of claim 7 , wherein the subject is a mammal.
9. The method of claim 1 , wherein the presence of the transactivation domain of MyoD increases the efficiency of iPSC production by greater than 40 fold as compared to when the transactivation domain of MyoD is absent.
10. The method of claim 1 , wherein the presence of the transactivation domain of MyoD increases the efficiency of iPSC production by greater than 50 fold as compared to when the transactivation domain of MyoD is absent.
11. The method of claim 1 , wherein the presence of the transactivation domain of MyoD increases the efficiency of iPSC production by greater than 100 fold as compared to when the transactivation domain of MyoD is absent.
12. A method to accelerate reprogramming cells to form iPSC comprising: a) introducing i) a nucleic acid encoding a fusion protein comprising a transactivation domain of MyoD fused to the N-terminus or C-terminus of Oct4 operably linked to a promoter, and ii) at least one nucleic acid encoding Sox2, Klf4, and optionally c-Myc operably linked to a promoter into a somatic cell, wherein the somatic cell expresses the nucleic acids of i) and ii); and b) culture said somatic cell for a time to reprogram into a pluripotent stem cell, where the fusion protein accelerates the reprogramming of the somatic cell into an iPSC.
13. The method of claim 11 , wherein the iPSC is present by day 5 of culturing.
14. The method of claim 11 , wherein the iPSC is present by day 7 of culturing.