Detection of human somatic cell reprogramming
The methods and kits described herein are based, in part, to the discovery of a phenotype representing a fully-reprogrammed iPS cell and several reprogramming intermediates. The methods and kits described herein permit identification of fully-reprogrammed iPS cells and further permits one of skill in the art to monitor the emergence of iPS cells during the reprogramming process. The methods/kits can also be performed using real time using live cell imaging. Also described herein are methods for screening candidate reprogramming agents by monitoring the emergence of fully-reprogrammed iPS cells in the presence and absence of such an agent.
1. A method for detecting complete somatic cell reprogramming in a culture, the method comprising:
(a) contacting a somatic cell induced to reprogram, or its progeny, with a plurality of detectably labeled binding moieties that bind Rex1, DNMT3B, and ABCG2; and
(b) detecting the binding of one or more moieties to the cell, or its progeny, to Rex1, DNMT3B, and ABCG2 expressed by a cell in the culture;
wherein binding indicates a fully-reprogrammed cell.
2. The method of claim 1 , further comprising detecting a stem cell marker selected from the group consisting of SSEA1, SSEA4, TRA160, CD9, Fbx15, Ecat1, Esg1, Eras, Gdf3, Fgf4, Cripto, Dax1, Zpf296, Slc2a3, Utf1, Oct4, SOX2, Nat1 and/or NANOG.
3. The method of claim 1 , further comprising detecting silencing of the expression of a viral reporter gene.
4. The method of claim 1 , wherein the somatic cell comprises a fibroblast.
5. The method of claim 4 , wherein expression of fibroblast marker CD13 is down-regulated.
6. The method of claim 1 , further comprising repeating steps (a)-(b) at a plurality of time points.
7. The method of claim 1 , wherein each of the detectably labeled moieties comprises a fluorescent moiety.
8. The method of claim 1 , wherein the reprogrammed cell is pluripotent.
9. The method of claim 1 , wherein the reprogrammed cell comprises a side population (SP) phenotype.
10. The method of claim 9 , wherein the SP phenotype comprises a HOECHST dim phenotype.
11. A method for identifying a fully reprogrammed induced pluripotent stem cell (iPS) cell from a culture of cells, the method comprising:
(a) contacting a somatic cell induced to reprogram, or its progeny, with a plurality of detectably labeled binding moieties that bind Rex1, DNMT3B, and ABCG2 expressed by a cell in the culture; and
(b) detecting the binding of one or more of the moieties to the cell, or its progeny, to Rex1, DNMT3B, and ABCG2 expressed by a cell in the culture;
wherein the presence of Rex1, DNMT3B, and ABCG2 indicates a fully reprogrammed iPS cell.
12. The method of claim 11 , further comprising detecting a stem cell marker selected from the group consisting of SSEA1, SSEA4, TRA160, CD9, Fbx15, Ecat1, Esg1, Eras, Gdf3, Fgf4, Cripto, Dax1, Zpf296, Slc2a3, Utf1, Oct4, SOX2, Nat1 and/or NANOG.
13. The method of claim 12 , wherein the stem cell markers further comprise TRA160.
14. A method for screening for an agent that promotes complete somatic cell reprogramming in a culture, the method comprising:
(a) contacting a somatic cell with a plurality of detectably labeled moieties that bind Rex1, DNMT3B, and ABCG2 expressed by a cell in the culture, in the presence and absence of a candidate reprogramming agent; and
(c) detecting the degree of cell labeling with one or more of the detectably labeled moieties of Rex1, DNMT3B, and/or ABCG2;
wherein if the level of expression of Rex1, DNMT3B, and ABCG2 in the presence of the agent is higher compared to the level of Rex1, DNMT3B, and ABCG2 in the absence of the agent, the agent enhances complete somatic cell reprogramming.
15. The method of claim 14 , further comprising detecting a stem cell marker selected from the group consisting of SSEA1, SSEA4, TRA160, CD9, Fbx15, Ecat1, Esg1, Eras, Gdf3, Fgf4, Cripto, Dax1, Zpf296, Slc2a3, Utf1, Oct4, SOX2, Nat1 and/or NANOG.
16. The method of claim 15 , wherein the stem cell markers comprise TRA160.