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