METHOD OF EFFECTING DE-DIFFERENTIATION OF A CELL

• US 20110165570A1
• Filed: 10/21/2008
• Published: 07/07/2011
• Est. Priority Date: 05/06/2008
• Status: Active Grant

• Current Assignee: Agency For Science Technology and Research
• Original Assignee: Agency For Science Technology and Research
• Inventors: Lufkin, Thomas, Hui Ng, Huck, Jiang, Jianming, Feng, Bo, Kraus, Petra
• Granted Patent: US 9,102,920 B2
• Time in Patent Office: Days
• US Class Current: 435/6.11
• CPC Class Codes: C12N 2501/38 with nuclear receptors
• CPC Class Codes: C12N 2501/602 Sox-2
• CPC Class Codes: C12N 2501/603 Oct-3/4
• CPC Class Codes: C12N 2501/606 c-Myc
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 5/0696 Artificially induced pluripotent stem cells, e.g. iPS

Abstract

The invention provides a method of effecting de-differentiation of an at least partially differentiated cell or of maintaining pluripotency and/or self-renewing characteristics of an undifferentiated cell. The method comprises increasing the amount or the activity of an Err protein, or a functional fragment thereof, in the cell.

Claims

1. A method of effecting de-differentiation of an at least partially differentiated cell or of maintaining at least one of pluripotency and self-renewing characteristics of an undifferentiated cell, the method comprising increasing the amount or the activity of an estrogen-related receptor (Err) protein, or a functional fragment thereof, in the cell.

2. The method of claim 1, wherein the Err protein is one of an Esrrb protein, an Esrrd protein and an Esrrg protein.

3. The method of claim 1, further comprising increasing the amount or the activity of one of Oct4 and Sox2.

4. The method of claim 1, wherein the at least partially differentiated cell is one of a terminally differentiated somatic cell, a precursor cell, a lineage-restricted stem cell, a somatic stem cell and a progenitor cell.

5. The method of claim 1, wherein the undifferentiated cell is one of a stem cell, a germ cell, an oocyte, a blastomer, and an inner cell mass cell.

6. The method of claim 1, wherein increasing the amount of the Err protein in the cell comprises increasing the expression of the Err protein.

7. The method of claim 6, wherein the expression of the Err protein is increased by means of a nucleic acid molecule.

8. The method of claim 7, wherein the nucleic acid molecule is a heterologous nucleic acid molecule.

9. The method of claim 7, wherein the nucleic acid molecule is RNA or DNA.

10. The method of claim 7, wherein the nucleic acid molecule comprises a sequence encoding an Err protein or a functional fragment thereof.

11. The method of claim 1, wherein increasing the activity of the Err protein in the cell comprises modulating the interaction of the Err protein with a nucleic acid target sequence.

12. The method of claim 11, wherein the nucleic acid target sequence comprises an estrogen response element or a palindromic thyroid hormone response element.

13. The method of claim 1, wherein increasing the activity of the Err protein in the cell comprises forming a complex between one of (i) the Err protein and a protein, (ii) the Err protein and a compound, and (iii) the Err protein, a protein and a compound.

14. The method of claim 13, wherein the compound is selected from the group consisting of a peptide, a peptoid, an inorganic molecule and a low molecular weight organic molecule.

15. The method of claim 14, wherein the Err protein is an Esrrb protein and wherein the low molecular weight organic molecule is a 4-hydroxybenzoic acid aryl hydrazide, a flavone phytoestrogen and an isoflavone phytoestrogen.

16. The method of claim 13, wherein the protein is one of GRIP 1, SRC-1a, ACTR and a PPAR gamma coactivator.

17. The method of claim 16, wherein the PPAR gamma coactivator is one of PGC-1α

and PGC-β

.

18. The method of claim 1, wherein increasing the activity of the Err protein in the cell comprises allowing an alteration of a posttranslational modification of the Err protein to occur.

19. The method of claim 18, wherein the posttranslational modification is one of phosphorylation, sumoylation and a combination thereof.

20. The method of claim 1, further comprising assessing one of:

(i) the amount or the activity of the Err protein, or a functional fragment thereof, in the cell, and

(ii) the presence of a marker of the differentiation status in the cell.

21. The method of claim 20, wherein the marker of the differentiation status in the cell is:

(i) the expression of one of Nanog, Oct4, Sox2, Sall4, Tcll, Tbx3, Eras, Klf2, Klf4, Klf5, Baf250a, BC031441, Eno3, Etv5, Gm1739, Gtf2h3, Hes6, Jub, Mtf2, Myodl, Nmycl, Notch4, Nr5a2, Nrg2, Otx2, Rab2b, Rbpsuh, Rest, Stat3, Utfl, Tcfap2c and Zfp553, or

(ii) the methylation status of the promoter of one of Nanog, Oct4, Sox2, Sall4, Tell, Tbx3, Eras, Klf2, Klf4, Klf5, Baf250a, BC031441, Eno3, Etv5, Gm1739, Gtf2h3, Hes6, Jub, Mtf2, Myodl, Nmycl, Notch4, Nr5a2, Nrg2, Otx2, Rab2b, Rbpsuh, Rest, Stat3, Utfl, Tcfap2c and Zfp553.

22. The method of claim 20, wherein assessing the amount and/or activity of the Err protein comprises assessing the amount and/or activity over a period of time.

23. The method of claim 20, wherein assessing the amount of the Err protein in the cell is achieved by measuring gene expression of the Err protein in the cell.

24. The method of claim 23, further comprising comparing the result of the measurement of Err gene expression with that of a control measurement.

25. The method of claim 24, wherein the control measurement comprises the use of conditions that do at least essentially not modulate Err gene expression.

26. The method of claim 20, wherein assessing the presence of a marker of the differentiation status in the cell comprises measuring the expression and/or the activity of the marker over a period of time.

27. The method of claim 1, wherein the cell is obtained from or is comprised in a host organism.

28. The method of claim 27, wherein the host organism is one of a microorganism, a fish, an amphibian, a bird and a mammal.

29. The method of claim 28, wherein the cell is a mammalian somatic cell selected from the group consisting of a fibroblast, a myeloid cell, a B lymphocyte, a T lymphocyte, a dendritic cell, a keratinocyte, an adipose cell, a mesenchymal cell, an epithelial cell, an epidermal cell, a chondrocyte, a hepatocyte, a cumulus cell, a neural cell, a glial cell, an astrocyte, a cardiac cell, an esophageal cell, a muscle cell, a pancreatic beta cell, a melanocyte, a hematopoietic cell, a macrophage, a monocyte, and a mononuclear cell.

30. The method of claim 1, wherein the cell is cultured.

31. The method of claims claim 27, comprising administering a compound that modulates at least one of the expression and the activity of an Err protein.

32. A dedifferentiated cell obtained by the method of claim 1.

33. A population of dedifferentiated cells of claim 32.

34. A method of identifying a candidate compound capable of effecting de-differentiation of an at least partially differentiated cell or of maintaining pluripotency and/or self-renewing characteristics of an undifferentiated cell, the method comprising introducing the compound into a cell capable of expressing an Err protein, or a functional fragment thereof, and determining the expression of the Err protein, wherein an increased expression of the Err protein is an indication that the compound is capable of effecting de-differentiation of an at least partially differentiated cell or of maintaining at least one of pluripotency and self-renewing characteristics of an undifferentiated cell.

35. The method of claim 34, wherein the Err protein is an Esrrb protein or an Esrrg protein.

36. An in-vitro method of identifying a compound capable of effecting de-differentiation of an at least partially differentiated cell or of maintaining pluripotency and/or self-renewing characteristics of an undifferentiated cell, the method comprising contacting the compound, an Err protein, or a functional fragment thereof, and at least one of Nanog and Oct4, wherein the facilitation of the formation of a complex between the Err protein and at least one of Nanog and Oct4 indicates that the compound is capable of effecting de-differentiation of an at least partially differentiated cell or of maintaining at least one of pluripotency and self-renewing characteristics of an undifferentiated cell.

37. The method of claim 36, comprising:

(a) adding to a test tube a compound that is suspected to be capable of modulating the complex formation of an Err protein, or a functional fragment thereof, and at least one of Nanog and Oct4,

(b) adding the Err protein, or a functional fragment thereof, to the test tube,

(c) adding at least one of Nanog and Oct4 to the test tube, and

(d) detecting the said complex formation.

38. The method of claim 37, wherein the detection is performed by a suitable spectroscopic, photochemical, photometric, fluorometric, radiological, enzymatic or thermodynamic method.

39. The method of claim 36, wherein the Err protein is one of an Esrrb protein, an Esrrd protein and an Esrrg protein.

40. The use of a nucleic acid molecule and/or a compound that increases the absolute quantity of an Err protein in a cell, in the manufacture of an agent for effecting de-differentiation of an at least partially differentiated cell or maintaining pluripotency and/or self-renewing characteristics of an undifferentiated cell.

41. The method of claim 40, wherein the Err protein is one of an Esrrb protein, an Esrrd protein and an Esrrg protein.