Methods for reprogramming somatic cells

• US 10,457,917 B2
• Filed: 07/09/2018
• Issued: 10/29/2019
• Est. Priority Date: 11/26/2003
• Status: Active Grant

• Current Assignee: Whitehead Institute for Biomedical Research
• Original Assignee: Whitehead Institute for Biomedical Research
• Inventors: Jaenisch, Rudolf, Hochedlinger, Konrad
• Primary Examiner(s): Noble, Marcia S
• Application Number:
  US16/030,815
• Publication Number:
  US 20190078057A1
• Time in Patent Office: 477 Days
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 67/0273 Cloned vertebrates
• CPC Class Codes: A01K 67/0275 Genetically modified vertebrates, e.g. transgenic
• CPC Class Codes: C07K 14/4702 Regulators; Modulating activity
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12N 15/8775 Murine embryos
• CPC Class Codes: C12N 2501/602 Sox-2
• CPC Class Codes: C12N 2501/603 Oct-3/4
• CPC Class Codes: C12N 2501/605 Nanog
• CPC Class Codes: C12N 2506/1307 from adult fibroblasts
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 2830/003 tet inducible
• CPC Class Codes: C12N 2830/006 tet repressible
• CPC Class Codes: C12N 5/0696 Artificially induced pluripotent stem cells, e.g. iPS

Abstract

The invention provides methods for reprogramming somatic cells to generate multipotent or pluripotent cells. Such methods are useful for a variety of purposes, including treating or preventing a medical condition in an individual. The invention further provides methods for identifying an agent that reprograms somatic cells to a less differentiated state.

Claims

1. A method of making a somatic cell more susceptible to reprogramming to a less differentiated state, comprising:

introducing an exogenous nucleic acid encoding an Oct 4 protein operably linked to at least one regulatory sequence into the somatic cell, thereby increasing expression of Oct4 protein in the somatic cell, wherein increased expression of Oct4 protein makes the cell more susceptible to reprogramming; and

wherein the exogenous nucleic acid is transiently transfected into the somatic cell.

2. The method of claim 1, wherein the somatic cell does not comprise a selectable marker.

3. The method of claim 1, wherein the somatic cell is a human cell or a mouse cell.

4. The method of claim 1, wherein the somatic cell is an adult stem cell.

5. The method of claim 4, wherein the adult stem cell is a hematopoietic stem cell, neural stem cell, or mesenchymal stem cell.

6. The method of claim 1, wherein the less differentiated state comprises pluripotent state.

7. The method of claim 1, further comprises:

contacting the somatic cell with a candidate agent of interest with respect to its potential to reprogram a somatic cell.

8. The method of claim 7, wherein the agent is Sox-2 or Nanog.

9. The method of claim 1, wherein the somatic cell does not comprise a selectable marker integrated into an endogenous locus of a pluripotency gene.

10. A method of making a somatic cell more susceptible to reprogramming to a cell having a less differentiated state, comprising:

obtaining a somatic cell that comprises an exogenously introduced polynucleic acid encoding Oct4 protein, and an exogenously introduced polynucleic acid encoding Sox2 or Nanog protein;

wherein the exogenously introduced polynucleic acids result in making the somatic cell more susceptible to reprogramming to a less differentiated state; and

wherein the exogenous polynucleic acids are transiently transfected into the cell.

11. The method of claim 10, the step of obtaining further comprises:

introducing an exogenous polynucleic acid encoding Oct4 protein, and an exogenously introduced polynucleic acid encoding Sox2 or Nanog protein into the somatic cell.

12. The method of claim 10 or 11, further comprising:

contacting the somatic cell with a candidate agent of interest with respect to its potential to reprogram a somatic cell.

13. The method of claim 10, wherein the exogenous polynucleic acids increase Oct4 expression in the somatic cell.

14. The method of claim 10, wherein the somatic cell is a mammalian cell.

15. The method of claim 10, wherein the somatic cell is a human cell or a mouse cell.

16. The method of claim 10, wherein the somatic cell is an adult stem cell.

17. The method of claim 16, wherein the adult stem cell is selected from the group consisting of:

a hematopoietic stem cell, a neural stem cell, and a mesenchymal stem cell.

18. The method of claim 10, wherein the less differentiated state comprises pluripotent state.