Methods for the production of IPS cells using non-viral approach
Methods and composition of induction of pluripotent stem cells and other desired cell types are disclosed. For example, in certain aspects methods for generating essentially vector-free induced pluripotent stem cells are described. Furthermore, the invention provides induced pluripotent stem cells and desired cell types essentially free of exogenous vector elements with the episomal expression vectors to express differentiation programming factors.
1. A method of producing human induced pluripotent stem (iPS) cells that are essentially free of differentiation programming vector genetic elements, the method comprising the steps of:
a) obtaining a starting population of human somatic cells;
b) obtaining one or more iPS Epstein-Barr virus (EBV)-based reprogramming vectors comprising a replication origin and one or more expression cassettes encoding iPS reprogramming factors comprising at least Sox-2, Oct-4, wherein one or more of said expression cassettes comprise a nucleotide sequence encoding a trans-acting factor that binds to the replication origin to replicate an extra-chromosomal template, and/or wherein cells of the starting population express such a trans-acting factor;
c) introducing the iPS reprogramming vector(s) into the human somatic cells;
d) culturing the human somatic cells that comprise the iPS EBV-based reprogramming vector(s) to effect expression of said reprogramming factors such that traits consistent with embryonic stem cells arise in at least a portion of cells in the cultured cells; and
e) further culturing cells having the embryonic stem cell traits for a sufficient number of generations in the absence of positive selection for the presence of the iPS reprogramming vector(s) to provide human iPS cells that are essentially free of iPS reprogramming vector genetic elements.
2. The method of claim 1 , wherein the starting cell is a fibroblast, a keratinocyte, a hematopoietic cell, a mesenchymal cell, a liver cell, a stomach cell, or a β cell.
3. The method of claim 1 , wherein the replication origin is a replication origin of a lymphotrophic herpes virus, an adenovirus, SV40, a bovine papilloma virus, or a yeast.
4. The method of claim 3 , the replication origin is a replication origin of a lymphotrophic herpes virus and corresponds to oriP of EBV.
5. The method of claim 4 , wherein the lymphotrophic herpes virus is Epstein Barr virus (EBV), Kaposi's sarcoma herpes virus (KSHV), Herpes virus saimiri (HS), or Marek's disease virus (MDV).
6. The method of claim 1 , wherein the trans-acting factor corresponds to EBNA-1 of EBV.
7. The method of claim 1 , wherein the trans-acting factor is a derivative of a wild-type protein corresponding to EBNA-1 of EBV, which derivative has a reduced ability to activate transcription from an integrated template as compared to wild-type EBNA-1.
8. The method of claim 1 , wherein the trans-acting factor is a derivative of a wild-type protein corresponding to EBNA-1 of EBV, which derivative activate transcription at levels at least 5% that of the corresponding wild-type protein from an extra-chromosomal template after the derivative binds the replication origin.
9. The method of claim 1 , wherein the trans-acting factor is a derivative of wild-type protein corresponding to EBNA-1 of EBV, which derivative has a deletion of residues corresponding to residues about 65 to about 89 of EBNA-1, and/or a deletion of residues corresponding to residues about 90 to about 328 of EBNA-1.
10. The method of claim 7 , wherein the derivative encodes a protein with at least 80% amino acid sequence identity to residues 1 to about 40 and residues about 328 to 641 of EBNA-1.
11. The method of claim 1 , further comprising a step of differentiating the target cell population.