Reprogramming immortalized B-cells to induced pluripotent stem cells
Methods and composition for providing induced pluripotent stem (iPS) cells are provided. For example, in certain aspects methods including reprogramming B lymphocytes transformed by episomal vectors such as Epstein-Barr virus-based vectors are described. Furthermore, the invention provides induced pluripotent stem cells essentially free of exogenous elements and having B cell immunoglobin variable region rearrangement.
1. A method for producing human induced pluripotent stem cells (iPS cells) from immortalized B cells, wherein the method comprises:
a) obtaining immortalized human B cells that have been immortalized by introducing one or more immortalizing Epstein-Barr virus (EBV) elements comprising EBNA into B cells;
b) reprogramming the immortalized B cells by introducing an exogenous reprogramming episomal vector or vectors encoding exogenous reprogramming factors comprising at least Oct-4 and Sox-2; and
c) culturing said cells under defined conditions in the absence of a feeder layer to effect expression of the exogenous reprogramming factors therein for sufficient time to produce iPS cells; and
d) removing the EBV and episomal vector elements from the iPS cells to provide human iPS cells that are essentially free of exogenous EBV and episomal vector elements.
2. The method of claim 1 , further comprising isolating or enriching iPS cells that are essentially free of the EBV elements.
3. The method of claim 1 , wherein the B cells prior to immortalization have been obtained from a blood sample.
4. The method of claim 3 , wherein the blood sample has a volume of from about 0.01 mL to about 5 mL.
5. The method of claim 4 , wherein the blood sample has a volume of from about 0.1 mL to about 0.5 mL.
6. The method of claim 1 , wherein the immortalized B cells are obtained by immortalizing B cells using Epstein-Barr virus (EBV) elements.
7. The method of claim 1 , wherein expression of the immortalizing EBV elements or reprogramming factors is under the control of an inducible.
8. The method of claim 1 , wherein the immortalized B cells are obtained from an established lymphoblastoid cell line.
9. The method of claim 1 , wherein reprogramming further comprises contacting the immortalized B cells with one or more signaling inhibitors including a glycogen synthase kinase 3 (GSK-3) inhibitor, a mitogen-activated protein kinase kinase (MEK) inhibitor, a transforming growth factor beta (TGF-β) receptor inhibitor, a p53 inhibitor, an NF-kappa B inhibitor, or a combination thereof.
10. The method of claim 1 , wherein reprogramming further comprises contacting the immortalized B cells with fibroblast growth factor (FGF) or leukemia inhibitory factor (LIF).
11. The method of claim 1 , wherein reprogramming does not include the introduction of an inhibitory nucleotide specific for Pax-5 into the immortalized B cells.
12. The method of claim 1 , wherein reprogramming does not include expression of exogenous C/EBPα in the immortalized B cells.