METHODS FOR CELL REPROGRAMMING AND GENOME ENGINEERING
Methods for producing engineered induced pluripotent stem (iPS) cells are provided comprising introducing a first nucleic acid into somatic cells for integration into their genome and reprogramming the cells to produce engineered iPS cells having the nucleic acid integrated into their genome. For example, in certain aspects the cells are reprogrammed by introduction of a genetic element that expresses one or more reprogramming factor and culturing of the cells under conditions sufficient to produce reprogrammed cells.
1 . A method for producing a population of human induced pluripotent stem (iPS) cells, comprising:
a) obtaining human somatic cells;
b) introducing into said human somatic cells a first vector comprising a first nucleic acid molecule for integration into the genome of the cells and at least a second vector comprising a second nucleic acid molecule comprising a genetic element that expresses one or more reprogramming factors sufficient when expressed in the somatic cell to convert the somatic cell to a pluripotent stem cell, wherein the second nucleic acid molecule is an episomal vector;
c) culturing said cells under reprogramming conditions; and
d) obtaining a population of iPS cells comprising said first nucleic acid integrated in their genome, wherein said first nucleic acid is expressible and wherein the second nucleic acid molecule is not present in the iPS cells.
2 . The method of claim 1 , further comprising:
d) obtaining a population of iPS cells comprising cells that (i) comprise said first nucleic acid integrated in their genome and cells which (ii) do not comprise the first nucleic acid integrated in their genome.
3 . The method of claim 1 , further comprising:
d) obtaining a first population of iPS cells which comprise said first nucleic acid integrated in their genome and a second population of iPS cells which do not comprise the first nucleic acid integrated in their genome.
4 . The method of claim 1 , further comprising:
d) screening or selecting iPS cells that comprise an expressible first nucleic acid integrated in their genome thereby obtaining the population of iPS cells wherein said first nucleic acid is expressible.
5 . The method of claim 1 , wherein the first nucleic acid is expressible upon differentiation of the iPS cells.
6 . The method of claim 1 , further defined as forming a composition comprising the somatic cells, the first nucleic acid molecule and the second nucleic acid molecule and culturing said composition.
7 . The method of claim 1 , wherein the first or second nucleic acid molecule is randomly integrated into the genome of the cells.
8 - 9 . (canceled)
10 . The method of claim 1 , wherein the first or second nucleic acid molecule is integrated into a selected genomic site of the cells.
11 - 13 . (canceled)
14 . The method of claim 1 , wherein the first nucleic acid molecule comprises a coding sequence of a screenable or selectable marker.
15 . The method of claim 1 , wherein the first nucleic acid molecule comprises a nucleic acid sequence selected from the group consisting of a sequence that corrects a genetic defect in the cells; a sequence that provides resistance to a pathogen infection; a sequence that provides resistance to a drug; a sequence that provides sensitivity to a drug; a sequence that alters immunogencity of the cells; and a sequence that provides a genetic tag in the cells.
16 . The method of claim 1 , wherein the somatic cell is a human fibroblast, keratinocyte, hematopoietic cell, mesenchymal cell, adipose cell, endothelial cell, epithelial cell, neural cell, muscle cell, mammary cell, liver cell, kidney cell, skin cell, digestive tract cell, cumulus cell, gland cell, or pancreatic islet cell.
17 - 19 . (canceled)
20 . The method of claim 1 , wherein the episomal vector comprises a replication origin and one or more expression cassettes for expression of reprogramming factors, wherein one or more of said expression cassettes further comprise a nucleotide sequence encoding a trans-acting factor that binds to the replication origin to replicate an extra-chromosomal template, and/or wherein the somatic cell expresses such a trans-acting factor.
21 . The method of claim 1 , wherein the reprogramming factor comprises one or more selected from the group consisting of Sox, Oct, Nanog, Lin-28, Klf4, C-myc, L-myc, a myc mutant or homolog that is deficient in transformation, and SV40LT.
22 . The method of claim 1 , wherein step (c) culturing said cells under reprogramming conditions comprises culturing the cells essentially free of feeder cells.
23 . The method of claim 1 , wherein step (c) culturing said cells under reprogramming conditions comprises culturing the cells in the presence of a matrix component.
24 . The method of claim 1 , wherein step (c) further comprises selecting or screening said cells for the presence of the first nucleic acid molecule.
25 - 28 . (canceled)
29 . The method of claim 1 , wherein step (c) culturing said cells under reprogramming conditions comprises culturing the cells for at least from about one day to fifteen days under reprogramming conditions.
30 - 34 . (canceled)
35 . The method of claim 1 , further comprising:
(e) culturing the iPS cells under expansion conditions.
36 - 40 . (canceled)
41 . A method for producing a population of induced pluripotent stem (iPS) cells and a population of engineered iPS cells, comprising:
a) obtaining somatic cells;
b) introducing into said cells a first nucleic acid molecule for integration into the genome of the cells and at least a second nucleic acid molecule comprising a genetic element that expresses one or more reprogramming factors sufficient when expressed in the somatic cell to convert the somatic cell to a pluripotent stem cell;
c) culturing said cells under reprogramming conditions; and
d) obtaining a first population of iPS cells which comprise said first nucleic acid integrated in their genome and a second population of iPS cells which do not comprise the first nucleic acid integrated in their genome.