Method of making induced pluripotent stem cell from adipose stem cells using minicircle DNA vectors
Human somatic cells are reprogrammed to become induced pluripotent stem cells (iPS cells) by the introduction of a minicircle DNA vector. Cells of interest include adipose stem cells.
1. A method of producing human iPS cells, comprising:
contacting a population of human adipose stem cells with one or more minicircle DNA vectors encoding a plurality of reprogramming factors operably linked to a promoter; wherein the plurality of reprogramming factors comprises Oct4, Sox2, Lin28, and Nanog, or Oct4, Sox2, c-Myc, and Klf4; and
maintaining the cells for a period of time sufficient to reprogram said human adipose stem cells to pluripotency in the absence of genomic integration of sequences of the one or more minicircle DNA vectors,
wherein each of the one or more minicircle DNA vectors is 0.3-10 Kb in length, lacks an origin of replication, and lacks a drug selectable marker.
2. The method of claim 1 , wherein a single minicircle DNA vector comprises the plurality of reprogramming factors sufficient to induce pluripotency of said somatic cells.
3. The method of claim 2 , wherein the coding sequences of said reprogramming factors are all operably linked to the same single promoter.
4. The method of claim 3 , wherein said coding sequences are separated sequences encoding self-cleaving peptides.
5. The method of claim 1 , wherein said adipose stem cells are reprogrammed to pluripotency in feeder-layer free culture.
6. A method of producing human iPS cells, comprising:
(a) contacting a population of at least 1×10 5 human adipose stem cells with a minicircle DNA vector encoding a plurality of reprogramming factors, wherein the plurality of reprogramming factors comprises Oct4, Sox2, Lin28, and Nanog; or Oct4, Sox2, c-Myc, and Klf4, wherein (i) the coding sequences of the reprogramming factors are all operably linked to the same single promoter, and are separated from each other by a sequence encoding a self-cleaving 2A peptide sequence, and (ii) the minicircle DNA vector is 0.3-10 Kb in length, lacks an origin of replication, and lacks a drug selectable marker; and
(b) maintaining the cells in feeder-layer free culture on a matrigel coated surface for a period of time sufficient to reprogram said adipose stem cells to pluripotency in the absence of genomic integration of sequences of the minicircle DNA vector.