Protein-induced pluripotent cell technology and uses thereof

• US 9,816,076 B2
• Filed: 05/02/2012
• Issued: 11/14/2017
• Est. Priority Date: 05/02/2011
• Status: Active Grant

• Current Assignee: Wayne State University
• Original Assignee: Wayne State University
• Inventors: Wang, Jianjun, Li, Qianqian
• Primary Examiner(s): Ton, Thaian N
• Application Number:
  US14/113,522
• Publication Number:
  US 20140242695A1
• Time in Patent Office: 2,022 Days
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/602 Sox-2
• CPC Class Codes: C12N 2501/603 Oct-3/4
• CPC Class Codes: C12N 2501/604 Klf-4
• CPC Class Codes: C12N 2501/605 Nanog
• CPC Class Codes: C12N 2501/606 c-Myc
• CPC Class Codes: C12N 2506/1307 from adult fibroblasts
• CPC Class Codes: C12N 5/0696 Artificially induced pluripotent stem cells, e.g. iPS

Abstract

A method of generating protein-induced pluripotent stem cells by delivering bacterially expressed reprogramming proteins into nuclei of starting somatic cells using the QQ-protein transduction technique, repeating several cell reprogramming cycles for creating reprogrammed protein-induced pluripotent stem cells, moving the reprogrammed cells into a feeder-free medium for expansion, and expanding and passaging the reprogrammed cells in a whole dish for generating homogeneous piPS cells. Also provided are the piPCS cells formed using this method and uses thereof.

Claims

1. A method of generating human protein-induced pluripotent stem cells (piPSC), comprising:

i) producing reprogramming proteins by bacterial expression, wherein the reprogramming proteins comprise the combination of Klf-4, Oct-4, Sox-2, c-myc (KOSM);

ii) modifying the bacterially expressed reprogramming proteins produced from step i) by mixing the proteins with a QQ-reagent comprising polyethylenimine (PEI) and DOTAP/DOPE;

iii) delivering the modified reprogramming proteins produced from step ii) into nuclei of starting human somatic cells, using a QQ-protein transduction technique;

iv) repeating several cell reprogramming cycles sufficient to create human reprogrammed piPSC;

v) moving the human reprogrammed piPSC from step iv into a feeder-free medium for expansion; and

vi) expanding and passaging the human reprogrammed piPSC from step v) as a whole dish of cells, with the proviso that colony picking is not performed, thereby generating homogeneous human piPSC.

2. The method according to claim 1, further including the step of in vitro labeling the reprogramming proteins with a small molecule fluorescence probe prior to said modifying step (ii).

3. The method according to claim 2, further including monitoring the efficiency of QQ-protein delivery into the nuclei of the starting human somatic cells.

4. The method according to claim 1, further comprising differentiating the piPSC into a cell type selected from the group consisting of:

specific adult stem cells, specific progenitor cells and a different somatic cell type compared to the starting human somatic cells.