Protein-induced pluripotent cell technology and uses thereof

• US 10,640,751 B2
• Filed: 02/08/2019
• Issued: 05/05/2020
• 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:
  US16/271,150
• Publication Number:
  US 20190161739A1
• Time in Patent Office: 452 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 piPS cells formed using this method and uses thereof.

Claims

1. A method of generating a homogenous population of mammalian protein-induced pluripotent stem cells (piPSC), comprising:

culturing mammalian somatic cells in the presence of a combination of QQ-reagent modified reprogramming proteins selected from;

     1) QQ-reagent modified Sox2, QQ-reagent modified Oct4, and QQ-reagent modified Nanog, and

     2) QQ-reagent modified Sox2, QQ-reagent modified Oct4, QQ-reagent modified Klf4 and QQ-reagent modified c-Myc, for at least a first or more reprogramming cycles, wherein each reprogramming cycle comprises culturing mammaliam cells with the combination of QQ-reagent modified reprogramming proteins for a period of about 3 hours to 5 days, wherein the QQ reagent comprises polyethylenimine (PEI) and DOTAP/DOPE, wherein the reprogramming proteins are denatured, wherein each QQ-reagent modified reprogramming protein is present in a concentration of greater than 2 nanograms/milliliter (ng/ml) and no greater than 500 ng/ml, wherein the cultured mammalian cells are passaged in a whole dish; and

passaging the cultured mammalian cells in a whole dish, with the proviso that colony picking is not performed, to generate a homogenous population of mammalian protein induced pluripotent stem cells.

2. The method of claim 1, further comprising a second reprogramming cycle comprising culturing the mammalian somatic cells in the presence of the combination of QQ-reagent modified reprogramming proteins, wherein each QQ-reagent modified reprogramming protein is present in a concentration of greater than 2 nanograms/milliliter (ng/ml) and no greater than 500 ng/ml.

3. The method of claim 2, wherein each QQ-reagent modified reprogramming protein is present in a reduced concentration during the second reprogramming cycle compared to the first reprogramming cycle.

4. The method of claim 2, further comprising a third reprogramming cycle comprising culturing the mammalian somatic cells in the presence of the combination of QQ-reagent modified reprogramming proteins, wherein each QQ-reagent modified reprogramming protein is present in a concentration of greater than 2 nanograms/milliliter (ng/ml) and no greater than 500 ng/ml.

5. The method of claim 4, wherein each QQ-reagent modified reprogramming protein is present in a reduced concentration during the third reprogramming cycle compared to the second reprogramming cycle.

6. The method of claim 1, wherein the mammalian somatic cells are selected from the group consisting of:

human primary cells, human cancer cells, human disease cells, non-human mammalian primary cells, non-human mammalian cancer cells, and non-human mammalian disease cells.

7. The method of claim 1, wherein the mammalian cells are converted to piPSC with a conversion efficiency of greater than 80%.