PROTEIN-INDUCED PLURIPOTENT CELL TECHNOLOGY AND USES THEREOF

• US 20190161739A1
• Filed: 02/08/2019
• Published: 05/30/2019
• Est. Priority Date: 05/02/2011
• Status: Active Grant

• Current Assignee: Wayne State University
• Original Assignee: Wayne State University
• Inventors: Wang, Jianjun, Li, Qianqian
• Granted Patent: US 10,640,751 B2
• Time in Patent Office: 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 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.

2. The method according to claim 1, further including producing reprogramming proteins using a bacterial expression system.

3. The method according to claim 2, further including modifying the reprogramming proteins using the QQ reagents.

4. The method according to claim 3, further including the step of in vitro labeling the reprogramming proteins with small molecule fluorescence probes prior to said modifying step.

5. The method according to claim 4, further including monitoring the efficiency of QQ-protein delivery into the nuclei of the starting somatic cells.

6. The method according to claim 1, providing a general protein-induced cell reprogramming method for generating piPS cells, specific adult stem cells, specific progenitor cells and a different somatic cell type via trans-differentiation from healthy and diseased animal and human somatic cells. Different protein cocktails have to be used when generate specific adult stem cells, specific progenitor cells and a different somatic cell types.

7. The piPSC method according to claim 1, for use in enabling direct use of the bacterially expressed cell reprogramming proteins for cell reprogramming of animal and human somatic cells to generate piPS cells.

8. The piPSC method according to claim 1, wherein said reprogramming step includes using different reprogramming protein cocktails of Yamanaka factors or the SON factors selected from the group consisting essentially of four reprogramming proteins (Oct4/Sox2/Klf4/c-Myc), three reprogramming proteins (Oct4/Sox2/Klf4 or Sox2/0ct4/Nanog), two reprogramming proteins (Oct4/Sox2 or Oct4/Nanog), and only one reprogramming proteins (Oct4 or Nanog).

9. The piPCS cells formed using the method according to claim 1.

10. The piPCS cells formed using the method according to claim 1, wherein said piPS cells can be efficiently differentiated into three major germ layer cells in vitro and in vivo for teratoma formation.

11. The piPSC cells according to claim 9, wherein said cells were derived from cells selected from the group consisting essentially of cells from different healthy animal and human somatic cells including, but not limited to, mouse primary fibroblasts, adult mouse fibroblasts, human newborn fibroblasts, human primary adult fibroblasts, human adult keratinocytes, and human amniotic fluid.

12. The piPSC cells according to claim 9, wherein said cells were derived from cells selected from the group consisting essentially of diseased animal and human somatic cells including, but not limited to:

mouse primary fibroblast from ALS mice, rat tumor cells, such as 9L-glioma cells, mouse metastatic breast cancer cells, such as 4T1-cells, human breast cancer cell lines, such as MDA-MB-231, human brain tumor cell lines, such as U87 and U251-glioma cells, human primary Stage 4 GBM cells, human primary fibroblasts from Alzheimer patients with apoE3 and apoE4 genotypes.

13. The piPSC cells according to claim 9, for use in human clinic applications selected from the group consisting essentially of, but not limited to, regeneration medicine and cell replacement therapy, generation of the iPS cell banks from individual patients with genetic disorders, disease models based on piPS cells from individual patients and testing of efficacy and toxicity of drugs, including, but not limited to, small molecule drugs, protein drugs, DNA drugs, RNA drugs, carbohydrate drugs, and lipid-based drugs.

14. The piPSC cells according to claim 9, for use in treating diseases selected from the group consisting essentially of, but not limited to, cancer, heart diseases, strokes, diabetes, obesity, Alzheimer'"'"'s diseases, Parkinson'"'"'s disease, amyotrophic lateral-sclerosis, myocardial infaction, muscular dystrophy, CMT-1A, spinal cord injury, traumatic brain injury, learning defects, missing teeth, wound healing, bone marrow translantation, osteoarthritis, rheumatoid arthritis, baldness, blindness, deafness, Crohn'"'"'s disease and genetic diseases, and other similar diseases.