GENERATION OF INDUCED PLURIPOTENT STEM CELLS FROM NORMAL HUMAN MAMMARY EPITHELIAL CELLS

• US 20160145642A1
• Filed: 07/11/2014
• Published: 05/26/2016
• Est. Priority Date: 07/12/2013
• Status: Active Grant

• Current Assignee: Cedars-Sinai Medical Center (Cedars Sinai Health System)
• Original Assignee: Cedars-Sinai Medical Center (Cedars Sinai Health System)
• Inventors: Cui, Xiaojiang, Dhruv, Sareen, Ornelas, Loren A.
• Granted Patent: US 10,738,323 B2
• Time in Patent Office: Days
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/235 Leukemia inhibitory factor [LIF]
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• 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 2501/608 Lin28
• CPC Class Codes: C12N 2501/727 Kinases (EC 2.7.)
• CPC Class Codes: C12N 2506/09 from epidermal cells, from skin cells, from oral mucosa cells
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 5/0696 Artificially induced pluripotent stem cells, e.g. iPS

Abstract

Described herein are reprogramming techniques allowing for production of mammary-derived iPSCs (“m-iPSCs”). The m-iPSCs described herein exhibit all the hallmarks of stem cell identity including round cluster, bright colony morphology, clonal expansion, and pluripotent marker expression (alkaline phosphatase expression, Oct-4, nanog, etc.) Further refined techniques allow for generation of m-iPSCs under essentially defined conditions.

Claims

1. A method of generating human mammary-derived induced pluripotent stem cells (m-iPSCs), comprising:

providing a quantity of human mammary cells;

transfecting the human mammary cells with at least one vector encoding at least one reprogramming factor;

plating the cells on a culture vessel coated with a substrate; and

culturing the cells in an induction media, wherein the transfection, plating and culturing generates colonies of human mammary-derived induced pluripotent stem cells (m-iPSCs).

2. The method of claim 1, wherein the mammary cells are from primary cells.

3. The method of claim 1, wherein the mammary cells are from a tumor.

4. The method of claim 1, wherein the mammary cells are from a cell line.

5. The method of claim 1, wherein the at least one vector encodes at least one reprogramming factor selected from the group consisting of:

Oct4, Sox2, Nanog, Kruppel-like Factor 4 (KLF4), L-Myc, Lin28, SV40 Large T Antigen (SV40LT) and p53 shRNA.

6. The method of claim 1, wherein the at least one vector comprises an episomal vector.

7. The method of claim 6, wherein the episomal vector comprises an oriP/EBNA1 vector.

8. The method of claim 1, wherein transfecting mammary cells comprises nucleofection or lipofection.

9. The method of claim 1, wherein plating the cells on a culture vessel coated with a substrate further comprises culturing in norm oxygen conditions.

10. The method of claim 1, wherein the substrate comprises Matrigel.

11. The method of claim 1, wherein the induction media comprises one or more of the following compounds:

HA-100, CHIR99021, PD0325901, and A83-01.

12. The method of claim 1, wherein culturing the cells in an induction media is for a period of 10-31 days.

13. A composition of m-iPSCs produced by the method of claim 1.

14. The composition of claim 13, wherein the m-iPSCs are capable of serial passaging as a cell line.

15. A multi-potent cell produced by culturing the composition of claim 14 in the presence of a differentiation agent.

16. A method of reprogramming a human mammary cell, comprising:

providing a quantity of human mammary cells;

culturing the human mammary cells in the presence of at least one vector and/or at least one reprogramming agent;

plating the cells on a culture vessel coated with a substrate; and

culturing the cells in an induction media, wherein the transfection, plating and culturing reprograms the human mammary cells to a less differentiated state.

17. The method of claim 16, wherein the mammary cells are from primary cells.

18. The method of claim 16, wherein the mammary cells are from a tumor.

19. The method of claim 16, wherein the mammary cells are from a cell line.

20. The method of claim 16, wherein the at least one vector encodes at least one reprogramming factor comprising Oct4, Sox2, Nanog, Kruppel-like Factor 4 (KLF4), L-Myc, Lin28, SV40 Large T Antigen (SV40LT) and p53 shRNA.

21. The method of claim 16, wherein the at least one vector encodes at least one reprogramming factor comprising a microRNA.

22. The method of claim 16, wherein the at least one reprogramming agent is a small molecule comprising HA-100, PD0325901, SB431542, CHIR99021, A83-01 and/or Y-27632.