Generation of induced pluripotent stem cells from normal human mammary epithelial cells

• US 10,738,323 B2
• Filed: 07/11/2014
• Issued: 08/11/2020
• 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.
• Primary Examiner(s): Marvich, Maria
• Application Number:
  US14/904,641
• Publication Number:
  US 20160145642A1
• Time in Patent Office: 2,223 Days
• Field of Search: None
• 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), the method comprising:

providing human mammary cells in culture;

transfecting the human mammary cells with vectors encoding Oct4, Sox2, Nanog, Kruppel-like Factor 4 (KLF4), L-Myc, Lin28, SV40 Large T Antigen (SV40LT) and p53 shRNA;

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

culturing the plated cells in an induction media, wherein the transfecting, the plating and the culturing generates colonies of the human m-iPSCs, wherein the induction media comprises HA-100, CHIR99021, PD0325901, and A83-01, wherein the plating of the transfected cells on the culture vessel coated with the substrate further comprises culturing in norm oxygen conditions, and wherein the plated mammary cells express the transfected Oct4, Sox2, Nanog, KLF4, L-Myc, Lin28, SV40LT and p53 shRNA.

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

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

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

5. The method of claim 1, wherein the vectors are episomal vectors.

6. The method of claim 5, wherein the episomal vectors are oriP/EBNA1 vectors.

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

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

9. The method of claim 1, wherein the induction media comprises HA-100, CHIR99021, PD0325901, A83-01 and Y-27632.

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

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

providing human mammary cells in culture;

expanding the human mammary cells to about 90% confluence;

transfecting the human mammary cells with vectors encoding Oct4, Sox2, Nanog, Kruppel-like Factor 4 (KLF4), L-Myc, Lin28, SV40 Large T Antigen (SV40LT) and p53 shRNA;

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

culturing the plated cells in an induction media, wherein the expanding, the transfecting, the plating and the culturing reprograms the human mammary cells to a less differentiated state, wherein the induction media comprises HA-100, CHIR99021, PD0325901, and A83-01, wherein the plating of the transfected cells on the culture vessel coated with the substrate further comprises culturing in norm oxygen conditions, and wherein the plated mammary cells express the transfected Oct4, Sox2, Nanog, KLF4, L-Myc, Lin28, SV40LT and p53 shRNA.

12. The method of claim 11, wherein the mammary cells are primary cells.

13. The method of claim 11, wherein the mammary cells are obtained from a tumor.

14. The method of claim 11, wherein the mammary cells are obtained from a cell line.

15. The method of claim 11, wherein the induction media comprises HA-100, PD0325901, SB431542, CHIR99021, A83-01 and Y-27632.