Markers for differentiation of stem cells into differentiated cell populations

• US 9,828,634 B2
• Filed: 01/22/2016
• Issued: 11/28/2017
• Est. Priority Date: 01/22/2015
• Status: Active Grant

• Current Assignee: Regenerative Medical Solutions, Inc.
• Original Assignee: Regenerative Medical Solutions, Inc.
• Inventors: Xu, Xiaofang, Odorico, Jon, Forsberg, Erik, Mael, Amber A., Tagliavia, Guy
• Primary Examiner(s): Ton, Thaian N
• Assistant Examiner(s): Montanari, David A
• Application Number:
  US15/004,188
• Publication Number:
  US 20160230225A1
• Time in Patent Office: 676 Days
• Field of Search: 435325
• CPC Class Codes: A61K 35/39 Pancreas; Islets of Langerhans Langerhans cells of epidermis A61K35/36
• CPC Class Codes: A61K 35/545 Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
• CPC Class Codes: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/15 Transforming growth factor beta (TGF-β)
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2501/16 Activin; Inhibin; Mullerian inhibiting substance
• 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 2501/606 c-Myc
• CPC Class Codes: C12N 2506/22 from pancreatic cells
• CPC Class Codes: C12N 2506/45 from artificially induced pluripotent stem cells
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 5/0676 Pancreatic cells
• CPC Class Codes: C12N 5/0696 Artificially induced pluripotent stem cells, e.g. iPS
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2600/158 Expression markers

Abstract

Provided herein are systems and methods for identifying cell-specific differentiation markers. In particular, provided herein are systems and methods for generating induced pluripotent cells (IPS) from human cells, differentiating the IPS into differentiated cells, and identifying differentiation specific markers.

Claims

1. A method of identifying differentiation markers expressed in pancreatic β

-cell lineage cells, comprising;

(a) culturing an isolated primary cell under conditions that comprise exogenously expressing Oct4, Sox2, cMyc, and Klf4 in said primary cell, such that said primary cell de-differentiates to generate an induced pluripotent stem cell (iPSC);

(b) treating said iPSC under conditions such that said iPSC differentiates into a pancreatic β

-cell, wherein said treating comprises;

(i) culturing the iPSC in a chemically defined medium comprising fibroblast growth factor, Activin A and bone morphogenetic protein,

(ii) culturing the cells produced from step (i) in the presence of chemically defined medium comprising insulin, transferrin, selenium, a fibroblast growth factor and nicotinamide,

(iii) culturing the cells produced from step (ii) in the presence of a chemically defined medium comprising insulin, transferrin, selenium, retinoic acid, a bone morphogenetic protein inhibitor and nicotinamide;

and (iv) culturing the cells produced from step (iii) in the presence of a serum-free medium comprising an insulin-like growth factor, insulin, nicotinamide, exendin-4 and/or GLP-1, a TGF-β

inhibitor, and an agent that increases cAMP; and

(c) identifying molecular markers expressed during differentiation, wherein said markers are indicative of differentiation of said iPS cell into a pancreatic β

-cell.

2. The method of claim 1, wherein said primary cell is a pancreatic beta cell.

3. The method of claim 1, wherein said culturing in step (a) further comprises expanding said cell.

4. The method of claim 1, wherein said culturing in step (a) further comprises inducing expression of one or more genes selected from the group consisting of nanog, LIN28, and Glis1.

5. The method of claim 1, further comprising:

(d) maintaining the cells of the pancreatic lineage for 1-50 days by culturing the cells from step (c) in the presence of a serum-free medium, an insulin-like growth factor, fibroblast growth factor, insulin, nicotinamide, exendin-4 and/or GLP-1, and an agent that increase cAMP.

6. The method of claim 1, wherein the fibroblast growth factor of step (b) is basic fibroblast growth factor (bFGF).

7. The method of claim 1, wherein the bone morphogenetic protein of step (b) is BMP4.

8. The method of claim 1, wherein the chemically defined medium comprising insulin, transferrin, and selenium of steps (b) and/or step (c) is ITS medium.

9. The method of claim 1, wherein the fibroblast growth factor of step (b) is FGF7.

10. The method of claim 1, wherein the bone morphogenetic protein inhibitor of step (b) is a BMP4 inhibitor.

11. The method of claim 1, wherein the serum-free medium of step (b) is B27.

12. The method of claim 1, wherein the primary and differentiated cells are the same or different cell types.

13. The method of claim 1, wherein step (a) has a duration of 2-10 days.

14. The method of claim 1, wherein said identifying comprises sequencing expressed genes in said primary cell, said iPSC, and said pancreatic β

-cell and comparing said expressed genes to identify gene expression markers indicative of differentiation into said pancreatic β

-cell.

15. The method of claim 14, wherein said comparing comprises the use of bioinformatics methods.