MARKERS FOR DIFFERENTIATION OF STEM CELLS INTO DIFFERENTIATED CELL POPULATIONS

• US 20160230225A1
• Filed: 01/22/2016
• Published: 08/11/2016
• 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
• Granted Patent: US 9,828,634 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• 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, comprising:

a) culturing a primary cell under conditions 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 differentiated cell; and

c) identifying molecular markers that are indicative of differentiation into said differentiated cell.

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

3. The method of claim 1, wherein said differentiated cell is a pancreatic beta cell.

4. The method of claim 1, wherein said culturing comprises expanding said cell.

5. The method of claim 1, wherein said culturing comprises inducing expression of one or more genes selected from the group consisting of Oct4 (Pou5f1), Sox2, cMyc, nanog, LIN28, Glis1, and Klf4.

6. The method of claim 1, wherein said treating comprises the steps of

(a) culturing the iPSCs in a chemically defined medium, fibroblast growth factor, Activin A, and bone morphogenetic protein;

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

(c) culturing the cells from step (b) in the presence of a chemically defined medium comprising insulin, transferrin, and selenium;

retinoic acid;

a bone morphogenetic protein inhibitor; and

nicotinamide; and

(d) culturing the cells from step (c) in the presence of a serum-free medium, an insulin-like growth factor, insulin, nicotinamide, exendin-4 and/or GLP-1, a TGF-beta inhibitor, and an agent that increase cAMP.

7. The method of claim 6, further comprising:

(e) maintaining the cells of the pancreatic lineage for 1-50 days by culturing the cells from step (d) 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.

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

9. The method of claim 6, wherein the bone morphogenetic protein of step (a) is BMP4.

10. The method of claim 6, wherein the chemically defined medium comprising insulin, transferrin, and selenium of steps (b) and/or step (c) is ITS medium.

11. The method of claim 6, wherein the fibroblast growth factor of step (b) and/or step (d) is FGF7.

12. The method of claim 6, wherein the bone morphogenetic protein inhibitor of step (c) is a BMP4 inhibitor.

13. The method of claim 6, wherein the serum-free medium of step (d) is B27.

14. The method of claim 6, wherein the primary and differentiated cells are the same or different cell types.

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

16. The method of claim 1, wherein said identifying comprises sequencing expressed genes in said primary cell, said iPSC, and said differentiated cell and comparing said expressed genes to identify gene expression markers indicative of differentiation into said differentiated cell.

17. The method of claim 16, wherein said comparing comprises the use of bioinformatics methods.

18. One or more differentiation markers identified by the method of claim 1.

19. A method of transplanting cells into a subject, comprising:

a) identifying therapeutically suitable differentiated cells that express one or more of the markers of claim 18; and

b) transplanting said cells into a subject in need thereof.

20. The method of claim 19, wherein said transplanting results in reduction or elimination of one or more symptoms of a disease or condition.