Method of Differentiating Stem Cells

• US 20180155693A1
• Filed: 07/17/2017
• Published: 06/07/2018
• Est. Priority Date: 01/31/2008
• Status: Active Application

• Current Assignee: ES Cell International Pte Ltd. (Lineage Cell Therapeutics, Inc.)
• Original Assignee: Biotime, Inc.
• Inventors: Chipperfield, Hiram, Dunn, Norris Ray
• Application Number:
  US15/652,215
• Publication Number:
  US 20180155693A1
• Time in Patent Office: Days
• CPC Class Codes: C12N 2500/44 Thiols, e.g. mercaptoethanol
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/119 Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
• 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 2501/385 of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
• CPC Class Codes: C12N 2501/41 Hedgehog proteins; Cyclopamine (inhibitor)
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 5/0603 Embryonic cells production of embryos, nuclear transfer A01K67/027; Embryoid bodies
• CPC Class Codes: C12N 5/0606 Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12N 5/0676 Pancreatic cells
• CPC Class Codes: C12N 5/0696 Artificially induced pluripotent stem cells, e.g. iPS

Abstract

There is provided an improved efficient method for differentiating stem cells into pancreatic endoderm cells and pancreatic hormone expressing and secreting cells which express Pdx-1 and C-peptide. The invention further provides screening methods for detecting factors of interest that will affect the differentiation of the stem cells into pancreatic endoderm cells.

Claims

1. A method for differentiating stem cells into cells expressing C-peptide comprising the steps of:

(a) culturing stem cells in the presence of at least one extracellular matrix;

(b) culturing the stem cells obtained from step (a) in at least one first medium comprising at least BMP4 and Activin A to obtain definitive endoderm cells;

(c) culturing the cells obtained from step (b) in at least one second medium comprising at least Activin A;

(d) culturing the cells obtained from step (c) in at least one third medium comprising at least one fibroblast growth factor (FGF), retinoic acid (RA) and at least one inhibitor of hedgehog signaling to obtain cells expressing Pdx-1; and

(e) culturing the cells obtained from step (d) in at least one fourth medium comprising at least one inhibitor of notch signaling to differentiate cells expressing Pdx-1 to C-peptide producing cells.

2. The method according to claim 1, wherein the method is a two dimensional method of differentiation.

3. The method according to claim 1, wherein the culturing of the cells in the first medium in step b is for at least 3 days.

4. The method according to claim 1, wherein the culturing of the cells in the second medium in step c is in the range from 4 days to 7 days.

5. The method according to claim 1, wherein the concentration of BMP4 is in the range of 10-200 ng/ml.

6. The method according to claim 1, wherein the concentration of Activin A is in the range of 10-200 ng/ml.

7. The method according to claim 1, wherein FGF is FGF2, FGF10 and/or FGF18.

8. The method according to claim 1, wherein the inhibitor of hedgehog signaling is SANT-1.

9. The method according to claim 1, wherein the inhibitor of notch signaling is Gamma Secretase Inhibitor X and/or DAPT.

10. The method according to claim 1, wherein the inhibitor of notch signaling is used at a concentration of at least 10 μ

M.

11. The method according to claim 1, wherein nicotinamide is added together with, before or after the addition of inhibitor of notch signaling.

12. The method according to claim 1, wherein the stem cells are selected from any of embryonic stem cells, fetal stem cells, or adult stem cells.

13. The method according to claim 1, wherein the stem cells are selected from any of human, mouse, primate or rat origin.

14. The method according to claim 1, wherein the extracellular matrix is selected from the group consisting of MATRIGEL ™

, growth factor-reduced MATRIGEL ™

, laminin, and fibronectin.

15. A method of identifying at least one factor that modulates the differentiation of stem cells differentiated into cells expressing markers characteristic of the endoderm lineage comprising the steps of:

(a) culturing at least one cell population according to any one of the preceding claims, in the presence of at least one factor to be tested; and

(b) comparing the differentiation of the cells in the presence and absence of the factor, wherein at least one difference in the differentiation in the presence of the factor is indicative of the identification of at least one factor that modulates the differentiation of the cells.

16. The method of claim 15, wherein the markers characteristic of the endoderm lineage comprise at least Sox17.

17. A method of identifying at least one factor that modulates the differentiation of stem cells differentiated into cells expressing Pdx-1 comprising the steps of:

(a) culturing at least one cell population according to claim 1, in the presence of at least one factor to be tested; and

(b) comparing the differentiation of the cells in the presence and absence of the factor, wherein at least one difference in the presence of the factor is indicative of the identification of at least one factor that modulates the differentiation of the cells.

18. A method of identifying at least one factor that modulates the differentiation of stem cells differentiated into cells expressing Pdx-1 and/or C-peptide comprising the steps of:

(a) culturing at least one cell population according to claim 1, in the presence of at least one factor to be tested; and

(b) comparing the differentiation of the cells in the presence and absence of the factor, wherein at least one difference in the presence of the factor is indicative of the identification of at least one factor that modulates the differentiation of the cells.