Method of deriving progenitor cell line

• US 9,018,005 B2
• Filed: 09/23/2010
• Issued: 04/28/2015
• Est. Priority Date: 09/02/2005
• Status: Active Grant

• Current Assignee: Paracrine Therapeutics Pte., Ltd.
• Original Assignee: Agency For Science Technology and Research
• Inventors: Lim, Sai Kiang, Lye, Elias
• Primary Examiner(s): Ton, Thaian N
• Application Number:
  US12/888,791
• Publication Number:
  US 20110014692A1
• Time in Patent Office: 1,678 Days
• Field of Search: 435/377, 435/366
• US Class Current: 435/377
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: A61P 17/02 for treating wounds, ulcers, burns, scars, keloids, or the like
• CPC Class Codes: A61P 25/16 Anti-Parkinson drugs
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/02 specific for leukemia
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/135 Platelet-derived growth factor [PDGF]
• CPC Class Codes: C12N 2501/734 Proteases (EC 3.4.)
• CPC Class Codes: C12N 2502/1352 Mesenchymal stem cells
• 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 5/0662 Stem cells
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: G01N 33/5044 involving specific cell types

Abstract

We disclose a method comprising: (a) providing an embryonic stem (ES) cell; and (b) establishing a progenitor cell line from the embryonic stem cell; in which the progenitor cell line is selected based on its ability to self-renew. Preferably, the method selects against somatic cells based on their inability to self-renew. Preferably, the progenitor cell line is derived or established in the absence of co-culture, preferably in the absence of feeder cells, which preferably selects against embryonic stem cells. Optionally, the method comprises (d) deriving a differentiated cell from the progenitor cell line.

Claims

1. A method of deriving a human mesenchymal progenitor cell line from a parental human embryonic stem cell, wherein the mesenchymal progenitor cell line comprises mesenchymal stem cells, the method comprising:

(a) providing the parental embryonic stem cell or descendents of the parental embryonic stem cell obtained by dispersing an embryonic stem cell colony with trypsin, wherein the parental embryonic stem cell is a human embryonic stem cell; and

(b) culturing the parental embryonic stem cell in the absence of feeder cells in rich media comprising (i) essential nutrients required for cell growth and (ii) serum or serum replacement, wherein the rich media does not comprise additional grown regulators of hormones that promote growth of embryonic stem cells, and wherein the culturing produces mesenchymal progenitor cells which self-renew;

thereby establishing in the absence of transformation a mesenchymal progenitor cell line from the mesenchymal progenitor cells which self-renew;

wherein the mesenchymal progenitor cell line is maintainable in cell culture for more than 20 generations, and wherein the mesenchymal progenitor cell line is lineage restricted compared to the parental embryonic stem cell.

2. The method according to claim 1, further comprising the step of selecting against somatic cells based on their inability to self-renew.

3. The method according to claim 1, further comprising the step of expanding the mesenchymal progenitor cell line.

4. The method according to claim 1, further comprising expanding the mesenchymal progenitor cell line in the absence of co-culture.

5. The method according to claim 1, further comprising the step of exposing the embryonic stem cell to conditions that enhance differentiation to a specific lineage.

6. The method according to claim 5, wherein the step of exposing the embryonic stem cell generates an embryoid body from the parental embryonic stem cell.

7. The method according to claim 5, further comprising the step of removing the embryonic stem cells from the conditions that enhance differentiation after pluripotency is lost.

8. The method according to claim 7, wherein the step of removing comprises disaggregating an embryoid body.

9. The method according claim 8, wherein said embryoid body has been grown from between about 3 days to about 6 days.

10. The method according to claim 1, wherein the mesenchymal progenitor cell line displays at least one of the following characteristics:

(a) has reduced expression of at least one of OCT4 or alkaline phosphatase activity;

(b) does not substantially express at least one of OCT4 or alkaline phosphatase activity;

(c) is substantially non-pluripotent;

(d) is substantially non-tumorigenic;

(e) has reduced expression of a pluripotency marker compared to an embryonic stem cell from which the mesenchymal progenitor cell line is derived, the pluripotency marker selected from the group consisting of Nanog, BMP4, FGF5, OCT4, Sox-2, and Utf1;

(f) is maintainable in cell culture for more than 40 generations;

(g) has a substantially stable karyotype or chromosome number when maintained in cell culture for at least 10 generations;

(h) has a substantially stable gene expression pattern from generation to generation;

or

(i) does not substantially induce formation of teratoma when transplanted to a recipient animal.

11. The method according to claim 1, further comprising the step of deriving a differentiated cell from the mesenchymal progenitor cell line.

12. The method according to claim 1, further comprising propagating the mesenchymal progenitor cell line for at least 5 generations.

13. The method according to claim 11, wherein the differentiated cell is an endothelial cell or a mesenchymal cell.

14. The method according to claim 11, wherein the differentiated cell is an adipocyte or an osteocyte.

15. The method according to claim 13, wherein expression of mesenchymal or endothelial markers of the differentiated cell is up-regulated.

16. The method according to claim 11, wherein expression of stem cell or pluripotency markers of the differentiated cell is down-regulated.

17. The method according to claim 10, wherein the recipient animal is immune compromised.

18. The method according to claim 10, wherein the mesenchymal progenitor cell line does not substantially induce formation of the teratoma after about three weeks.

19. The method according to claim 10, wherein the mesenchymal progenitor cell line does not substantially induce formation of teratoma after about 2 months to about 9 months.

20. The method according to claim 1, wherein the mesenchymal progenitor cell line is maintainable in cell culture for more than 25 generations.

21. The method according to claim 1, wherein the mesenchymal progenitor cell line is maintainable in cell culture for more than 30 generations.

22. The method according to claim 1, wherein the mesenchymal progenitor cell line is maintainable in cell culture for more than 35 generations.

23. The method according to claim 1, wherein the rich media is DMEM with D-glucose, fetal calf serum, non-essential amino acids, L-glutamine, and β

-mercaptoethanol.

24. The method according to claim 1, wherein the mesenchymal progenitor cells which self-renew are CD29⁺, CD44⁺, CD105⁺, CD166⁺, CD34⁻

, and CD45⁻

.