Method of deriving progenitor cell line

• US 9,005,897 B2
• Filed: 03/07/2013
• Issued: 04/14/2015
• Est. Priority Date: 09/02/2005
• Status: Active Grant

• Current Assignee: Paracrine Therapeutics Pte., Ltd.
• Original Assignee: Agency For Science Technology and Research (Government of Singapore)
• Inventors: Lim, Sai Kiang, Lye, Elias
• Primary Examiner(s): Ton, Thaian N
• Application Number:
  US13/788,627
• Publication Number:
  US 20130280719A1
• Time in Patent Office: 768 Days
• Field of Search: 435/6.13, 435/366
• US Class Current: 435/6.13
• 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 screening a candidate molecule for its effect on a human mesenchymal progenitor cell, the method comprising:

(a) providing a parental embryonic stem cell or descendants of a 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 or 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;

(c) culturing cells from the mesenchymal progenitor cell line in the presence of a candidate molecule; and

(d) determining the effect of a candidate molecule on cells from the mesenchymal progenitor cell line treated with the candidate molecule compared with untreated cells from the mesenchymal progenitor cell line or cells from the mesenchymal progenitor cell line treated with an inert compound.

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

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

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

5. The method according to claim 1, wherein the mesenchymal progenitor cell line is maintainable in cell culture for more than 40 generations.

11. 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.

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

, and CD45⁻

.

6. A method of screening a candidate molecule for its effect on a cell differentiated from a human mesenchymal progenitor cell line, the method comprising:

(a) providing a parental embryonic stem cell or descendants of a 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 or 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;

(c) deriving a differentiated cell from the mesenchymal progenitor cell line;

(d) treating the differentiated cell with a candidate molecule;

and

(e) determining the effect of a candidate molecule on the differentiated cell treated with the candidate molecule compared with untreated differentiated cells or differentiated cells treated with an inert compound.

7. The method according to claim 6, wherein the mesenchymal progenitor cell line is maintainable in cell culture for more than 25 generations.

8. The method according to claim 6, wherein the mesenchymal progenitor cell line is maintainable in cell culture for more than 30 generations.

9. The method according to claim 6, wherein the mesenchymal progenitor cell line is maintainable in cell culture for more than 35 generations.

10. The method according to claim 6, wherein the mesenchymal progenitor cell line is maintainable in cell culture for more than 40 generations.

12. The method according to claim 6, wherein the rich media is DMEM with D-glucose, fetal calf serum, non-essential amino acids, L-glutamine, and β

-mercaptoethanol.

14. The method according to claim 6, wherein the mesenchymal progenitor cells which self-renew are CD29⁺, CD44⁺, CD105⁺, CD166⁺, CD34⁻

, and CD45⁻

.