Method of Deriving Mesenchymal Stem Cells

• US 20080219957A1
• Filed: 08/15/2006
• Published: 09/11/2008
• 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, Elias, Lye
• Granted Patent: US 8,962,318 B2
• Time in Patent Office: Days
• US Class Current: 424/93.7
• 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 obtaining a mesenchymal stem cell (MSC), the method comprising providing a cell obtained by dispersing ani embryonic stem (ES) cell colony, or a descendent thereof, and propagating the cell in the absence of co-culture in a serum free medium comprising FGF2.

2. A method according to claim 1, in which the embryonic stem cell colony is a human embryonic stem cell colony (hESC).

3. A method according to claim 1, in which the embryonic stem cell colony is dispersed with trypsin.

4. A method according to claim 1, in which the method comprises dispersing a confluent plate of human embryonic stem cells (hESC) with trypsin prior to plating the cells out.

5. A method according to claim 3, in which the absence of co-culture comprises culturing the cells without a feeder cell layer.

6. A method according to claim 1, in which the cell is propagated on a culture substrate.

7. A method according to claim 6, in which the culture substrate comprises a gelatinized plate.

8. A method according to claim 1, in which the serum free medium comprises FGF2 at a concentration of about 5 ng/ml.

9. A method according to claim 8, in which the serum free medium further comprises PDGF AB at a concentration of about 5 ng/ml.

10. A method according to claim 1, in which the serum free medium is supplemented with serum replacement media.

11. A method according to claim 3, in which the cells are split 1:

4 when confluenit by trypsinising.

12. A method according to claim 1, which further comprises the step of selecting a mesenchlymal stem cell from the propagated cells based on expression of a cell surface marker.

13. A method according to claim 12, in which the mesenchymal stem cell is obtained by selecting a cell with an elevated expression of one or more surface markers selected from the group consisting of:

ANPEP (CD13;

LAP1;

PEPN;

gp150;

NM_—

001150.1);

ENG (END;

ORW;

HHT1;

ORW1;

CD105;

NM_—

000118.1);

SCN9A (PN1;

NE-NA;

NM_—

002977.1);

TRPV2 (VRL;

VRL1;

VRL-1;

MGC12549;

NM_—

016113.3);

RAMP1 (;

NM_—

005855.1);

F2RL2 (PAR3;

NM_—

004101.2);

NTSR1 (NTR;

NM_—

002531.1);

GABRA2 (;

NM_—

000807.1);

SLC16A4 (MCT4;

NM_—

004696.1);

ITGA4 (CD49D;

NM_—

000885.2);

NCAM2 (NCAM21;

MGC51008;

NM_—

004540.2);

IL1R1 (P80;

IL1R;

IL1RA;

CD121A;

D2S1473;

IL-1R-alpha;

NM_—

000877.2);

PDGFRA (CD140A;

PDGFR2;

NM_—

006206.2);

VCAM1 (INCAM-100;

NM_—

080682.1);

SSFA2 (CS1;

CS-1;

KRAP;

SPAG13;

KIAA1927;

NM_—

006751.3);

TRHDE (PAP-II;

NM_—

013381.1);

EDG2 (LPA1;

edg-2;

vzg-1;

Gpcr26;

Mrec1.3;

rec.1.3;

NM_—

001401.3);

NT5E (eN;

NT5;

NTE;

eNT;

CD73;

E5NT;

NM_—

002526.1);

FLRT2 (KIAA0405;

NM_—

013231.2); and

FAP (FAPA;

DPPIV;

SEPRASE;

NM_—

004460.2).

14. A method according to claim 12, in which the surface marker comprises CD105 (Accession Number NM_—

000118.1).

15. A method according to claim 12, in which the surface marker comprises CD73 (Accession Number NM_—

002526.

     1).

16. A method according to claim 12, in which the mesenchymal stem cell is obtained by selecting a cell with a reduced expression of one or more surface markers selected from the group consisting of:

ITGB1BP3 (MIBP;

NM_—

014446.1);

PTPRZ1 (PTPZ;

HPTPZ;

PTP18;

PTPRZ;

RPTPB;

NM_—

002851.1);

CNTN1 (F3;

GP135;

NM_—

175038.1);

PCDH1 (PC42;

PCDH42;

MGC45991;

NM_—

002587.3);

PODXL (PCLP;

Gp200;

NM_—

005397.2);

GPR64 (HE6;

TM7LN2;

NM_—

005756.1);

PROM1 (AC133;

CD133;

PROML1;

NM_—

006017.1);

GPRC5C (RAIG3;

RAIG-3;

NM_—

022036.2);

CD24 (CD24A;

NM_—

013230.1);

CLDN3 (RVP1;

HRVP1;

CPE-R2;

CPETR2;

NM_—

001306.2);

TACSTD1 (EGP;

KSA;

M4S1;

MK-1;

EGP40;

MIC18;

TROP1;

Ep-CAM;

hEGP-2;

CO17-1A;

GA733-2;

NM_—

002354.1);

HTR3A (HTR3;

NM_—

000869.1);

FGFR4 (TIU;

JTK2;

NM_—

022963.1);

ADCY1 (;

NM_—

021116.1);

FGFR3 (ACH;

CEK2;

JTK4;

HSFGFR3EX;

NM_—

022965.1);

IL17RB (CRL4;

EV127;

IL17BR;

IL17RH1;

MGC5245;

NM_—

018725.2);

SORL1 (LR11;

LRP9;

SORLA;

gp250;

SorLA-1;

NM_—

003105.3);

GPM6B (M6B;

NM_—

005278.2);

KCNS3 KV9.3;

MGC9481;

NM_—

002252.3); and

FZD3 (Fz-3;

hFz3;

NM_—

017412.2).

17. A method according to claim 12, in which the surface marker comprises CD24 (Accession Number NM_—

013230.1).

18. A method according to claim 12, in which the mesenchymal stem cell is obtained by selecting for cells which are CD105+ CD24−

.

19. A method according to claim 12, in which the mesenchymal stem cell is selected by labelling the cell with an antibody against the surface antigen.

20. A method according to claim 12, in which the mesenchymal stem cell is selected by fluorescence activated cell sorting (FACS).

21. A method according to claim 1, in which the cell is passaged for fewer than 35 generations.

22. A method according to claim 1, in which the cells is propagated for about 7 to about 14 days.

23. A method according to claim 1, in which the embryonic stem cell (hESC) colony comprises a huES9 colony.

24. A method according to claim 1, in which the embryonic stem cell (hESC) colony comprises a H1 ESC colony.

25. A method according to claim 1, in which the mesenchymal stem cell (MSC) obtained displays a surface antigen profile which is at least one of CD29+, CD44+, CD49a and e+, CD105+, CD166+ and CD34−

, CD45−

.

26. A method according to claim 1, in which the mesenchymal stem cell (MSC) obtained displays a reduced expression of at least one of HESX1, POUFL5, SOX-2, UTF-1 and ZFP42.

27. A method according to claim 1, in which the mesenchymal stem cell (MSC) obtained displays a reduced expression of at least one of OCT4, NANOG and SOX2.

28. A method according to claim 1, in which the mesenchymal stem cell (MSC) obtained displays no detectable alkaline phosphatase activity.

29. A method according to claim 1, in which the mesenchymal stem cell (MSC) obtained is not teratogenic when implanted in SCID mice.

30. A method according to claim 6, in which a cell culture obtained is negative for mouse-specific c-mos repeat sequences and positive for human specific alu repeat sequences.

31. A method according to claim 1, in which the mesenchymal stem cell (MSC) obtained is capable of undergoing osteogenesis, adipogenesis or chondrogenesis.

32. A method according to claim 6, in which a cell culture obtained comprises huES9.E1, huES9.E3 or H1.E2.

33. A method according to claim 2, in which at least two mesenchymal stem cells selected by the method exhibit substantially identical gene expression profiles.

34. A method according to claim 2, in which a gene expression correlation coefficient between any two or more isolates of mesenchymal stem cells obtained by the method is greater than 0.9.

35. A method according to claim 2, in which at least two isolates of mesenchymal stem cells obtained by the method are substantially similar with each other.

36. A method according to claim 2, in which a gene expression correlation coefficient between a mesenchymal stem cell obtained by the method and cells of a parental culture is greater than 0.8.

37. A method according to claim 12, further comprising deriving a cell line from the cell selected therefrom.

38. A method according to claim 12, further comprising differentiating the stem cell.

39. A method according to claim 38, in which the differentiated mesenchymal cell is an osteocyte, an adipocyte or a chondrocyte.

49. A method according to claim 1, further comprising the step of obtaining a polypeptide from the mesenchymal stem cell, mesenchymal stem cell line or differentiated mesenchymal stem cell.

55. A method according to claim 31, in which the MSC is capable of differentiating into osteocytes, adipocytes or chondrocytes.

56. A method according to claim 37, further comprising differentiating the cell line.

40. A cell obtained by providing a cell obtained by dispersing an embryomic cell colony or a descendent thereof and propagating the cell in the absence of co-culture in a serum free medium comprising FGF2

57. A cell as in claim 40 wherein said embryonic cell colony is a human embryonic cell colony.

58. A cell as in claim 40 wherein said embryonic cell colony is dispersed with trypsin.

59. A cell as in claim 40 wherein the cell has a cell surface marker selected from the group consisting of CD105 and CD73.

41. A method of deriving a cell culture from an embryonic stem cell, the method comprising:

(a) providing a embryonic stem cell (hESC) colony;

(b) dispersing the hESC colony;

(c) propagating the dispersed cells;

(i) in the absence of a feeder layer (ii) in serum free media in the presence of FGF2; and

(d) deriving a cell culture therefrom.

64. A method according to claim 41, further comprising differentiating the cell culture.

65. A method according to claim 41, further comprising dispersing a confluent plate of human embryonic stem cells with trypsin prior to plating the cells out.

66. A method according to claim 41, further comprising selecting a mesenchymal stem cell from the propagated cells based on expression of a cell surface marker.

42. (canceled)

43. A method of treatment of a disease comprising obtaining a mesenchymal stem cell, mesenchymal stem cell line or differentiated mesenchymal stem cell and administering the mesenchymal stem cell, mesenchymal stem cell line or the differentiated cell into a patient, wherein said step of obtaining comprises providing a cell obtained by dispersing an embryonic stem cell colony or a descendent thereof and propagating the cell in the absence of co-culture in a serum free medium comprising FGF2.

46. A method according to claim 43, in which the disease is selected from the group consisting of:

cardiac failure, bone marrow disease, skin disease, burns and degenerative diseases such as diabetes, Alzheimer'"'"'s disease, Parkinson'"'"'s disease and cancer.

67. A method of treatment according to claim 43, further comprising the step of deriving a differentiated cell from the mesenchymal stem cell or mesenchymal stem cell line.

44. Use of mesenchymal stem cell to prepare a medicament for the treatment or prevention of a disease in an individual, wherein said mesenchymal stem cell is obtained by providing a cell obtained by dispersing an embryonic cell colony or a descendent thereof and propagating the cell in the absence of co-culture in a serum free medium comprising FGF2.

68. A method according to claim 44, in which the disease is selected from the group consisting of:

cardiac failure, bone marrow disease, skin disease, burns and degenerative diseases such as diabetes, Alzheimer'"'"'s disease, Parkinson'"'"'s disease and cancer.

45. A pharmaceutical composition comprising a mesenchymal stem cell, mesenchymal stem cell line or differentiated mesenchymal stem cell and a pharmaceutically acceptable excipient or carrier, wherein said mesenchymal stem cell is obtained by providing a cell obtained by dispersing an embryonic cell colony or a descendent thereof and propagating the cell in the absence of co-culture in a serum free medium comprising FGF2.

47. A method of conditioning a cell culture medium, the method comprising obtaining a cell by dispersing an embryonic cell colony or a descendent thereof, propagating the cell in the absence of co-culture in a serum free medium comprising FGF2. and culturing a mesenchymal stem cell, mesenchymal stem cell line or differentiated mesenchymal stem cell in a cell culture medium.

69. A method according to claim 47 further comprising the step of isolating the cell culture medium.

48. A conditioned medium obtained by obtaining a cell by dispersing an embryonic cell colony or a descendent thereof, propagating the cell in the absence of co-culture in a serum free medium comprising FGF2, and culturing a mesenchymal stem cell, mesenchymal stem cell line or differentiated mesenchymal stem cell in a cell culture medium.

50. A conditioned medium according to claim 48 wherein said medium is used to treat a disease in an individual.

51-54. -54. (canceled)

60. A cell line obtained by providing a cell obtained by dispersing an embryonic cell colony or a descendent thereof, propagating the cell in the absence of co-culture in a serum free medium comprising FGF2, and selecting a mesenchymal stem cell from the propagated cells based on expression of a cell surface marker, and deriving a cell line from the cell selected therefrom.

61. A cell as in claim 60 wherein said embryonic cell colony is a human embryonic cell colony.

62. A cell as in claim 60 wherein said embryonic cell colony is dispersed with trypsin.

63. A cell as in claim 60 wherein the cell has a cell surface marker selected from the group consisting of CD105 and CD73.