Method of deriving mesenchymal stem cells from ES cells using FGF2

• US 8,962,318 B2
• Filed: 08/15/2006
• Issued: 02/24/2015
• 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): Lankford, Blaine
• Application Number:
  US12/065,551
• Publication Number:
  US 20080219957A1
• Time in Patent Office: 3,115 Days
• US Class Current: 435/375
• 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 establishing, from an embryonic stem (ES) cell, a mesenchymal progenitor cell line comprising mesenchymal stem cells (MSCs), the method comprising:

(a) propagating an ES cell in the absence of co-culture with a feeder cell layer, in a serum free medium comprising FGF2,

(b) selecting mesenchymal stem cells from the propagated cells based on expression of one or more cell surface markers, and

(c) growing the mesenchymal stem cells selected in step (b) in medium comprising serum;

thereby establishing a mesenchymal progenitor cell line comprising MSCs, wherein the mesenchymal progenitor cell line maintains self-renewal without transformation, and wherein the mesenchymal progenitor cell line is lineage restricted compared to the ES cell.

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

3. A method according to claim 1, in which the embryonic stem cell is obtained by dispersing an ES cell colony with trypsin.

4. A method according to claim 1, in which the ES cell is obtained by splitting a ES cell culture.

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

6. A method according to claim 5, in which the culture substrate comprises a gelatinized plate.

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

8. A method according to claim 7, in which the serum free medium further comprises PDGF AB at a concentration of about 5 ng/ml.

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

10. A method according to claim 1, in which the propagated cells are split 1:

4 when confluent by trypsinising.

11. A method according to claim 1, 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;

Gper26;

Mrec1.3;

rec.1.3;

NM_—

001401.3);

NTSE (eN;

NT5;

NTE;

eNT;

CD73;

E5NT;

NM_—

002526.1);

FLRT2 (KIAA0405;

NM_—

013231.2); and

FAP (FAPA;

DPPIV;

SEPRASE;

NM_—

004460.2).

12. A method according to claim 1, in which the surface marker comprises CD105 (Accession Number NM_—

000118.1).

13. A method according to claim 1, in which the surface marker comprises CD73 (Accession Number NM_—

002526.1).

14. A method according to claim 1, 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).

15. A method according to claim 1, in which the surface marker comprises CD24 (Accession Number NM_—

013230.1).

16. A method according to claim 1, in which the mesenchymal stem cell is obtained by selecting for cells which are CD105+ CD24−

.

17. A method according to claim 1, in which the mesenchymal stem cell is selected by labelling the cell with an antibody against the surface antigen.

18. A method according to claim 1, in which the mesenchymal stem cell is selected by fluorescence activated cell sorting (FACS).

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

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

21. A method according to claim 1, in which the ES cell is from a huES9 colony.

22. A method according to claim 1, in which the ES cell is from a H1 ESC colony.

23. A method according to claim 1, in which the MSCs display a surface antigen profile which is at least one of CD29+, CD44+, CD49a and e+, CD105+, CD166+ and CD34−

, CD45−

.

24. A method according to claim 1, in which the MSCs display reduced expression of at least one of HESX1, POUFL5, SOX-2, UTF-1 and ZFP42.

25. A method according to claim 1, in which the MSCs display reduced expression of at least one of OCT4, NANOG and SOX2.

26. A method according to claim 1, in which the MSCs display no detectable alkaline phosphatase activity.

27. A method according to claim 1, in which the MSCs are not teratogenic when implanted in SCID mice.

28. A method according to claim 1, in which the MSCs are negative for mouse-specific c-mos repeat sequences and positive for human specific alu repeat sequences.

29. A method according to claim 1, in which the MSCs are capable of undergoing osteogenesis, adipogenesis or chondrogenesis.

30. A method according to claim 2, in which the mesenchymal progenitor cell line comprises huES9.E1, huES9.E3 or H1.E2.

31. A method according to claim 1, in which at least two mesenchymal stem cells selected by the method exhibit substantially identical gene expression profiles.

32. A method according to claim 1, 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.

33. A method according to claim 1, in which at least two isolates of mesenchymal stem cells obtained by the method are substantially similar with each other.

34. A method according to claim 1, 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.

35. A method according to claim 1, further comprising deriving a cell line from the cell selected therefrom.

36. A method according to claim 1, further comprising differentiating the mesenchymal stem cell.

37. A method according to claim 36, in which the differentiated mesenchymal cell is an osteocyte, an adipocyte or a chondrocyte.

39. A method according to claim 29, in which the MSC is capable of differentiating into osteocytes, adipocytes or chondrocytes.

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

(a) providing an embryonic stem cell (ESC) colony;

(b) dispersing the ESC colony with trypsin;

(c) propagating the dispersed cells;

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

(d) selecting cells from the propagated cells based on expression of one or more cell surface markers, and

(e) growing the cells selected in step (d) in medium comprising serum;

thereby deriving a cell culture therefrom an embryonic stem cell.

40. A method according to claim 38, further comprising differentiating the cell line.

41. A method according to claim 38, further comprising differentiating the cell culture.

42. A method according to claim 38, wherein the dispersed ESC colony of step (b) is propagated to confluence and dispersed with trypsin prior to step (c).

43. A method according to claim 38, further comprising selecting a mesenchymal stem cell from the propagated cells based on expression of a cell surface marker.

44. A method according to claim 38, wherein the ESC colony is a human ESC.