Conditioned media for propagating human pluripotent stem cells

• US 6,642,048 B2
• Filed: 07/06/2001
• Issued: 11/04/2003
• Est. Priority Date: 01/11/2000
• Status: Expired due to Term

• Current Assignee: Asterias Biotherapeutics Incorporated (Lineage Cell Therapeutics, Inc.)
• Original Assignee: Geron Corporation
• Inventors: Xu, Chunhui, Gold, Joseph D.
• Primary Examiner(s): Crouch, Deborah
• Assistant Examiner(s): TON, THAIAN N
• Application Number:
  US09/900,752
• Publication Number:
  US 20020022268A1
• Time in Patent Office: 851 Days
• Field of Search: 435/325, 435/363, 435/366, 435/377, 435/374, 435/383, 435/384
• US Class Current: 435/366
• CPC Class Codes: A61K 35/12 Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells vaccines or medicinal preparations containing antigens or antibodies A61K39/00
• CPC Class Codes: C12N 15/1034 Isolating an individual clone by screening libraries
• CPC Class Codes: C12N 15/1072 Differential gene expression library synthesis, e.g. subtracted libraries, differential screening
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• CPC Class Codes: C12N 2500/14 Calcium; Ca chelators; Calcitonin
• CPC Class Codes: C12N 2500/30 Organic components metal chelators C12N2500/10; calcitonin C12N2500/14; transferrin C12N2500/24
• CPC Class Codes: C12N 2500/38 Vitamins
• CPC Class Codes: C12N 2500/44 Thiols, e.g. mercaptoethanol
• CPC Class Codes: C12N 2500/62 DMSO
• CPC Class Codes: C12N 2501/105 Insulin-like growth factors [IGF]
• CPC Class Codes: C12N 2501/13 Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• 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 2501/39 Steroid hormones
• CPC Class Codes: C12N 2501/999 Small molecules not provided for elsewhere
• CPC Class Codes: C12N 2502/13 connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
• CPC Class Codes: C12N 2502/99 genetically modified cells
• CPC Class Codes: C12N 2503/02 Drug screening
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 2510/04 Immortalised cells
• CPC Class Codes: C12N 2533/90 : Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
• 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

Abstract

This invention provides media that support the growth of primate pluripotent stem cells in feeder-free culture, and cell lines useful for producing such media and other purposes. Conventionally, it has been necessary to grow pluripotent embryonic cells on feeder layers of primary embryonic fibroblasts, in order to prevent them from differentiating. It has now been discovered that standard culture media conditioned by special cell lines can be used to support proliferation of pluripotent stem cells while inhibiting differentiation in an environment free of feeder cells. This invention includes mesenchymal and fibroblast-like cell lines obtained from embryonic tissue or differentiated from embryonic stem cells. Methods for deriving such cell lines, processing media, and growing stem cells using the conditioned media are described and illustrated in this disclosure.

Claims

1. A method for proliferating human blastocyst-derived pluripotent stem cells in a substantially undifferentiated form, comprising culturing the stem cells in a growth environment that is essentially free of feeder cells, but that contains an extracellular matrix and a conditioned medium produced by a method comprising:

a) culturing cells in a tissue culture medium, thereby conditioning the medium; and

then

b) harvesting the conditioned medium from the culture;

wherein the cells used to condition the medium are from a non-malignant mouse cell line immortalized by genetic engineering, or a non-malignant human cell line that proliferates in culture for at least 60 days.

2. The method of claim 1, wherein the pluripotent stem cells are a line of human embryonic stem (hES) cells.

3. The method of claim 1, wherein the cell line used to condition the medium is an immortalized mouse cell line.

4. The method of claim 1, wherein the cell line used to condition the medium is a human cell line.

5. The method of claim 1, wherein the cell line used to condition the medium is a mesenchymal cell line.

6. The method of claim 1, wherein the cell line used to condition the medium is a fibroblast line.

7. The method of claim 4, wherein the cell line used to condition the medium has been obtained by differentiating human embryonic stem cells ex vivo.

8. The method of claim 1, wherein the cell line used to condition the medium is euploid.

9. The method of claim 1, wherein the cell line used to condition the medium has been genetically altered to express telomerase reverse transcriptase (TERT) at an elevated level.

10. A method for preparing a conditioned medium for proliferating primate pluripotent stem (pPS) cells, comprising:

a) culturing cells in a tissue culture medium, thereby conditioning the medium; and

then

b) harvesting the conditioned medium from the culture;

wherein the cells used to condition the medium are from a human cell line immortalized with telomerase reverse transcriptase (TERT); and

wherein pPS cells proliferate without differentiation when cultured in an environment essentially free of feeder cells but comprising the conditioned medium, an extracellular matrix, and a growth factor.

11. The method of claim 10, further comprising adding a fibroblast growth factor to the medium before or after harvesting.

12. The method of claim 11, wherein the fibroblast growth factor is basic FGF or FGF-4.

13. The method of claim 10, wherein the cells used to condition the medium are irradiated.

14. The method of claim 10, wherein the cells used to condition the medium are mesenchymal cells.

15. The method of claim 10, wherein the cells used to condition the medium are fibroblasts.

16. The method of claim 10, wherein the cells used to condition the medium has been obtained by differentiating human embryonic stem cells ex vivo.

17. The method of claim 10, wherein the cells used to condition the medium have been genetically altered to express telomerase reverse transcriptase (TERT) at an elevated level.

18. A composition of undifferentiated pluripotent stem cells obtained from a human blastocyst, proliferating in a growth environment containing an extracellular matrix and a medium conditioned by:

a) culturing cells in a tissue culture medium, thereby conditioning the medium; and

then

b) harvesting the conditioned medium from the culture;

wherein the cells used to condition the medium are from a non-malignant mouse cell line immortalized by genetic engineering, or a human cell line that proliferates in culture for at least 60 days.

19. The composition of claim 18, wherein the cells used to condition the medium are an immortalized mouse cell line.

20. The composition of claim 18, wherein the cells used to condition the medium are human cells.

21. The composition of claim 18, wherein the pluripotent stem cells proliferating in the composition are a line of human embryonic stem (hES) cells.

22. The composition of claim 18, wherein the cells used to condition the medium are from a line of human cells that proliferates in culture for at least 60 days.

23. The composition of claim 18, wherein the cells used to condition the medium have been obtained by differentiating a culture of hES cells, and then selecting mesenchymal or fibroblast-like cells from the culture.

24. The composition of claim 18, wherein the cells used to condition the medium have been immortalized.

25. A method for preparing a conditioned medium for proliferating human blastocyst-derived pluripotent stem cells, comprising:

a) culturing cells of a telomerized mammalian cell line in a tissue culture medium, thereby conditioning the medium;

b) harvesting the conditioned medium from the culture; and

then

c) adding a growth factor to the medium;

wherein pPS cells proliferate without differentiation when cultured in an environment essentially free of feeder cells, but comprising an extracellular matrix and the conditioned medium.

26. The method of claim 25, wherein the cells used to condition the medium are mesenchymal cells.

27. The method of claim 25, wherein the cells used to condition the medium are fibroblasts.

28. The method of claim 25, wherein cells used to condition the medium were obtained by differentiating hES cells ex vivo.

29. The method of claim 25, wherein the cells used to condition the medium have are human.

30. A conditioned medium for proliferating human blastocyst-derived pluripotent stem cells, produced by a method comprising:

a) culturing cells of a telomerized mammalian cell line in a tissue culture medium, thereby conditioning the medium;

b) harvesting the conditioned medium from the culture; and

then

c) adding a growth factor to the medium;

wherein pPS cells proliferate without differentiation when cultured in an environment essentially free of feeder cells, but comprising an extracellular matrix and the conditioned medium.

31. The conditioned medium of claim 30, wherein the cells used to condition the medium are mesenchymal cells.

32. The conditioned medium of claim 30, wherein the cells used to condition the medium are fibroblasts.

33. The conditioned medium of claim 30, wherein cells used to condition the medium were obtained by differentiating hES cells ex vivo.

34. The conditioned medium of claim 30, wherein the cells used to condition the medium are human.

35. A method for determining whether a non-malignant human cell line is capable of producing conditioned medium according to claim 30, comprising:

a) providing a growth environment essentially free of feeder cells, but containing an extracellular matrix and a medium that has been conditioned by culturing with the human cell line; and

b) determining whether pPS cells cultured in the growth environment proliferate without differentiating.