Conditioned media for propagating human pluripotent stem cells

• US 20020022268A1
• Filed: 07/06/2001
• Published: 02/21/2002
• Est. Priority Date: 01/11/2000
• Status: Active Grant

• Current Assignee: Asterias Biotherapeutics Incorporated (Lineage Cell Therapeutics, Inc.)
• Original Assignee: Geron Corporation
• Inventors: Gold, Joseph D., Xu, Chunhui
• Granted Patent: US 6,642,048 B2
• Time in Patent Office: Days
• 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
• 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 culturing primate pluripotent stem (pPS) cells, comprising culturing pPS cells in a growth environment essentially free of feeder cells but containing 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 cell line from a non-malignant source that can proliferate in culture for at least 60 days.

2. The method of claim 1, wherein the pPS cells are 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 cell line.

7. The method of claim 1, 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 has been genetically altered to express telomerase reverse transcriptase (TERT) at an elevated level.

10. A method for preparing a conditioned medium suitable for culturing primate pluripotent stem (pPS) cells in a growth environment essentially free of feeder 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 from a non-malignant source that can proliferate in culture for at least 60 days; and

wherein pPS cells can be caused to proliferate without differentiation in an environment essentially free of feeder cells but comprising the medium and a growth factor.

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

14. The human cell line of claim 13, wherein the cell line is a fibroblast cell line.

15. The human cell line of claim 13, which has been genetically altered to express telomerase reverse transcriptase (TERT) at an elevated level.

16. The human cell line of claim 13, which can be used to produce conditioned medium by a method comprising:

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

then

b) harvesting the conditioned medium from the culture;

wherein pPS cells can be caused to proliferate without differentiation in an environment essentially free of feeder cells but comprising the medium and a growth factor.

17. A method for preparing a conditioned medium suitable for culturing primate pluripotent stem (pPS) cells in feeder-free culture, comprising:

a) culturing the cell line of claim 13 in a tissue culture medium, thereby conditioning the medium; and

then

b) harvesting the conditioned medium from the culture.

18. A conditioned medium to support culturing primate pluripotent stem (pPS) cells without differentiation in feeder-free culture, produced by a method comprising:

a) culturing the cell line of claim 13 in a tissue culture medium, thereby conditioning the medium; and

then

b) harvesting the conditioned medium from the culture.

20. A device for preparing conditioned medium according to claim 18, comprising:

a) a culture chamber containing a plurality of cells from a cell line that can proliferate in culture for at least 60 days;

whereby the cells condition medium in the device in such a manner that renders the medium able to support growth of pPS cells in feeder-free culture without differentiation; and

b) a port for withdrawing medium from the culture chamber after conditioning by the cells.

12. A composition of proliferating pPS cells, comprising undifferentiated pPS cells in a growth environment that includes 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 cell line from a non-malignant source that can proliferate in culture for at least 60 days.

13. A human cell line obtained by differentiating a culture of human embryonic stem (hES) cells, and then selecting mesenchymal or fibroblast-like cells from the culture;

wherein medium conditioned by culturing with the selected cells supports growth of pPS cells without differentiation in a culture environment essentially free of feeder cells.

19. A method for screening cells suitable for producing conditioned medium that supports growth of primate pluripotent stem (pPS) cells without differentiation in feeder-free culture, comprising:

a) providing a growth environment essentially free of feeder cells, wherein growth of pPS cells in the environment in a substantially undifferentiated state is promoted by medium conditioned by primary mouse embryonic fibroblasts (mEF), but wherein a test medium conditioned by cells being screened according to the method is used in the growth environment instead of the mEF conditioned medium; and

b) determining whether pPS cells cultured in the growth environment containing the test medium grow without differentiating.