Human feeder cells that support proliferation of undifferentiated pluripotent stem cells

• US 20020072117A1
• Filed: 12/07/2001
• Published: 06/13/2002
• Est. Priority Date: 01/11/2000
• Status: Abandoned Application

• Current Assignee: Asterias Biotherapeutics Incorporated (Lineage Cell Therapeutics, Inc.)
• Original Assignee: Asterias Biotherapeutics Incorporated (Lineage Cell Therapeutics, Inc.)
• Inventors: Gold, Joseph D., Xu, Chunhui
• Application Number:
  US10/013,205
• Publication Number:
  US 20020072117A1
• 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 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 for other purposes, such as use as a primary feeder layer. 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 feeder cells or conditioned media are described and illustrated in this disclosure.

Claims

1. A composition of cells comprising:

substantially undifferentiated human embryonic stem (hES) cells; and

cells from a human cell line that are capable of maintaining human embryonic stem cells in a substantially undifferentiated state through at least 4 passages when the cells are cultured together.

2. The composition of claim 1, wherein the human cell line is a line of mesenchymal cells or fibroblasts from a non-malignant source.

3. The composition of claim 1, wherein the human cell line has been obtained by differentiating human pluripotent stem cells.

4. The composition of claim 3, wherein the pluripotent stem cells from which the human cell line has been obtained are embryonic stem cells.

5. The composition of claim 1, wherein the human cell line is an immortalized cell line.

6. The composition of claim 1, wherein the human cell line has been genetically altered to express telomerase reverse transcriptase (TERT) at an elevated level.

7. The composition of claim 3, wherein the human cell line has the same genome as that of the hES cells in the composition.

8. The composition of claim 3, wherein the human cell line has a different genome from that of the hES cells in the composition.

9. The composition of claim 1, wherein the cells of the human cell line that are present in the composition have been inactivated to prevent them from proliferating.

10. The composition of claim 9, wherein the inactivating comprises irradiation or treatment with mitomycin c.

11. The composition of claim 1, wherein the human cell line has the property that culturing it in a medium conditions the medium such that hES cells can be maintained in a substantially undifferentiated state in a growth environment essentially free of feeder cells but containing the conditioned medium.

12. The composition of claim 1, wherein at least ˜

60% of the hES cells in the composition are undifferentiated.

13. A method of maintaining hES cells in a substantially undifferentiated state, comprising culturing the composition of claim 1.

15. A method of obtaining a differentiated cell population, comprising culturing the composition of claim 1, and subsequently causing progeny of the hES cells to differentiate.

16. The method of claim 15, wherein the differentiated cell population is a population of hepatocytes, neuronal cells, glial cells, cardiomyocytes, or osteoblasts.

14. A method of maintaining hES cells in a substantially undifferentiated state, comprising coculturing the hES cells through at least 4 passages with an immortalized human cell line differentiated from human embryonic stem cells.