Use of human embryonic stem cells for drug screening and toxicity testing

• US 7,041,438 B2
• Filed: 10/23/2001
• Issued: 05/09/2006
• 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: Inokuma, Margaret S., Xu, Chunhui, Carpenter, Melissa K.
• Primary Examiner(s): Woitach, Joseph
• Assistant Examiner(s): Ton, Thaian N.
• Application Number:
  US10/039,956
• Publication Number:
  US 20020137204A1
• Time in Patent Office: 1,659 Days
• Field of Search: 435/325, 435/363, 435/366, 435/374, 435/404, 435/405, 435/4, 435/29, 435/377, 435/383, 435/394
• US Class Current: 435/4
• 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 disclosure provides an improved system for culturing human pluripotent stem (pPS) cells in the absence of feeder cells. The role of the feeder cells can be replaced by supporting the culture on an extracellular matrix, and culturing the cells in a conditioned medium. Permanent cell lines are provided that can produce conditioned medium on a commercial scale. Methods have also been discovered to genetically alter pPS cells by introducing the cells with a viral vector or DNA/lipid complex. The system described in this disclosure allows for bulk proliferation of pPS cells for use in studying the biology of pPS cell differentiation, and the production of important products for use in human therapy.

Claims

1. A method of screening a substance, comprising:

a) obtaining a composition comprising undifferentiated pPS cells proliferating on an extracellular matrix instead of feeder cells, but in a medium conditioned by fibroblast feeder cells;

b) optionally causing or permitting the pPS cells to differentiate;

then

c) combining the cells with the substance; and

d) determining any effect of the substance on the cells.

2. The method of claim 1, wherein the extracellular matrix upon which the undifferentiated pPS cells are cultured is Matrigel®

basement membrane matrix, laminin, or collagen.

3. The method of claim 1, wherein the cells are undifferantiated when contacted with the substance.

4. The method of claim 1, wherein the cells have been caused or permitted to differentiate before being contacted with the substance.

5. The method of claim 4, wherein the cells have been caused to differentiate by a process comprising replating them onto a surface that promotes differentiation.

6. The method of claim 4, wherein the cells have been caused to differentiate by adding component(s) to the medium that promote differentiation towards a particular cell lineage.

7. The method of claim 4, comprising causing the cells to differentiate into cells having characteristics of neuronal cells, glial cells, or neural precursors.

8. The method of claim 4, comprising causing the cells to differentiate into cells having characteristics of hepatocytes.

9. The method of claim 1, wherein the pPS cells are human embryonic stem (hES) cells.

10. The method of claim 1, comprising determining the effect of the substance on growth of the cells.

11. The method of claim 1, comprising determining whether the substance affects differentiation of the cells.

12. The method of claim 1, comprising determining whether the substance affects expression of a marker or receptor by the cells.

13. The method of claim 1, comprising determining whether the substance affects release of a mwter or enzyme from the cells.

14. The method of claim 1, comprising determining whether the substance affects DNA synthesis or repair in the cells.

15. The method of claim 1, comprising analyzing the cells by metaphase spread.

16. The method of claim 1, comprising determining whether the substance is toxic to the cells.

17. A method of screening a substance for its effect on undifferentiated human embryonic stem (hES) cells, comprising:

a) obtaining a composition comprising undifferentiated pPS cells proliferating on an extracellular matrix instead of feeder cells, but in a medium conditioned by fibroblast feeder cells;

b) combining the undifferentiated hES cells with the substance; and

c) determining any effect of the substance on the cells.

18. The method of claim 17, comprising determining the effect of the substance on growth of the cells.

19. The method of claim 17, comprising determining whether the substance affects differentiation of the cells.

20. The method of claIm 17, comprising determining whether the substance affects expression of a marker or receptor by the cells.

21. The method of claim 17, comprising determining whether the substance is toxic to the cells.

22. A method of screening a substance, comprising:

a) obtaining a composition comprising undifferentiated hES cells proliferating on an extracellular matrix instead of feeder cells, in a medium conditioned by fibroblast feeder cells;

b) differentiating said hES cells;

c) contacting the population of differentiated cells with the substance;

d) determining any phenotypic or metabolic change in the cell that results from contact with the substance, and

e) correlating the change with cellular toxicity or modulation.

23. The method of claim 22, comprising causing the cells to differentiate into cells having characteristics of neuronal cells, glial cells, or neural precursors.

24. The method of claim 22, comprising causing the cells to differentiate into cells having characteristics of hepatocytes.

25. The method of claim 22, comprising determining the effect of the substance on growth of the cells.

26. The method of claim 22, comprising determining whether the compound affects expression of a marker or receptor by the cells.

27. The method of claim 22, comprising determining whether the compound is toxic to the cells.