Culture system for rapid expansion of human embryonic stem cells

• US 20080020458A9
• Filed: 09/04/2002
• Published: 01/24/2008
• Est. Priority Date: 01/10/2001
• Status: Active Grant

• Current Assignee: Asterias Biotherapeutics Incorporated (Lineage Cell Therapeutics, Inc.)
• Original Assignee: Geron Corporation
• Inventors: Carpenter, Melissa, Gold, Joseph, Xu, Chunhui, Mandalam, Ramkumar
• Granted Patent: US 7,410,798 B2
• Time in Patent Office: Days
• US Class Current: 435/366
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C12N 11/04 entrapped within the carrier, e.g. gel or hollow fibres
• CPC Class Codes: C12N 15/1034 Isolating an individual clone by screening libraries
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• CPC Class Codes: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2500/98 Xeno-free medium and culture conditions
• CPC Class Codes: C12N 2500/99 Serum-free medium
• CPC Class Codes: C12N 2501/065 Modulators of histone acetylation
• CPC Class Codes: C12N 2501/105 Insulin-like growth factors [IGF]
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/119 Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
• CPC Class Codes: C12N 2501/125 Stem cell factor [SCF], c-kit ligand [KL]
• 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 2501/145 Thrombopoietin [TPO]
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2501/2306 Interleukin-6 (IL-6)
• CPC Class Codes: C12N 2501/235 Leukemia inhibitory factor [LIF]
• CPC Class Codes: C12N 2501/237 Oncostatin M [OSM]
• CPC Class Codes: C12N 2501/26 Flt-3 ligand (CD135L, flk-2 ligand)
• CPC Class Codes: C12N 2501/998 : Proteins 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 2506/02 : from embryonic cells
• CPC Class Codes: C12N 2510/00 : Genetically modified cells
• CPC Class Codes: C12N 2510/04 : Immortalised cells
• CPC Class Codes: C12N 2533/50 : Proteins
• CPC Class Codes: C12N 2533/90 : Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
• CPC Class Codes: C12N 5/0062 : General methods for three-dimensional culture
• CPC Class Codes: C12N 5/0068 : General culture methods using substrates for specific animal cell type C12N5/06
• CPC Class Codes: C12N 5/0075 : using microcarriers
• CPC Class Codes: C12N 5/0603 : Embryonic cells production of embryos, nuclear transfer A01K67/027; Embryoid bodies
• 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
• CPC Class Codes: C12N 5/0607 : Non-embryonic pluripotent stem cells, e.g. MASC induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12N 5/0619 : Neurons
• CPC Class Codes: C12N 5/0622 : Glial cells, e.g. astrocytes, oligodendrocytes; Schwann cells
• CPC Class Codes: C12N 5/0662 : Stem cells
• CPC Class Codes: C12N 5/0671 : Three-dimensional culture, tissue culture or organ culture; Encapsulated cells
• CPC Class Codes: C12N 5/0672 : Stem cells; Progenitor cells; Precursor cells; Oval cells
• CPC Class Codes: C12N 5/0678 : Stem cells; Progenitor cells; Precursor cells
• CPC Class Codes: C12N 5/068 : Stem cells; Progenitors
• CPC Class Codes: C12N 5/0692 : Stem cells; Progenitor cells; Precursor cells
• CPC Class Codes: C12N 5/0693 : Tumour cells; Cancer cells
• CPC Class Codes: C12N 5/0696 : Artificially induced pluripotent stem cells, e.g. iPS

Abstract

This disclosure provides an improved system for culturing human pluripotent stem cells. Traditionally, pluripotent stem cells are cultured on a layer of feeder cells (such as mouse embryonic fibroblasts) to prevent them from differentiating. In the system described here, the role of feeder cells is replaced by components added to the culture environment that support rapid proliferation without differentiation. Effective features are a suitable support structure for the cells, and an effective medium that can be added fresh to the culture without being preconditioned by another cell type. Culturing human embryonic stem cells in fresh medium according to this invention causes the cells to expand surprisingly rapidly, while retaining the ability to differentiate into cells representing all three embryonic germ layers. This new culture system allows for bulk proliferation of pPS cells for commercial production of important products for use in drug screening and human therapy.

Claims

1. A method for expanding a population of pluripotent stem cells, comprising:

a) obtaining a population of pluripotent stem cells that have been isolated or propagated from a human blastocyst;

b) culturing the cell population in a culture environment containing the following components;

i) an extracellular matrix made from isolated extracellular matrix components;

ii) a fresh isotonic culture medium comprising added protein or amino acids, added nucleosides, and added lipids; and

iii) a fibroblast growth factor; and

c) optionally passaging the cell population one or more times into a new culture environment containing said components;

wherein all the cells in each culture environment have the same genotype;

until there is at least a 10-fold expansion of the number of cells in the population, such that at least 50% of the cells are undifferentiated, and can be caused to differentiate into derivatives of the endoderm, mesoderm, and ectoderm.

2. The method of claim 1, wherein the cell population is cultured until there is at least a 50-fold expansion of the number of cells in the population;

such that at least 50% of the cells in the expanded cell population are undifferentiated and capable of generating progeny representing all three germ layers.

3. The method of claim 1, wherein the cell population is cultured until there is at least a 20-fold expansion of the number of cells in the population;

such that at least 70% of the cells in the expanded cell population are undifferentiated and capable of generating progeny representing all three germ layers.

4. The method of claim 1, comprising passaging the cell population at least three times into new culture environments containing said components during the expansion.

5. The method of claim 4, wherein the cells are cultured after each passage until there is at least a 2-fold expansion in the number of undifferentiated cells since the last passage.

6. The method of claim 4, wherein after passaging and culturing the cells according to the method, there is at least a 100-fold expansion in the number of pluripotent stem cells originally obtained;

and after the 100-fold expansion, at least 50% of the cells are undifferentiated and capable of generating progeny representing all three germ layers.

7. The method of claim 1, wherein the culturing and optional passaging causes the cell population to proliferate at a rate that is at least 1.5-fold faster than if the same cell population were cultured on mouse fetal fibroblast feeder cells.

8. The method of claim 1, wherein the expanded cell population expresses telomerase reverse transcriptase (TERT) or OCT-4 at a level within 5-fold that of what it would be if the same cell population were cultured on mouse fetal fibroblast feeder cells.

11. A method of producing genetically altered stem cells, comprising:

a) providing a population of proliferating undifferentiated pluripotent stem cells that have been isolated or propagated from a human blastocyst;

b) transfecting cells in the population with a DNA-lipid complex;

c) selecting cells that have been genetically altered with the complex; and

d) expanding the cell population according to the method of any of claims 1-8, before or after they have been genetically altered.

14. The method of claim 1, wherein the culture environment comprises an extracellular matrix that contains laminin or is made from Engelbreth-Holm-Swarm cells.

15. The method of claim 1, wherein protein is added to the medium in the form of fetal calf serum, albumin, or serum replacement.

16. The method of claim 1, wherein lipids are added to the medium in the form of high or low density lipoproteins.

17. The method of claim 1, wherein the fibroblast growth factor (FGF) is basic FGF, FGF-4, or an antibody or ligand that binds to the receptor for either basic FGF or FGF-4.

18. The method of claim 1, wherein the pluripotent stem cells are progeny of an established line of human embryonic stem (hES) cells.

19. The method of claim 1, further comprising causing differentiation of the expanded cell population (or a subpopulation thereof) into a cell population of differentiated cells in which at least 95% of the cells represent the same germ layer.

20. The method of claim 1, wherein 95% of the differentiated cells are neural cells, hepatocytes, cardiomyocytes, mesenchymal cells, or osteoblasts.

9. A culture environment for expanding a population of pluripotent stem cells, comprising:

i) an extracellular matrix made from isolated extracellular matrix components;

ii) a fresh isotonic culture medium comprising added protein or amino acids, added nucleosides, and added lipids; and

iii) a fibroblast growth factor;

wherein when pluripotent stem cells that have been isolated or propagated from a human blastocyst are;

a) cultured in the culture environment; and

b) optionally passaged one or more times into a new culture environment containing said components)

wherein all the cells in each culture environment have the same genotype;

until there is at least a 10-fold expansion of the number of cells in the population;

a cell population is obtained in which at least 50% of the cells are undifferentiated, and can be caused to differentiate into derivatives of the endoderm, mesoderm, and ectoderm.

10. A composition for expanding human pluripotent stem cells, comprising:

a) a population of pluripotent stem cells that have been isolated or propagated from a human blastocyst; and

b) a culture environment containing the following components;

i) an extracellular matrix made from isolated extracellular matrix components;

ii) a fresh isotonic culture medium comprising added protein or amino acids, added nucleosides, and added lipids; and

iii) a fibroblast growth factor;

wherein all the cells in each culture environment have the same genotype;

and wherein culturing the composition (and optionally passaging the cell population one or more times into a new culture environment containing said components) until there is at least a 10-fold expansion of the number of cells in the population,

produces a cell population in which at least 50% of the cells are undifferentiated and can be caused to differentiate into derivatives of the endoderm, mesoderm, and ectoderm.

12. A method of producing genetically altered stem cells, comprising:

a) providing a composition according to claim 10 comprising proliferating undifferentiated pluripotent stem cells;

b) transfecting cells in the population with a DNA-lipid complex; and

c) selecting cells that have been genetically altered with the complex; and

13. A rapid expansion method for generating pluripotent stem cells without feeder cells, comprising:

culturing pluripotent stem cells that have been isolated or propagated from a human blastocyst in a suitable culture environment that is essentially free of feeder cells and that has a medium containing sufficient fibroblast growth factor (FGF) to cause the cells to expand with a doubling time of less than about 24 hours into a cell population at least 10-fold larger in which at least 50% of the cells are undifferentiated.