Using fibroblast growth factor to establish a line of embryonic stem cells

• US 20050153444A1
• Filed: 12/10/2004
• Published: 07/14/2005
• Est. Priority Date: 10/23/1998
• 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, Mandalam, Ramkumar, Carpenter, Melissa K.
• Application Number:
  US11/009,571
• Publication Number:
  US 20050153444A1
• 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 establishing a line of human embryonic stem cells, comprising:

a) isolating cells from the inner cell mass of a human blastocyst;

b) forming colonies comprising undifferentiated cells from the isolated blastocyst cells;

c) passaging the colonies into a culture environment that contains an extracellular matrix instead of feeder cells; and

d) culturing the colonies in the culture environment in a nutrient medium containing fibroblast growth factor, thereby establishing a line of human embryonic stem cells;

wherein the embryonic stem cell line can proliferate in culture for at least 64 days without differentiation, while maintaining a normal karyotype and the potential to differentiate to cells of endoderm, mesoderm, and ectoderm tissues.

2. The method of claim 1, wherein the extracellular matrix is a fibroblast matrix, prepared by culturing fibroblasts in said culture environment.

3. The method of claim 1, wherein the extracellular matrix is made from one or more extracellular matrix components selected from laminin, fibronectin, proteoglycan, and entactin.

4. The method of claim 1, wherein the nutrient medium is a serum-free medium.

5. The method of claim 1, wherein the medium is a fresh medium not previously conditioned by culturing with feeder cells.

6. The method of claim 1, wherein the fibroblast growth factor is recombinant basic human fibroblast growth factor.

7. The method of claim 1, wherein the medium also contains added stem cell factor (SCF).

8. The method of claim 1, wherein the medium also contains added Flt3 ligand.

9. The method of claim 1, wherein the embryonic stem cell line expresses SSEA-4, telomerase reverse transcriptase (TERT), and OCT-4.

10. The method of claim 1, further comprising culturing and passaging the embryonic stem cell line in the culture environment for at least 40 days so that the cells proliferate without differentiation and while maintaining a normal karyotype.

11. The method of claim 1, wherein cells in the established stem cell line comprise a genetic alteration.

12. The method of claim 11, wherein the cells have been genetically altered by transfecting with a DNA-lipid complex;

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

expanding the selected cells in a feeder-free culture environment that contains an extracellular matrix and fibroblast growth factor.

14. The method of claim 11, wherein the extracellular matrix is made from one or more extracellular matrix components selected from laminin, fibronectin, proteoglycan, and entactin.

15. The method of claim 11, wherein the medium is a fresh medium not previously conditioned by culturing with feeder cells.

16. The method of claim 11, wherein the fibroblast growth factor is recombinant basic human fibroblast growth factor.

17. The method of claim 11, wherein the medium also contains added stem cell factor (SCF).

18. The method of claim 11, wherein the medium also contains added Flt3 ligand.

13. A method for producing a population of differentiated cells, comprising:

a) providing a line of human embryonic stem cells that have been established in culture without using feeder cells;

b) growing undifferentiated embryonic stem cells from said line in a culture environment that contains an extracellular matrix instead of feeder cells in a nutrient medium containing fibroblast growth factor; and

then

c) causing the cells grown in b) to differentiate into the population of differentiated cells.