System for expanding and differentiating human embryonic stem cells

• US 20050153445A1
• 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:
  US11/010,140
• Publication Number:
  US 20050153445A1
• 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 obtaining a population of differentiated 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 cells from the colonies onto an extracellular matrix in a culture environment that is essentially free of feeder cells;

d) culturing the passaged cells on the extracellular matrix in a nutrient medium containing fibroblast growth factor; and

e) differentiating the cultured cells into a population comprising lineage restricted cells or terminally differentiated cells.

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, further comprising genetically altering the cells before they are differentiated.

10. The method of claim 9, wherein the cells are genetically altered by transfecting cells in the population with a DNA-lipid complex;

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

11. The method of claim 1, wherein the cells are differentiated by culturing the cells in the absence of serum or serum replacement.

12. The method of claim 1, wherein the cells are differentiated by adding retinoic acid, butyrate, DMSO, or a growth factor to the growth environment.

13. The method of claim 1, wherein the cells are differentiated by culturing on a solid surface that promotes differentiation.

14. The method of claim 1, wherein the cells are differentiated by a process that comprises forming embryoid bodies.

15. The method of claim 1, wherein the cells are differentiated into neural precursor cells, neuronal cells or glial cells.

16. The method of claim 1, wherein the cells are differentiated into cardiomyocytes.

17. The method of claim 1, wherein the cells are differentiated into hepatocytes.

18. The method of claim 1, wherein the population obtained is at least about 80% lineage restricted cells or terminally differentiated cells.

19. The method of claim 1, wherein the population obtained is at least about 50% neuronal cells.

20. The method of claim 10, wherein the population obtained is at least about 50% cardiomyocyte lineage cells.