Techniques for growth and differentiation of human pluripotent stem cells

• US 20020090723A1
• Filed: 11/26/2001
• Published: 07/11/2002
• 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: Inokuma, Margaret S., Xu, Chunhui, Carpenter, Melissa K.
• Application Number:
  US09/994,440
• Publication Number:
  US 20020090723A1
• 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
• 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 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 into a culture environment that is essentially free of feeder cells;

d) culturing the passaged cells in the culture environment that is essentially free of feeder cells; 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 cells are differentiated by withdrawing serum, serum replacement, or a growth factor from the growth environment.

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

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

5. The method of claim 1, wherein the cells are differentiated through the formation of embryoid bodies.

6. The method of claim 1, wherein the cells are differentiated without forming embryoid bodies.

7. The method of claim 1, wherein the cells are differentiated into neural precursor cells.

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

9. The method of claim 1, wherein the cells are differentiated into fibroblasts.

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

11. The method of claim 1, wherein the population obtained is at least ˜

80% lineage restricted cells or terminally differentiated cells.

12. The method of claim 7, wherein the population obtained is at least ˜

50% neural cells.

13. The method of claim 8, wherein the population obtained is at least 50% neuronal cells.

14. The method of claim 9, wherein the population obtained is at least ˜

50% fibroblasts.

15. The method of claim 10, wherein the population obtained is at least ˜

50% hepatocytes.