Direct differentiation of human pluripotent stem cells and characterization of differentiated cells

• US 20020019046A1
• Filed: 06/21/2001
• Published: 02/14/2002
• 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: Thies, R. Scott, Carpenter, Melissa K., Funk, Walter D.
• Application Number:
  US09/888,309
• Publication Number:
  US 20020019046A1
• Time in Patent Office: Days
• US Class Current: 435/368
• 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 invention provides a system for efficiently producing differentiated cells from pluripotent cells, such as human embryonic stem cells. Rather than permitting the cells to form embryoid bodies according to established techniques, differentiation is effected directly in monolayer culture on a suitable solid surface. The cells are either plated directly onto a differentiation-promoting surface, or grown initially on the solid surface in the absence of feeder cells and then exchanged into a medium that assists in the differentiation process. The solid surface and the culture medium can be chosen to direct differentiation down a particular pathway, generating a cell population that is remarkably uniform. The methodology is well adapted to bulk production of committed precursor and terminally differentiated cells for use in drug screening or regenerative medicine.

Claims

1. A method for producing differentiated cells from a donor culture of undifferentiated primate pluripotent stem (pPS) cells, comprising:

a) preparing a suspension of pPS cells from the undifferentiated donor culture;

b) replating and culturing the suspended cells on a solid surface so that they differentiate without forming embryoid bodies; and

c) harvesting differentiated cells from the solid surface.

3. The method of claim 1, wherein the donor culture comprises both undifferentiated pPS cells and feeder cells, which are replated onto a solid surface without adding fresh feeder cells.

4. The method of claim 1, wherein the donor culture is essentially free of feeder cells, which are replated on a solid surface without any extracellular matrix.

5. The method of claim 1, wherein the solid surface bears a polycation.

6. The method of claim 5, wherein the polycation is polyomithine.

7. The method of claim 1, wherein the cells are cultured after replating in a medium containing a factor that promotes differentiation.

8. The method of claim 7, wherein the factor is Brain Derived Neurotrophic Factor (BDNF) or Neutrotrophin-3 (NT-3).

12. The method of claim 1, whereby cells cultured on the solid surface differentiate to precursor cells committed to a restricted cell lineage and capable of proliferation.

16. The method of claim 1, whereby cells cultured on the solid surface become fully differentiated cells.

21. A method of screening a compound for cellular toxicity or modulation, comprising combining the compound with a committed or differentiated cell prepared according to the method of claim 1, determining any phenotypic or metabolic changes in the cell that result from contact with the compound, and correlating the change with cellular toxicity or modulation.

22. A method for obtaining a polynucleotide comprising a nucleotide sequence contained in an mRNA that is expressed at a different level in committed or differentiated cells prepared according to the method of claim 1, compared with undifferentiated primate pluripotent stem (pPS) cells, the method comprising:

a) determining the level of expression of a plurality of mRNAs in committed or differentiated cells, in comparison to the level of expression of the same mRNAs in undifferentiated pPS cells;

b) identifying an mRNA expressed at a different level in the committed or differentiated cells, relative to the undifferentiated pPS cells; and

c) preparing a polynucleotide comprising a nucleotide sequence of at least 30 consecutive nucleotides contained in the identified mRNA.

2. A method for obtaining differentiated cells from a donor culture of undifferentiated primate pluripotent stem (pPS) cells, comprising:

a) culturing the pPS cells on a solid surface in an environment essentially free of feeder cells;

b) changing medium used to culture the cells so that they differentiate before there is overgrowth or formation of colonies; and

c) harvesting differentiated cells from the solid surface.

9. The method of claim 2, wherein the changed medium is essentially free of fibroblast growth factor.

10. The method of claim 2, wherein the changed medium contains Brain Derived Neurotrophic Factor (BDNF) or Neutrotrophin-3 (NT-3).

11. The method of claim 2, wherein the changed medium contains noggin or follistatin.

13. The method of claim 12, wherein the precursor cells are ectodermal cells.

14. The method of claim 13, wherein the precursor cells are committed to the neuroectoderm lineage.

15. The method of claim 14, wherein the precursor cells are cells of the mesoderm, endoderm or visceral endoderm.

17. The method of claim 16, wherein the fully differentiated cells are neurons or glial cells.

18. The method of claim 17, wherein at least ˜

10% of the cells staining positive for MAP-2 are also positive for tyrosine hydroxylase.

19. Committed precursor cells prepared according to the method of claim 12.

20. Fully differentiated cells prepared according to the method of claim 16.