cDNA libraries reflecting gene expression during growth and differentiation of human pluripotent stem cells

• US 6,667,176 B1
• Filed: 10/10/2000
• Issued: 12/23/2003
• Est. Priority Date: 01/11/2000
• Status: Expired due to Term

• Current Assignee: Asterias Biotherapeutics Incorporated (Lineage Cell Therapeutics, Inc.)
• Original Assignee: Geron Corporation
• Inventors: Inokuma, Margaret S., Carpenter, Melissa K., Gold, Joseph D., Xu, Chunhui, Funk, Walter D.
• Primary Examiner(s): Crouch, Deborah
• Assistant Examiner(s): TON, THAIAN N
• Application Number:
  US09/688,031
• Time in Patent Office: 1,169 Days
• Field of Search: 435/6, 435/320.1, 435/325, 435/455, 435/363, 435/366, 435/377, 536/23.1
• US Class Current: 435/363
• 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 a system for obtaining expression libraries from primate pluripotent stem (pPS) cells. pPS cells can be maintained in vitro without requiring a layer of feeder cells to inhibit differentiation. The role of the feeder cells is replaced by several other culture conditions provided in a suitable combination. Conditions that promote pPS cell growth without differentiation include supporting the culture on an extracellular matrix, and culturing the cells in a medium conditioned by another cell type. The cDNA libraries from such cultures are devoid of transcripts of feeder cell origin, relatively uncontaminated by transcripts from differentiated cells, and can have a high proportion of full-length transcripts. Subtraction libraries can also be produced that are enriched for transcripts modulated during differentiation.

Claims

1. A method of obtaining an mRNA preparation or a cDNA library from undifferentiated primate pluripotent stem (pPS) cells or their differentiated progeny, comprising:

a) providing undifferentiated pPS cells cultured in a suitable nutrient medium on an extracellular matrix but essentially free of feeder cells,

b) optionally permitting the pPS cells to differentiate, and

c) isolating mRNA from the undifferentiated or differentiated cells.

2. The method of claim 1, which is a method for obtaining mRNA from undifferentiated pPS cells, comprising isolating mRNA from pPS cells in a culture essentially free of feeder cells.

3. The method of claim 1, which is a method for obtaining a cDNA library from undifferentiated pPS cells, comprising isolating mRNA from pPS cells in a culture essentially free of feeder cells, and recombining cDNA copies of the mRNA into a cloning vector.

4. The method of claim 3, wherein the cloning vector is an expression vector in which cDNA is operatively linked to a transcriptional regulatory control element that promotes transcription of the cDNA in undifferentiated pPS cells.

5. The method of claim 4, wherein the regulatory control element is a phosphoglycerate kinase (PGK) promoter.

6. The method of claim 1, wherein the culture of pPS cells essentially free of feeder cells has been maintained in a culture environment comprising an extracellular matrix, and a nutrient medium conditioned by culturing feeder cells in the medium, and then harvesting the medium for use in culturing the pPS cells.

7. The method of claim 6, wherein the feeder cells are a euploid human cell line that can be maintained in culture for at least 60 days.

8. The method of claim 1, wherein the pPS cells are human embryonic stem (hES) cells.

9. The method of claim 1, which is a method for obtaining mRNA from differentiated cells, comprising obtaining pPS cells from a culture essentially free of feeder cells, permitting the pPS cells to differentiate, and isolating mRNA from the differentiated cells.

10. The method of claim 1, which is a method for obtaining a cDNA library from differentiated cells, comprising isolating mRNA from cells differentiated from pPS cells obtained from a culture essentially free of feeder cells, and recombining cDNA copies of the mRNA into a cloning vector.

11. The method of claim 1, which is a method for obtaining a cDNA library enriched for transcripts differently expressed upon differentiation of pPS cells, comprising incubating together preparations of mRNA (or cDNA copies thereof) obtained from undifferentiated pPS cells in feeder-free culture, and from cells differentiated from pPS cells, under conditions that permit polynucleotides present from each preparation to cross-hybridize;

and then recombining polynucleotides that have not cross-hybridized into a cloning vector.

12. A cDNA subtraction library enriched for transcripts that alter expression during differentiation, produced according to the method of claim 11.

13. The method of claim 1, which is a method for obtaining a cDNA library enriched for transcripts differently expressed in two differentiated cell types, comprising differentiating pPS cells from feeder-free culture into two differentiated cell types;

incubating together preparations of mRNA (or cDNA copies thereof) obtained from the two cell types under conditions that permit polynucleotides present from each preparation to cross-hybridize; and

then recombining polynucleotides that have not cross-hybridized into a cloning vector.

14. The method of claim 1, which is a method for obtaining a cDNA library enriched for transcripts that undergo altered expression as a consequence of exposure to a compound, comprising incubating together two preparations of mRNA (or cDNA copies thereof) obtained from pPS cells in feeder-free culture under conditions that permit polynucleotides present from each preparation to cross-hybridize, wherein the first preparation is from pPS cells that have been exposed to the compound and the second preparation is from pPS that have not been exposed to the compound;

and then recombining polynucleotides that have not cross-hybridized into a cloning vector.

26. A method for producing a polynucleotide containing a sequence of an mRNA expressed in undifferentiated or differentiated pPS cells, comprising determining nucleotide sequence from an mRNA or cDNA obtained according to claim 1, and manufacturing a polynucleotide containing the determined sequence, wherein the mRNA is upregulated or downregulated when pPS cells differentiate.

15. A cDNA library of undifferentiated primate pluripotent stem (pPS) cells, which can be obtained by providing undifferentiated pPS cells cultured in a suitable nutrient medium on an extracellular matrix but essentially free of feeder cells, and isolating mRNA from the undifferentiated cells.

16. A host cell containing the cDNA library of claim 15.

25. A method for producing a polynucleotide containing a sequence of an mRNA expressed in undifferentiated or differentiated pPS cells, comprising:

a) obtaining the cDNA library of claim 15,

b) determining nucleotide sequence from a cDNA contained in the library; and

c) manufacturing a polynucleotide containing the determined sequence.

17. A cDNA library comprising cDNA from at least 1,000 genes expressed at the mRNA level, wherein the library has been obtained from undifferentiated pPS cells, and is essentially free of cDNA of other vertebrates.

18. The cDNA library of claim 17, cloned in an expression vector in which cDNA is operatively linked to a transcriptional regulatory control element that promotes transcription of the cDNA in undifferentiated pPS cells.

19. The cDNA library of claim 18, wherein the regulatory control element is a phosphoglycerate kinase (PGK) promoter.

20. The DNA library of claim 17, wherein at least 30% of cDNA segments in the library comprise the entire encoding region of the corresponding mRNA.

21. The cDNA library of claim 17, wherein the pPS cells are human embryionic stem (hES) cells. kinase (PGK) promoter.

22. A host cell containing the cDNA library of claim 17.

27. A method for producing a polynucleotide containing a sequence of an mRNA expressed in undifferentiated pPS cells, comprising determining nucleotide sequence of a cDNA in the cDNA library of claim 17, and manufacturing a polynucleotide containing the determined sequence.

23. A cDNA library of at least 1,000 genes in which at least 50% of the cDNA is from genes that are modulated during differentiation of pPS cells, essentially free of cDNA of other vertebrates.

24. The cDNA library of claim 23 enriched for transcripts that alter expression while forming embryoid bodies from hES cells.