Pluripotent cell lines and methods of use thereof

• US 10,233,422 B2
• Filed: 08/31/2016
• Issued: 03/19/2019
• Est. Priority Date: 06/24/2008
• Status: Active Grant

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.), Tyler Medical Research LLC
• Original Assignee: Parkinson's Institute, Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Inventors: Reijo Pera, Renee Ann, Langston, J. William, Schule, Birgitt, Palmer, Theodore D., Byers, Blake, Nguyen, Ha Nam, Byrne, James Anthony, Cord, Branden John
• Primary Examiner(s): Long, Scott
• Application Number:
  US15/253,737
• Publication Number:
  US 20170114324A1
• Time in Patent Office: 930 Days
• 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 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2500/46 Amines, e.g. putrescine
• CPC Class Codes: C12N 2501/01 Modulators of cAMP or cGMP, e.g. non-hydrolysable analogs, phosphodiesterase inhibitors, cholera toxin
• 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 2501/13 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 2501/15 Transforming growth factor beta (TGF-β)
• CPC Class Codes: C12N 2501/392 Sexual steroids
• CPC Class Codes: C12N 2501/41 Hedgehog proteins; Cyclopamine (inhibitor)
• CPC Class Codes: C12N 2501/50 Cell markers; Cell surface determinants
• CPC Class Codes: C12N 2501/602 Sox-2
• CPC Class Codes: C12N 2501/603 Oct-3/4
• CPC Class Codes: C12N 2501/604 Klf-4
• CPC Class Codes: C12N 2501/70 Enzymes
• CPC Class Codes: C12N 2501/71 Oxidoreductases (EC 1.)
• CPC Class Codes: C12N 2503/02 Drug screening
• CPC Class Codes: C12N 2506/1307 from adult fibroblasts
• CPC Class Codes: C12N 2506/45 from artificially induced pluripotent stem cells
• CPC Class Codes: C12N 2740/15032 Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
• CPC Class Codes: C12N 5/0619 : Neurons
• CPC Class Codes: C12N 7/00 : Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00
• CPC Class Codes: C12Q 1/6883 : for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2600/136 : Screening for pharmacological compounds
• CPC Class Codes: G01N 2333/47 : Assays involving proteins of known structure or function as defined in the subgroups
• CPC Class Codes: G01N 33/5023 : on expression patterns
• CPC Class Codes: G01N 33/5058 : Neurological cells

Abstract

Methods of generating cell lines with a sequence variation or copy number variation of a gene of interest, methods of use thereof, and cell lines with a sequence variation or copy number variation of a gene of interest are provided.

Claims

1. A neural cell line prepared from a subject with Parkinson'"'"'s Disease or a Parkinson'"'"'s-like disease, wherein cells of the neural cell line are capable of proliferating in vitro, the neural cell line produced by:

a) obtaining somatic cells from the subject with Parkinson'"'"'s Disease or the Parkinson'"'"'s-like disease, wherein the subject carries a genetic mutation which causes Parkinson'"'"'s Disease or the Parkinson'"'"'s-like disease;

b) de-differentiating the somatic cells, wherein the dedifferentiating comprises expressing at least one differentiating factor selected from (i) Sox2, (ii) Oct3 or Oct4, and (iii) Klf4, in the somatic cell, thereby producing induced pluripotent stem cells; and

c) differentiating the induced pluripotent stem cells towards a neural cell fate, thereby producing differentiated induced pluripotent stem cells, and

d) selecting and growing at least a portion of the differentiated induced pluripotent stem cells that express a neural cell marker and display a sign of pathology related to the neurodegenerative disorder, thereby producing the cell line.

2. The neural cell line of claim 1, wherein differentiating comprises contacting the induced pluripotent stem cells with at least one differentiating agent selected from fibroblast growth factor 8, sonic hedgehog, transforming growth factor P3, brain derived neurotrophic factor and glial derived neurotrophic factor.

3. The neural cell line of claim 1, wherein the neural cell marker is selected from Map2, type III beta tubulin, doublecortin, NeuN, glial fibrillary acidic protein, S100-beta, NG2, GalC, tyrosine hydroxylase, aromatic amino acid decarboxylase, aromatic amino acid decarboxylase, glutamate decarboxylase, glutamate transporter, and dopamine beta hydroxylase, and combinations thereof.

4. The neural cell line of claim 1, wherein the sign of pathology is a protein aggregate or a cellular aggregate.

5. The neural cell line of claim 4, wherein the protein aggregate comprises alpha synuclein.

6. The neural cell line of claim 1, wherein the sign of pathology is selected from apoptosis, necrosis, oxidative stress, and mitochondrial dysfunction.

7. The method of claim 6, wherein mitochondrial dysfunction is characterized by increased production of glutathione, reactive oxygen species, or 4-hydroxy-2-nonenal, as compared to healthy controls.

8. The neural cell line of claim 1, wherein the de-differentiating comprises transfecting the somatic cells with at least one nucleic acid encoding Sox2, Oct4, and Klf4.

9. The neural cell line of claim 1, wherein the de-differentiating comprises infecting the somatic cells with a virus comprising at least one nucleic acid encoding Sox2, Oct4, and Klf4.

10. The neural cell line of claim 9, wherein the virus is a retrovirus.

11. The neural cell line of claim 9, wherein the virus is a lentivirus, and the at least one nucleic acid is integrated in a DNA molecule of the neural cell line.

12. The neural cell line of claim 1, wherein the de-differentiating comprises incubating the somatic cells with Sox2 protein, Oct 3 protein or Oct4 protein, and Klf4 protein.

13. The neural cell line of claim 1, wherein the somatic cells comprise at least one cell selected from a neuroectodermal cell, neuronal cell, neuroendocrine cell, dopaminergic cell, cholinergic cell, serotonergic (5-HT) cell, glutamatergic cell, GABAergic cell, adrenergic cell, noradrenergic cell, sympathetic neuronal cell, parasympathetic neuronal cell, sympathetic peripheral neuronal cell, microglial cell, astrocyte, oligodendrocyte, ependymal cells, radial glial cell;

Schwann cell, and satellite cell.

14. The neural cell line of claim 1, wherein the somatic cells are selected from neuronal cells and glial cells.

15. The neural cell line of claim 1, wherein the somatic cells are selected from autonomic cells and sympathetic cells.

16. The neural cell line of claim 1, wherein the somatic cells are selected from dopaminergic neurons, serotonergic neurons, cholinergic neurons, GABAergic neurons, glutamatergic neurons, and peptidergic, neurons.

17. The neural cell line of claim 1, wherein the dedifferentiating comprises expressing (i) Sox2, (ii) Oct3 or Oct4, and (iii) Klf4, in the somatic cell.

18. The neural cell line of claim 1, wherein the somatic cells are dopaminergic neurons.

19. The neural cell line of claim 1, wherein the subject has Parkinson'"'"'s Disease.