Induction of motor neurons from pluripotent stem cells

• US 10,519,421 B2
• Filed: 03/24/2014
• Issued: 12/31/2019
• Est. Priority Date: 03/21/2013
• Status: Active Grant

• Current Assignee: Kyoto University
• Original Assignee: Kyoto University
• Inventors: Inoue, Haruhisa, Imamura, Keiko, Kondo, Takayuki
• Primary Examiner(s): Ton, Thaian N
• Application Number:
  US14/778,670
• Publication Number:
  US 20160046905A1
• Time in Patent Office: 2,108 Days
• CPC Class Codes: A61P 21/02 Muscle relaxants, e.g. for tetanus or cramps
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2500/42 Organic phosphate, e.g. beta glycerophosphate
• 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 2501/39 Steroid hormones
• CPC Class Codes: C12N 2501/392 Sexual steroids
• CPC Class Codes: C12N 2501/41 Hedgehog proteins; Cyclopamine (inhibitor)
• CPC Class Codes: C12N 2501/60 Transcription factors
• CPC Class Codes: C12N 2503/02 Drug screening
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2506/115 from monocytes, from macrophages
• CPC Class Codes: C12N 2506/45 from artificially induced pluripotent stem cells
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• 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 5/0641 Erythrocytes
• CPC Class Codes: C12N 5/0645 Macrophages, e.g. Kuepfer cells in the liver; Monocytes
• CPC Class Codes: G01N 2500/04 : Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
• CPC Class Codes: G01N 2500/10 : involving cells
• CPC Class Codes: G01N 2800/2835 : Movement disorders, e.g. Parkinson, Huntington, Tourette
• CPC Class Codes: G01N 2800/52 : Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
• CPC Class Codes: G01N 33/5073 : Stem cells
• CPC Class Codes: G01N 33/56966 : Animal cells

Abstract

The present invention aims to provide a method of producing motor neurons/neurons that sufficiently reproduce intrinsic properties of motor neurons/neurons, especially of the motor neurons/neurons in patients, from pluripotent stem cells promptly and synchronically, and a pluripotent stem cell capable of differentiating into a neuron or a motor neuron promptly and synchronically after a drug treatment.

A method of generating a motor neuron from a pluripotent stem cell, comprising the following steps in order from (1) to (2):

(1) introducing one or more nucleic acids encoding Lhx3, Ngn2, and Isl1 into a pluripotent stem cell; and

(2) maintaining expression of the Lhx3, Ngn2, and Isl1 for three days or more.

Claims

1. A method of generating a motor neuron from a pluripotent stem cell in vitro, comprising in order from (1) to (3):

(1) introducing a vector comprising an inducible promoter operably linked to Lhx3, Ngn2, and Isl1 genes into the pluripotent stem cell;

(2) inducing expression of the Lhx3, Ngn2, and Isl1 genes in the pluripotent stem cell from (1) in KSR media or N3 media containing a drug capable of inducing an activation of the promoter; and

(3) culturing the pluripotent stem cell from (2) and maintaining expression of the Lhx3, Ngn2, and Isl1 genes for three days or more in said cell, thereby producing a motor neuron.

2. The method according to claim 1, wherein Lhx3, Ngn2, and Isl1 genes are polycistronically expressed from the one or more nucleic acids.

3. The method according to claim 1, wherein expression of the Lhx3, Ngn2, and Isl1 genes is maintained for 7 days or more in step (2).

4. The method according to claim 1, wherein said pluripotent stem cell is an induced pluripotent stem cell derived from a human.

5. The method according to claim 1, wherein said pluripotent stem cell has one or more mutant SOD1 genes.

6. The method according to claim 1, wherein in step (2), expression of the Lhx3, Ngn2, and Isl1 genes in the pluripotent stem cell from (1) is induced in N3 media containing the drug capable of inducing an activation of the promoter.

7. The method according to claim 1, wherein in step (2), expression of the Lhx3, Ngn2, and Isl1 genes in the pluripotent stem cell from (1) is induced in KSR media containing the drug capable of inducing an activation of the promoter.