Method for inducing dopaminergic neuron progenitor cells

Patent metadata

Field	Value
Publication number	US-2023114089-A1
Application number	US-202217824603-A
Country	US
Kind code	A1
Filing date	May 25, 2022
Priority date	Sep 5, 2013
Publication date	Apr 13, 2023
Grant date	—

Abstract

The present invention provides a method for producing dopaminergic neuron progenitor cells from pluripotent stem cells, which method comprises the steps of: (i) performing adherent culture of pluripotent stem cells on an extracellular matrix in a medium containing a reagent(s) selected from the group consisting of BMP inhibitor, TGFβ inhibitor, SHH signal-stimulating agent, FGF8, and GSK3β inhibitor; (ii) collecting Corin- and/or Lrtm1-positive cells from the cells obtained in Step (i) using a substance which binds to Corin and/or a substance which binds to Lrtm1; and (iii) performing suspension culture of the cells obtained in Step (ii) in a medium containing a neurotrophic factor.

First claim

1 .- 16 . (canceled)

17 . A method for producing dopaminergic neuron progenitor cells from pluripotent stem cells, said method comprising the steps of:
(i) culturing pluripotent stem cells on an extracellular matrix in a medium containing a reagent(s) selected from the group consisting of BMP inhibitor, TGFβ inhibitor, SHH signal-stimulating agent, FGF8, and GSK3β inhibitor during a period enough for appearance of Corin- and/or leucine-rich repeats and transmembrane domains 1(Lrtm1) positive cells; (ii) collecting Corin- and/or Lrtm1-positive cells from the cells obtained in Step (i) using a substance which binds to Corin and/or a substance which binds to Lrtm1; and (iii) culturing the cells obtained in Step (ii) in suspension in a medium containing a neurotrophic factor, wherein Step (iii) is carried out for at least 7 days.

18 . The method according to claim 17 , wherein Step (i) comprises the steps of:
(a) culturing pluripotent stem cells on an extracellular matrix in a medium containing a BMP inhibitor and a TGFβ inhibitor for at least one day; (b) replacing the medium from Step (a) with a medium containing a BMP inhibitor, a TGFβ inhibitor, a SHH signal-stimulating agent, and FGF8, and further culturing the cells for at least one day; (c) replacing the medium from Step (b) with a medium containing a BMP inhibitor, a TGFβ inhibitor, a SHH signal-stimulating agent, FGF8, and a GSK3β inhibitor and further culturing the cells for at least one day; (d) replacing the medium from Step (c) with a medium containing a BMP inhibitor and a GSK3β inhibitor and further culturing the cells for at least one day.

19 . The method according to claim 17 , wherein the BMP inhibitor is selected from a group consisting of Chordin, Noggin, Follistatin, Dorsomorphin and LDN193189.

20 . The method according to claim 17 , wherein the TGFβ inhibitor is selected from a group consisting of Lefty-1, SB431542, SB202190, SB505124, NPC30345, SD093, SD908 and SD208, LY2109761, LY364947, LY580276 and A83-01.

21 . The method according to claim 17 , wherein the SHH signal-stimulating agent is selected from a group consisting of SHH, Hh-Ag1.5, SAG, 20ahydroxycholesterol and purmorphamine.

22 . The method according to claim 17 , wherein the GSK3β inhibitor is selected from a group consisting of BIO, 6-bromoindirubin-3′-oxime), SB216763, GSK-3β inhibitor VII, L803-mts and CHIR99021.

23 . The method according to claim 17 , wherein the BMP inhibitor is LDN193189 or Dorsomorphin; the TGFβ inhibitor is SB-431542 or A83-01; the SHH signal-stimulating agent is purmorphamine or SAG; and the GSK3β inhibitor is BIO or CHIR99021.

24 . The method according to claim 17 , wherein the neurotrophic factor comprises BDNF and GDNF.

25 . The method according to claim 17 , wherein the medium in Step (iii) further comprises B27 supplement, ascorbic acid, and dibutyryl cyclic AMP.

26 . The method according to claim 17 , wherein the Step (i) is carried out for at least 10 days.

27 . The method according to claim 17 , wherein the Step (i) is carried out for 12 days to 21 days.

28 . The method according to claim 17 , wherein the Step (iii) is carried out for 14 days to 30 days.

29 . The method according to claim 17 , wherein the extracellular matrix is laminin or a fragment thereof.

30 . The method according to claim 17 , wherein the extracellular matrix is laminin 511E8.

31 . The method according to claim 17 , wherein the substance which binds to Corin or the substance which binds to Lrtm1 is an antibody or an aptamer which binds to Corin or Lrtm1.

32 . A cell aggregate comprising dopaminergic neuron progenitor cells, wherein the cell aggregate,
(1) comprises about 70% or more of Foxa2 positive cells, (2) has high expression of ALCAM, SHH, WNT5A and CORIN and low expression of PAX6, (3) can be matured into the cell aggregate which comprises
(i) about 37% or more of TH positive cells, and
(ii) Foxa2, Nurrr1, Pitx3 and Girk2 positive cells; and
which shows a property wherein the quantification of dopamine and 3, 4-dihydroxyphenyl acetic acid released due to stimulation by high potassium chloride is greater than that of serotonin.

33 . The cell aggregate according to claim 32 , wherein the cell aggregate can be administered by 22-gauge needle.

34 . A method of treating Parkinson's disease, comprising a step of administering the cell aggregate described in claim 32 to a patient in need thereof.

35 . A method of treating Parkinson's disease, comprising steps:
(1) preparing a cell aggregate according to claim 32 , (2) transplanting the cell aggregate obtained by Step (1) into a brain of a patient suffering from Parkinson's disease.

36 . The method according to claim 35 , wherein the Step (1) comprising the following steps:
(i) culturing pluripotent stem cells on an extracellular matrix in a medium containing a reagent(s) selected from the group consisting of BMP inhibitor, TGFβ inhibitor, SHE signal-stimulating agent, FGF8, and GSK3β inhibitor during a period enough for appearance of Corin- and/or leucine-rich repeats and transmembrane domains 1(Lrtm1) positive cells; (ii) collecting Corin- and/or Lrtm1-positive cells from the cells obtained in Step (i) using a substance which binds to Corin and/or a substance which binds to Lrtm1; and (iii) culturing the cells obtained in Step (ii) in suspension in a medium containing a neurotrophic factor, wherein Step (iii) is carried out for at least 7 days.

Assignees

• Univ Kyoto
• Univ Osaka
• Eisai R&D Man Co Ltd

Inventors

• Takahashi Jun
• Doi Daisuke
• Samata Bumpei
• Sekiguchi Kiyotoshi
• Ono Yuichi

Classifications

• C12N2501/727

  Kinases (EC 2.7.) · CPC title

• C12N2501/41

  Hedgehog proteins; Cyclopamine (inhibitor) · CPC title

• A61K35/30 Primary

  Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue · CPC title

• C12N2501/119

  Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10 · CPC title

• C12N5/0623 Primary

  Stem cells · CPC title