Method for producing dopaminergic neuron progenitor cell

Patent metadata

Field	Value
Publication number	US-12516286-B2
Application number	US-202318539589-A
Country	US
Kind code	B2
Filing date	Dec 14, 2023
Priority date	Apr 22, 2016
Publication date	Jan 6, 2026
Grant date	Jan 6, 2026

Abstract

A cell population comprising Corin- and/or Lrtm1-positive cells was produced by the following steps (1) and (2), from which Corin positive and/or Lrtm1 positive cells are collected using a substance that binds to Corin and/or a substance that binds to Lrtm1, and dopaminergic neuron progenitor cells are produced by performing suspension culture of the Corin positive and/or Lrtm1 positive cells in a culture solution containing one or more nutritional factors: (1) a step of performing adhesion culture of pluripotent stem cells in a medium for maintaining undifferentiated state containing a Sonic hedgehog (SHH) signal stimulant, and an undifferentiated state-maintaining factor in the absence of feeder cells but in the presence of an extracellular matrix, and(2) a step of culturing the cell population obtained in the step (1) in a culture solution containing one or more differentiation-inducing factors.

First claim

What is claimed is:

1 . A method for producing a cell population comprising Corin positive and/or Lrtm1 positive cells, said method comprising
(i) culturing human pluripotent stem cells on an extracellular matrix coated plate in the absence of feeder cells in a culture medium supplemented with a Sonic hedgehog (SHH) signal stimulating agent and an FGF signal transduction pathway agonist for a period of 18 to 48 hours to maintain the stem cells in an undifferentiated state, wherein the extracellular matrix is a laminin, (ii) culturing pluripotent stem cells obtained in step (i) on the laminin in a medium comprising TGF beta inhibitor and BMP inhibitor: (iii) culturing the cells obtained in step (ii) on the laminin in a medium comprising TGF beta inhibitor, BMP inhibitor, SHH signal stimulating agent and FGF8 for at least one day; (iv) culturing the cells obtained in (iii) on the laminin in a medium comprising TGF beta inhibitor, BMP inhibitor, SHH signal-stimulating agent, FGF8 and GSK3b inhibitor for at least one day; (v) culturing the cells obtained in (iv) on the laminin in a medium comprising BMP inhibitor and GSK3 beta inhibitor; (vi) collecting Corin- and/or leucine-rich repeats and transmembrane domains 1(Lrtm1)-positive cells from the cells obtained in step (v) using an antibody or aptamer which binds to Corin and/or an antibody or aptamer which binds to Lrtm1 to obtain a cell population comprising Corin positive and/or Lrtm1 positive cells.

2 . The method according to claim 1 , wherein the SHH signal stimulating agent is SAG (N-methyl-N′-(3-pyridinylbenzyl)-N′-(3-chlorobenzo[b]thiophene-2-carbonyl)-1,4-diaminocyclohexane), shh protein or a fragment thereof, Purmorphamine, or a combination thereof.

3 . The method according to claim 1 , wherein the medium for maintaining undifferentiated state in (i) further comprises a TGFβ inhibitor or a BMP inhibitor.

4 . The method according to claim 3 , wherein the TGFβ inhibitor is selected from a group consisting of A83-01, Lefty-1, Lefty-2, SB431542, SB202190, SB505124, NPC30345, SD093, SD908, SD208, LY2109761, LY364947, and LY570276 and the BMP inhibitor is selected from a group consisting of LDN 193189, Chordin, Noggin, Follistatin, and Dorsomorphin and a derivative thereof.

5 . The method according to claim 1 , wherein the FGF signal transduction pathway agonist is bFGF.

6 . The method according to claim 1 , wherein the medium in (ii) further comprises a ROCK inhibitor.

7 . The method according to claim 1 , wherein the percentage of Corin positive and/or Lrtm1 positive cells in the cell population is 10% or more.

8 . The method according to claim 1 , wherein the laminin is selected form a group consisting of laminin 511, laminin 511 E8, and laminin 111.

9 . A method for producing dopaminergic neuron progenitor cells comprising
a cell population comprising Corin positive and/or Lrtm1 positive cells, said method comprising
(i) culturing human pluripotent stem cells on an extracellular matrix coated plate in the absence of feeder cells in a culture medium supplemented with a Sonic hedgehog (SHH) signal stimulating agent and an FGF signal transduction pathway agonist for a period of 18 to 48 hours to maintain the stem cells in an undifferentiated state, wherein the extracellular matrix is a laminin,
(ii) culturing pluripotent stem cells obtained in step (i) on the laminin in a medium comprising TGF beta inhibitor and BMP inhibitor:
(iii) culturing the cells obtained in step (ii) on the laminin in a medium comprising TGF beta inhibitor, BMP inhibitor, SHH signal stimulating agent and FGF8 for at least one day;
(iv) culturing the cells obtained in (iii) on the laminin in a medium comprising TGF beta inhibitor, BMP inhibitor, SHH signal stimulating agent, FGF8 and GSK3 beta inhibitor for at least one day;
(v) culturing the cells obtained in (iv) on the laminin in a medium comprising BMP inhibitor and GSK3 beta inhibitor;
(vi) collecting Corin- and/or leucine-rich repeats and transmembrane domains 1 (Lrtm1)-positive cells from the cells obtained in step (v) using an antibody or aptamer which binds to Corin and/or an antibody or aptamer which binds to Lrtm1 to obtain a cell population comprising Corin positive and/or Lrtm1 positive cells, and
(vii) performing suspension culture of the cell population comprising Corin positive and/or Lrtm1 positive cells in a medium comprising one or more neurotrophic factors to produce dopaminergic neuron progenitor cells.

10 . The method according to claim 9 , wherein the substance that binds to Corin or the substance that binds to Lrtm1 is an antibody or aptamer that binds to Corin or Lrtm1.

11 . The method according to claim 9 , wherein the neurotrophic factors are BDNF and GDNF.

12 . The method according to claim 9 , wherein the medium comprising a neurotrophic factor further comprises ascorbic acid, and a cAMP analog.

13 . The method according to claim 12 , wherein the CAMP analogue is Dibutyryl cyclic AMP.

14 . The method according to claim 9 , wherein the suspension culture in a medium comprising a neurotrophic factor is performed for at least 7 days.

15 . The method according to claim 9 , wherein the laminin is selected form a group consisting of laminin 511, laminin 511 E8, and laminin 111.

Assignees

• Univ Kyoto
• Racthera Co Ltd

Inventors

• Takahashi Jun
• Doi Daisuke
• Yoshida Kenji
• Kuwahara Atsushi
• Takahashi Masayo

Classifications

• C12N2533/90

  Substrates of biological origin, e.g. extracellular matrix, decellularised tissue · CPC title

• C12N2506/45

  from artificially induced pluripotent stem cells · CPC title

• C12N2501/999

  Small molecules not provided for elsewhere · CPC title

• C12N2501/13

  Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins · CPC title

• C12N2501/115

  Basic fibroblast growth factor (bFGF, FGF-2) · CPC title