Eisai R&d Management Co., Ltd.

Kyoto University

Osaka University

Eisai Company, Limited

Yuichi Ono

Yasuko Nakagawa

Yoshimasa Sakamoto

Tomoya Nakatani

Toshio Imai

Yoshimi Takai

Eri Mizuhara

Jun Takahashi

Miyuki Nishimura

Masakazu Takeuchi

Daisuke Doi

Bumpei Samata

Mayumi Asano

Koji Morimoto

Yoko Inoue

Eiji Inoue

Kiyotoshi Sekiguchi

Norimasa Miyamoto

Shunsuke Ito

Akio Yamada

Aki Nakatani

Toshifumi Hirayama

Kana Namiki

Yoshitoshi Kasuya

Ryota Taguchi

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, TGF&#x3b2; inhibitor, SHH signal-stimulating agent, FGF8, and GSK3&#x3b2; 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.

Method for inducing dopaminergic neuron progenitor cells

It is intended to provide an anti-EphA4 antibody or an EphA4-binding fragment thereof which is capable of binding to EphA4 and inhibiting the binding between EphA4 and its ligand, and a pharmaceutical composition comprising the anti-EphA4 antibody or the EphA4-binding fragment thereof as an active ingredient. A mouse anti-EphA4 antibody having binding affinity for EphA4 was obtained, and the sequences of complementarity-determining regions (CDRs) of the mouse anti-EphA4 antibody were identified. This allowed for preparation of a humanized antibody comprising the CDR sequences of the mouse anti-EphA4 antibody in heavy chain variable region and light chain variable region.

Anti-EphA4 antibody

The present invention relates to polynucleotide probes and antibodies for detecting Lrp4/Corin dopaminergic neuron progenitor cell markers, which enable the efficient separation of dopaminergic neuron progenitor cells; and methods for selecting the progenitor cells by the use thereof.

Lrp4/corin dopamine-producing neuron precursor cell marker

The present invention provides a neural stem cell having increased passage ability and a method for manufacturing a neural stem cell having increased passage ability. A neural stem cell in which the N-type calcium channel gene is knocked out or the influx of Ca2+ via the N-type calcium channel is substantially absent can be passaged for at least 4 generations and maintains the differentiation potential into a nerve cell even after passage for 4 generations.

Neural stem cell having increased passage ability, method for manufacturing neural stem cell having said increased passage abiility, and method for culturing neural stem cells for increasing passage ability of neural stem cells

The present invention provides a method for selecting dopaminergic neuron progenitor cells, which comprises detecting any one or more of markers selected from the group consisting of CD15 (SSEA-1), CD24, CD46, CD47, CD49b, CD57, CD58, CD59, CD81, CD90, CD98, CD147, CD184, Disalogangliosid GD2, SSEA-4, CD49f, SERINC4, CCR9, PHEX, TMPRSS11E, HTR1E, SLC25A2, Ctxn3, Ccl7, Chrnb4, Chrna3, Kcnv2, Grm2, Syt2, Lim2, Mboat1, St3gal6, Slc39a12, Tacr1, Lrtm1, Dscam and CD201.

Markers for dopaminergic neuron progenitor cells

The present invention provides markers that can selectively distinguish pancreatic progenitor cells. The present invention also provides methods for distinguishing pancreatic progenitor cells by using the markers as an indicator, and reagents to be used in the methods. The present inventors successfully identified the surface marker Nephrin-like 3 (Neph3) which is specifically expressed in pancreatic progenitor cells, and isolated pancreatic progenitor cells using the marker as an indicator or such. Viable pancreatic progenitor cells can be selected by using Neph3 as an indicator without using translated products and transcripts of any foreign genes. The marker is useful in preparing and identifying pancreatic progenitor cells which are applied to regenerative medicine or such for treatment of pancreatic diseases.

Method for obtaining pancreatic progenitor cell using NEPH3

The present inventors identified a selective marker 65B13 for GABA neuron progenitor cells of the spinal dorsal horn and cerebellum, and successfully isolated GABA neuron progenitor cells using antibodies that bind to a protein encoded by the gene. 65B13 was demonstrated to be useful as a marker to isolate GABA-producing neuron progenitor cells in the spinal dorsal horn and cerebellum. GABA neuron progenitor cells can be efficiently identified or isolated by using the identified marker as an indicator.

GABA neuron progenitor cell marker 65B13

An object of the present invention is to provide a probe, a primer, a primer set and an antibody for use in the detection or selection of a dopaminergic neuron progenitor cell. The present invention provides a probe, a primer and a primer set for use in the detection or selection of a mesencephalon dopaminergic neuron progenitor cell, and preferably a dopaminergic neuron proliferative progenitor cell, which can hybridize with a nucleotide sequence of a 187A5 gene, or a complementary sequence thereto, and an antibody for use in the detection or selection of a mesencephalon dopaminergic neuron progenitor cell, and preferably a dopaminergic neuron progenitor cell, which is capable of binding to a 187A5 protein.

Dopaminergic neuron progenitor cell marker 187A5

The present invention identified Lmx1a genes, which are expressed in dopaminergic neurons at all differentiation stages, from proliferating dopaminergic neuron progenitor cells before cell cycle exit to cells after cell cycle exit. Lmx1a expression in cells can be used as an indicator when selecting cells suitable for transplantation therapy for neurodegenerative diseases such as Parkinson's disease, and is useful as a marker for screening agents involved in the induction of dopaminergic neuron differentiation.

Specific marker Lmx1a on dopaminergic neurons

In neuron transplantation therapy, in terms of safety, it is preferable to use a cell population consisting only of a desired type of cells, and to use postmitotic neurons in consideration to avoid the risk of tumorigenesis. Moreover, greater therapeutic effects would be expected through the use of earlier progenitor cells in consideration of post-transplantation viability, proper network formation ability, and such. According to the present invention, Lrp4, a gene that is specifically expressed in dopaminergic neuron proliferative progenitor cells prior to cell cycle exit, was identified. The use of Lrp4 expression in cells as an index allows for the isolation. of cells suitable for transplantation therapy of neurodegenerative diseases such as Parkinson's disease in terms of safety, survival rate, and network formation ability.

Methods of selecting a dopaminergic neuron proliferative progenitor cells using Lrp4/Corin markers

The present invention is a probe, a primer, and an antibody, for detecting a dopaminergic neuron proliferative progenitor cell. According to the present invention, there is provided a polynucleotide probe and a polynucleotide primer for use in the detection or selection of a dopaminergic neuron proliferative progenitor cell, which can hybridize with a polynucleotide consisting of a nucleotide sequence of a Nato3 gene, or a complementary sequence thereto, and an antibody against a Nato3 protein, or a part thereof for use in the detection or selection of a dopaminergic neuron proliferative progenitor cell.

Dopaminergic neuron proliferative progenitor cell marker Nato3

A novel gene 65B13 expressed specifically and transiently in dopaminergic neuron precursor cells immediately after cell cycle exit was obtained by the present invention. The cellular expression of 65B13 can be used as an index to select cells that are suitable in terms of their safety, survival rate, and network formation ability, for transplant therapy of neurodegenerative diseases such as Parkinson's disease.

Gene specifically expressed in postmitotic dopaminergic neuron precursor cells

A mouse cDNA library from gene fragments encoding proteins localizing at cell-cell junctions was screened by a technique visualizing localization of a protein to identify a junction-enriched and -associated protein, JEAP. GenBank homology search was performed based on the sequence. Based on the obtained sequence, a mouse cDNA library was screened to identify JEAP-2. By using prepared antibodies against these proteins, it was revealed that these proteins express specifically at tight junctions, in particular, tight junctions in exocrine glands.

Growing community of inventors

Kobe, Japan

Yuichi Ono