Corneal endothelial cell marker

Patent metadata

Field	Value
Publication number	US-2016266114-A1
Application number	US-201414908036-A
Country	US
Kind code	A1
Filing date	Jul 28, 2014
Priority date	Jul 30, 2013
Publication date	Sep 15, 2016
Grant date	—

Abstract

The purpose of the present invention is to provide a method of purification and preparation of cultured corneal endothelial cells, and in particular, to provide cell surface markers for use in corneal endothelial cells not including transformed cells. Provided are cell markers for distinguishing normal cells and transformed cells, in particular normal and transformed corneal endothelium cells. These cell markers relate to specific cell surface markers, for example, to a normal corneal endothelial surface marker such as CD166, and a transformed cell surface marker such as CD73. By using the transformed cell surface marker such as CD73 to remove transformed cells by sorting, it becomes possible to improve purity of a normal cultured corneal endothelium. By using normal corneal endothelial surface marker such as CD166, or by combined use with the transformed cell surface marker, it becomes possible to provide a means for verifying the purity of a prepared corneal endothelium.

First claim

1 . A method for identifying a normal cell and a transformed cell in corneal endothelial cells in a sample comprising corneal endothelial cells, comprising the step of detecting in the sample at least one marker selected from
a normal corneal endothelial cell marker comprising a nucleic acid of a gene selected from the group consisting of CD166, HLA-A2, CD66a, CD66c, CD66d, CD66e, CD98, CD59, CD54, CD340, CD47, EGF-R, CD29, CD74, CD165, CD221, CD49a, SSEA-4, CD130, and CD49f and/or a protein encoded by the nucleic acid, and a transformed corneal endothelial cell marker comprising a nucleic acid of a gene selected from the group consisting of CD26, CD9, CD49b, CD49e, CD13, CD99, CD105, CD63, CD58, CD201, CD56, CD44, CD55, CD71, CD73, CD91, HLA-DQ, CD164, CD49d, CD49c, CD90, MICA/B, CD46, CD140b, CD146, CD147, CD81, CD151, CD200, and CD10 and/or a protein encoded by the nucleic acid.

2 . The method of claim 1 , wherein the marker is a cell surface marker.

3 . The method of claim 1 , wherein the marker comprises at least one transformed corneal endothelial cell marker and at least one normal corneal endothelial cell marker.

4 . The method of claim 1 , wherein the marker comprises at least one molecule selected from the group consisting of CD98, CD166, and CD340 as the normal corneal endothelial cell marker.

5 . The method of claim 1 , wherein the marker comprises at least one molecule selected from the group consisting of CD9, CD49e, CD44, and CD73 as the transformed corneal endothelial cell marker.

6 . The method of claim 1 , wherein the marker comprises at least one molecule selected from the group consisting of CD98, CD166, and CD340 as the normal corneal endothelial cell marker and at least one molecule selected from the group consisting of CD9, CD49e, CD44, and CD73 as the transformed corneal endothelial cell marker.

7 . The method of claim 1 , wherein the marker comprises at least one marker selected from the group consisting of CD166 and CD73.

8 . The method according to claim 1 , wherein the step of detecting comprises using a detection agent for identifying a normal corneal endothelial cell and a transformed corneal endothelial cell, comprising a substance that binds to the marker.

9 . A method for diagnosis of a corneal endothelial cell, comprising: applying a substance that binds to the marker according to claim 1 to a sample comprising the corneal endothelial cell; and detecting a bond between the substance to the cell, wherein a normal corneal endothelial cell and a transformed corneal endothelial cell are identified by determining the presence of the bond.

10 . The method for diagnosis of claim 9 , wherein the corneal endothelial cells are cells present in an organism.

11 . The method for diagnosis of claim 9 , wherein the diagnosis comprises diagnosis of a corneal endothelial disease.

12 . The method for diagnosis of claim 11 , wherein the corneal endothelial disease is a disease associated with a fibroblast.

13 . The method for diagnosis of claim 12 , wherein the corneal endothelial disease is selected from the group consisting of bullous keratopathy, corneal endothelial disorders due to trauma or intraocular surgery, and corneal dystrophy including Fuchs corneal endothelial dystrophy and posterior polymorphous corneal endothelial dystrophy.

14 . The method for diagnosis of claim 9 , wherein a transformed corneal endothelial cell is determined to be comprised when at least one of the transformed corneal endothelial cell marker is expressed.

15 . An method for diagnosis of a corneal endothelial cell, comprising a substance that binds to the marker according to claim 1 ; and detecting a binding between the substance and the corneal endothelial cell, wherein the corneal endothelial cell is diagnosed to be suffering from irreversible dystrophy when at least one of the transformed corneal endothelial cell marker is expressed.

16 . The method for diagnosis according to claim 9 , wherein the substance that binds to the marker further comprises an agent for staining the substance that binds to the marker.

17 . (canceled)

18 . A method of preparing a corneal endothelial cell sample with an elevated ratio of normal corneal endothelial cells, comprising,
a) providing a sample comprising corneal endothelial cells, and b) elevating a ratio of normal corneal endothelial cells while using the marker according to claim 1 as an indicator in the sample.

19 . A method for determining purity when making a sample comprising corneal endothelial cells, comprising the step of: applying a substance that binds to the marker according to claim 1 to a sample comprising the corneal endothelial cell; and detecting a binding between the substance and the cell, wherein a normal corneal endothelial cell and a transformed corneal endothelial cell are identified by determining the presence of the binding.

20 . (canceled)

21 . A method of preparing purified corneal endothelial cells, wherein the method comprises:
inducing corneal endothelial cells from undifferentiated cells; and elevating a ratio of the corneal endothelial cells while using the marker according to claim 1 as an indicator.

22 . The method of claim 21 , wherein the undifferentiated cells are induced pluripotent stem (iPS) cells or embryonic stem (ES) cells.

23 . A method of therapy or prevention of a corneal endothelial disease in a subject, comprising the steps of:
A) providing a corneal endothelial cell preparation with an elevated ratio of normal corneal endothelial cells, comprising,
a) providing corneal endothelial cells, and
b) elevating a ratio of normal corneal endothelial cells while using the marker according to claim 1 as an indicator in the corneal endothelial cells; and
B) administering the cell the preparation to the subject.

24 .- 25 . (canceled)

26 . A method of therapy or prevention of a corneal endothelial disease in a subject, comprising the steps of:
A) providing a corneal endothelial cell preparation with an elevated ratio of normal corneal endothelial cells, comprising,
(a) inducing corneal endothelial cells from undifferentiated cells; and
(b) elevating a ratio of the corneal endothelial cells while using the marker according to claim 1 as an indicator in the corneal endothelial cells; and
B) administering the cell the preparation to the subject.

Assignees

• Kyoto Prefectural Public Univ Corp
• The Doshisha
• Senju Pharma Co

Inventors

• Koizumi Noriko
• Okumura Naoki
• Hirano Hiroatsu
• Kinoshita Shigeru
• Ueno Morio

Classifications

• G01N2333/70596

  Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705 · CPC title

• G01N2333/705

  Assays involving receptors, cell surface antigens or cell surface determinants · CPC title

• G01N33/56966 Primary

  Animal cells · CPC title

• A61K35/30

  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

• C12N5/0621

  Eye cells, e.g. cornea, iris pigmented cells (photoreceptors C12N5/062) · CPC title