Method and kit for single cell protein expression profiling of floorplate mesencephalic dopaminergic progenitor cells

Patent metadata

Field	Value
Publication number	US-12013388-B2
Application number	US-201816632229-A
Country	US
Kind code	B2
Filing date	Jul 16, 2018
Priority date	Jul 17, 2017
Publication date	Jun 18, 2024
Grant date	Jun 18, 2024

Abstract

The present invention provides an in-vitro method for analyzing a cell composition comprising human floorplate mesDA progenitor cells, the method comprising a) contacting the cells of said cell composition or the cells of a sample thereof with antigen binding molecules specific for the antigens FOXA2, OTX2, PAX6, and NKX6.1, thereby labeling the cells of said cell composition or of said sample, b) determining the percentage of said cells that are labelled with said antigen binding molecules for each of said antigens, and wherein the cells of said cell composition qualify as human floorplate mesDA progenitor cells if the protein expression profile of said cells is: 80-100% of said cells are positive for FOXA2, 80-100% of said cells are positive for OTX2, less than 10% of said cells are positive for PAX6, and less than 10% of said cells are positive for NKX6.1. A kit comprising said antigen binding molecules for use in said method is also provided.

First claim

The invention claimed is:

1. A method for obtaining and characterizing a composition of human dopaminergic progenitor cells, the method comprising:
a) differentiating a population of human pluripotent stem cells or progeny thereof into a preparation of neural progenitor cells;
b) maturing the preparation of neural progenitor cells into a cell composition that contains dopaminergic progenitor cells;
c) determining what percentage of cells in the composition expresses each of a panel of markers consisting of FOXA1, OTX2, PAX6, and NKX6.1, thereby obtaining a protein expression profile;
whereby the cell composition is characterized as having a desirable phenotype if the protein expression profile is as follows:
80 to 100% of said cells are positive for FOXA2,
80 to 100% of said cells are positive for OTX2,
less than 10% of said cells are positive for PAX6, and
less than 10% of said cells are positive for NKX6.1.

2. The method of claim 1 wherein the percentage of cells in the composition that express each of said markers is determined by flow cytometry or immunocytochemistry of single cells in the cell composition using fluorophore-conjugated antigen binding molecules that are specific for each of the markers.

3. The method of claim 1 , wherein said human pluripotent stem cells are induced pluripotent stem (iPS) cells or human embryonic stem cells.

4. The method of claim 2 , wherein said antigen binding molecules are antibodies or antigen binding fragments thereof.

5. The method of claim 1 , wherein step (a) comprises inhibiting glycogen synthase kinase 3 (GSK3) and activating sonic hedge hog (SHH) signaling in an adherent culture of the stem cells or progeny.

6. The method of claim 5 , wherein the adherent culture in step (a) is done in a medium comprising CHIR99021 and SHH protein.

7. The method of claim 5 , wherein step (b) comprises culturing the preparation of neural progenitor cells in a medium comprising a plurality of maturation factors selected from bone derived neurotrophic factor (BDNF), glial cell derived neurotrophic factor (GDNF), and fibroblast growth factor 8b (FGF8b).

8. A method for administering to a subject in need thereof a composition of dopaminergic progenitor cells, the method comprising:
obtaining a composition of human dopaminergic progenitor cells produced and characterized according to the method of claim 1 ; and
administering said cell composition to the subject if the composition qualifies as having a desirable phenotype according to the protein expression profile determined in step (c) of claim 1 .

9. A kit for analyzing the protein expression profile of a preparation of dopaminergic progenitor cells to determine if cells in the preparation have a desirable phenotype,
wherein the kit comprises a panel of antigen binding molecules that consists of antigen binding molecules specific for each of the antigens FOXA2, OTX2, PAX6, and NKX6.1, optionally in combination with other reagents,
wherein the kit is configured and the antigen binding molecules are formulated whereby a user of the kit may determine on a single cell basis the percentage of cells in said preparation of dopaminergic progenitor cells that express each of said antigens, thereby obtaining said protein expression profile.

10. The kit according to claim 9 , wherein said antigen binding molecules are antibodies or antigen binding fragments thereof.

11. The kit according to claim 9 , wherein said antigen binding molecules are conjugated to fluorophores.

12. The kit according to claim 9 , wherein said antigen binding molecules are formulated and configured to determine said protein expression profile by flow cytometry.

Assignees

• Miltenyi Biotec Bv & Co Kg

Inventors

• Jungblut Melanie
• Bosio Andreas
• Knöbel Sebastian
• Kirkeby Agnete
• Parmar Malin

Classifications

• G01N33/58

  involving labelled substances (G01N33/53 takes precedence) · CPC title

• C12N2501/60

  Transcription factors · CPC title

• C12N2501/599

  with CD designations not provided for elsewhere · CPC title

• C12N5/0623

  Stem cells · CPC title

• G01N33/5005 Primary

  involving human or animal cells (immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025) · CPC title