Methods for detection of rare subpopulations of cells and highly purified composition of cells

Patent metadata

Field	Value
Publication number	US-12545945-B2
Application number	US-202318347930-A
Country	US
Kind code	B2
Filing date	Jul 6, 2023
Priority date	Jul 23, 2010
Publication date	Feb 10, 2026
Grant date	Feb 10, 2026

Abstract

Methods are provided for detection of a target cell type within a cell population, and compositions are provided comprising cells and an indicator that indicates the number of cells of the target cell type in the cell population. Examples are provided in which these methods are used to detect human embryonic stem cells within a differentiated cell population with exquisite sensitivity. Differentiated cells produced from embryonic stem cells can be characterized by these methods before transplantation into a recipient, thereby providing further assurance of safety.

First claim

What is claimed is:

1 . A method of producing a population of retinal pigment epithelium (RPE) cells comprising:
(a) generating a preparation of differentiated RPE cells by culturing pluripotent stem cells in vitro in a differentiation medium for a sufficient duration for RPE cells to appear; (b) isolating an aliquot of cells from the preparation of differentiated RPE cells; (c) culturing the aliquot of cells in a stem cell medium, wherein the stem cell medium maintains pluripotent stem cells in their pluripotent state; and (d) examining the aliquot of cells by microscopic observation for the presence of pluripotent stem cells.

2 . The method of claim 1 , wherein step (d) comprises:
(i) applying a first stain and a second stain to the aliquot of cells, wherein the first stain detects a first embryonic stem cell marker and the second stain detects a second embryonic stem cell marker, wherein the first embryonic stem cell marker is different from the second embryonic stem cell marker; and (ii) identifying by microscopic observation any cells that are positive for the first stain and the second stain.

3 . The method of claim 1 , wherein the pluripotent stem cells are human cells.

4 . The method of claim 1 , wherein the pluripotent stem cells are selected from the group consisting of: embryonal carcinoma embryonal carcinoma (EC) cells; embryonic stem (ES) cells; and induced pluripotent stem (iPS) cells.

5 . The method of claim 2 , wherein the first stain is observable under visible light and the second stain is observable under ultraviolet light.

6 . The method of claim 2 , wherein the first stain comprises an alkaline phosphatase substrate selected from the group consisting of: napthol AS-BI phosphate; 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and Nitro Blue Tetrazolium (NBT); BCIP reagent and INTX reagent; naphthol AS-BI and fast red violet LB; tetrazolium salts; diazo compounds; and p-Nitrophenylphosphate (pNPP).

7 . The method of claim 2 , wherein the first stain comprises a first primary antibody that specifically binds to the first embryonic stem cell marker.

8 . The method of claim 7 , wherein the first primary antibody is directly or indirectly coupled to: a fluorescent label; a reagent that is observable under visible light; an enzyme that catalyzes a reaction that produces a product that is observable under visible light; or gold particles.

9 . The method of claim 2 , wherein the second stain comprises a second primary antibody that specifically binds to the second embryonic stem cell marker.

10 . The method of claim 9 , wherein the second primary antibody is directly or indirectly coupled to: a fluorescent label; a reagent that is observable under visible light; an enzyme that catalyzes a reaction that produces a product that is observable under visible light; or gold particles.

11 . The method of claim 2 , wherein each of the first embryonic stem cell marker and the second embryonic stem cell marker is selected from the group consisting of: alkaline phosphatase, Oct-4, Nanog, Stage-specific embryonic antigen-3 (SSEA-3), SSEA-4, TRA-1-60, TRA-1-81, TRA-2-49/6E, Sox2, growth and differentiation factor 3 (GDF3), reduced expression 1 (REX 1), fibroblast growth factor 4 (FGF4), embryonic cell-specific gene 1 (ESG1), developmental pluripotency-associated 2 (DPPA2), DPPA4, telomerase reverse transcriptase (hTERT), SALL4, E-CADHERIN, Cluster designation 30 (CD30), Cripto (TDGF-1), GCTM-2, Genesis, Germ cell nuclear factor, and Stem cell factor (SCF or c-Kit ligand).

12 . The method of claim 1 , further comprising determining the approximate number of cells in the RPE cell population.

13 . The method of claim 1 , wherein the RPE cell population contains at least 10 5 RPE cells.

14 . The method of claim 2 , further comprising:
applying a third stain to the aliquot of cells, wherein the third stain detects a third embryonic stem cell marker; and identifying by microscopic observation any cells that are positive for the third stain.

15 . The method of claim 14 , wherein each of the first embryonic stem cell marker, the second embryonic stem cell marker, and the third embryonic stem cell marker is selected from the group consisting of: alkaline phosphatase, Oct-4, Nanog, Stage-specific embryonic antigen-3 (SSEA-3), SSEA-4, TRA-1-60, TRA-1-81, TRA-2-49/6E, Sox2, growth and differentiation factor 3 (GDF3), reduced expression 1 (REX 1), fibroblast growth factor 4 (FGF4), embryonic cell-specific gene 1 (ESG1), developmental pluripotency-associated 2 (DPPA2), DPPA4, telomerase reverse transcriptase (hTERT), SALL4, E-CADHERIN, Cluster designation 30 (CD30), Cripto (TDGF-1), GCTM-2, Genesis, Germ cell nuclear factor, and Stem cell factor (SCF or c-Kit ligand).

16 . The method of claim 1 , wherein step (a) comprises: providing pluripotent stem cells; culturing the pluripotent stem cells to form a multilayer population of pluripotent stem cells or embryoid bodies comprising pluripotent stem cells; culturing the multilayer population or embryoid bodies in the differentiation medium for a sufficient duration for RPE cells to appear in the culture of cells; and isolating the RPE cells from the culture.

Assignees

• Astellas Inst For Regenerative Medicine

Inventors

• Klimanskaya Irina
• Gay Roger

Classifications

• G01N33/5005

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

• G01N33/56966 Primary

  Animal cells · CPC title

• C12Q1/42 Primary

  involving phosphatase · CPC title