Method of isolating a lineage specific stem cell in vitro

• US 5,639,618 A
• Filed: 05/13/1994
• Issued: 06/17/1997
• Est. Priority Date: 05/13/1994
• Status: Expired due to Term

• Current Assignee: Plurion, Inc. (Stem Cell Innovations, Inc.)
• Original Assignee: Plurion, Inc. (Stem Cell Innovations, Inc.)
• Inventors: Gay, David A.
• Primary Examiner(s): Feisee, Lila
• Assistant Examiner(s): Gambel, Phillip
• Application Number:
  US08/242,547
• Time in Patent Office: 1,131 Days
• Field of Search: 435/2, 435/172.3, 435/240.1, 435/240.2, 435/240.21, 435/7.1, 435/7.21, 435/7.2
• US Class Current: 435/7.21
• CPC Class Codes: C12N 15/65 using markers enzymes used as markers C12N15/52
• CPC Class Codes: C12N 2501/60 Transcription factors
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 5/0623 Stem cells
• CPC Class Codes: G01N 33/56966 Animal cells

Abstract

The present invention provides a method of isolating a lineage specific stem cell in vitro, comprising a) transfecting a pluripotent embryonic stem cell with a construct comprising a regulatory region of a lineage specific gene operably linked to a DNA encoding a reporter protein, b) culturing the pluripotent embryonic stem cell under conditions such that the pluripotent embryonic stem cell differentiates into a lineage specific stem cell and c) separating the cells which express the reporter protein from the other cells in the culture, the cell which expresses the reporter protein being an isolated lineage specific stem cell. A lineage specific stem cell can also be identified utilizing this method.

Claims

1. A method of isolating a lineage specific stem cell in vitro, comprising:

a. transfecting in vitro a pluripotent embryonic stem cell with a construct comprising a regulatory region of a lineage specific gene operably linked to a DNA encoding a reporter protein;

b. culturing the pluripotent embryonic stem cell in vitro under conditions such that the pluripotent embryonic stem cell differentiates into a lineage specific stem cell; and

c. separating the cells fluorescent activated cell sorting or by immunoaffinity procedures which express the reporter protein from the other cells in culture, the cell which expresses the reporter protein being an isolated lineage specific stem cell.

2. The method of claim 1 wherein the regulatory region of the lineage specific gene is selected from the group consisting of an Otx, Dlx, NIx, Emx, Wnt, En, Hox and ACHRβ

.

3. The method of claim 1 wherein the reporter protein is a protein expressed on the cell surface.

4. The method of claim 1 wherein the reporter protein is LacZ.

5. A method of isolating a lineage specific stem cell in vitro, comprising:

a. transfecting in vitro a pluripotent embryonic stem cell with a construct comprising a regulatory region of a lineage specific gene operably linked to a DNA encoding a reporter protein;

b. culturing the pluripotent embryonic stem cell in vitro under conditions which allow the formation of aggregates of differentiating cells; and

c. separating the cells by fluorescent activated cell sorting or by immunoaffinity procedures in the aggregates which express the reporter protein from the other cells, the cell which expresses the reporter protein being an isolated lineage specific stem cell.

6. The method of claim 5 wherein the aggregates are cultured to form embryoid bodies prior to separating the cells.

7. The method of claim 5 wherein the regulatory region of the lineage specific gene is selected from the group consisting of an Otx, Dlx, Nlx, Emx, Wnt, En, Hox and ACHRβ

.

8. The method of claim 5 wherein the reporter protein is expressed on the cell surface.

9. The method of claim 5 wherein the reporter protein is LacZ.