Methods of identifying small molecules for renewal, survival and migration of cardiac progenitors

• US 20080108090A1
• Filed: 05/02/2007
• Published: 05/08/2008
• Est. Priority Date: 02/02/2004
• Status: Abandoned Application

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Qyang, Yibing, Evans, Sylvia, Chen, Ju, Moretti, Alessandra, Chien, Ken, Laugwitz, Karl, Lin, Lizhu
• Application Number:
  US11/799,839
• Publication Number:
  US 20080108090A1
• Time in Patent Office: Days
• US Class Current: 435/7.21
• CPC Class Codes: A01K 2217/052 inducing gain of function
• CPC Class Codes: A01K 2217/072 maintaining or altering function, i.e. knock in
• CPC Class Codes: A01K 2217/203 Animal model comprising inducible/conditional expression system, e.g. hormones, tet
• CPC Class Codes: A01K 2217/206 Animal model comprising tissue-specific expression system, e.g. tissue specific expression of transgene, of Cre recombinase
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 2267/0375 Animal model for cardiovascular diseases
• CPC Class Codes: A01K 67/0275 Genetically modified vertebrates, e.g. transgenic
• CPC Class Codes: C07K 14/4702 Regulators; Modulating activity
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2502/99 genetically modified cells
• CPC Class Codes: C12N 2800/30 Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
• CPC Class Codes: C12N 2840/203 having an IRES
• CPC Class Codes: C12N 5/0657 Cardiomyocytes; Heart cells
• CPC Class Codes: C12N 5/0662 Stem cells
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2600/136 Screening for pharmacological compounds
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: G01N 33/5073 Stem cells
• CPC Class Codes: G01N 33/56966 Animal cells

Abstract

The present invention relates to a small molecule high-throughput screening assay consisting of detectably labeled cardiac progenitor cells. The invention also describes a method of identifying small molecules from the high-throughput assay affecting cardiogenesis and/or modulating cardiac progenitor cell development. Also described are methods of stimulating maturation of cardiac progenitor cells using a GSK-3β inhibitor.

Claims

1. A high-throughput screening assay comprising:

(a) injecting tamoxifen into a transgenic non-human animal having a tamoxifen-dependent Cre-recombinase in the isl1 locus (isl1-mER-Cre-mER) and an Cre reporter; and

(b) isolating at least one cell fraction from the animal, wherein the cells express a detectably labeled marker.

2. The assay of claim 1, wherein the labeled marker is β

-galactosidase.

3. The assay of claim 1, wherein the labeled marker is LacZ.

4. The assay of claim 1, wherein the non-human animal is a rat or mouse.

5. A method of identifying a small molecule affecting cardiogenesis comprising:

(a) labeling a cell fraction isolated from a transgenic non-human animal having a tamoxifen-dependent Cre-recombinase in the isl1 locus (isl1-mER-Cre-mER) and an Cre reporter (R16R) with a fluorescent label, wherein the cell fraction contains detectably labeled cardiac progenitor cells;

(b) identifying a test molecule from the cell fraction having increased fluorescence as compared to a control molecule;

(c) contacting the test molecule with the detectably labeled cardiac progenitor cells; and

(d) determining the expression of the isl1+ transcription factor,

thereby identifying a small molecule affecting cardiogenesis.

6. The method of claim 5, wherein the non-human animal is a rat or mouse.

7. The method of claim 5, further comprising detecting expression of one or more growth factors selected from the group consisting of BMP4, BMP7, and FGF10.

8. A method of identifying a small molecule which regulates cardiogenesis comprising:

(a) labeling a cell fraction isolated from a transgenic non-human animal having a tamoxifen-dependent Cre-recombinase in the isl1 locus (isl1-mER-Cre-mER) and an Cre reporter with a fluorescent label, wherein the cell fraction contains detectably labeled cardiac progenitor cells;

(b) identifying a test molecule from the cell fraction having increased fluorescence as compared to a control molecule;

(c) contacting the test molecule with the detectably labeled cardiac progenitor cells;

(d) determining the expression of the isl1+ transcription factor; and

(e) identifying a factor which is modulates isl1+ expression,

thereby identifying a small molecule which regulates cardiogenesis.

9. The method of claim 8, wherein the non-human animal is a rat or mouse.

10. The method of claim 8, further comprising detecting expression of one or more growth factors selected from the group consisting of BMP4, BMP7, and FGF10.

11. A method for stimulating maturation of cardiac progenitor cells comprising contacting the cells with an effective amount of a GSK-3β

inhibitor, thereby stimulating maturation of cardiac progenitor cells.

12. The method of claim 11, wherein the inhibitor is 6-bromoindirubin-3′

-oxime (BIO).

13. A method for generating an Isl1 lineage-traced cell comprising contacting an undifferentiated progenitor cell that expresses Isl1 with a GSK-3 inhibitor that activates or enhances expression of Isl1 in the cell, thereby generating a Isl1 lineage-traced cell.

14. The method of claim 13, wherein the inhibitor is 6-bromoindirubin-3′

-oxime (BIO).

15. The method of claim 13, wherein the activation or enhancement of Isl1 expression causes the cell to differentiate into a cell selected from the group consisting of cardiomyocyte, endothelial cell, and smooth muscle cell.

16. The method of claim 13, wherein the undifferentiated progenitor cell is selected from the group consisting of embryonic heart muscle cell, post-natal heart muscle cells, embryonic stem cells, progenitors from cord blood, and adult progenitor cells.

17. The method of claim 16, wherein the adult progenitor cells are bone marrow cells or adipose tissue cells.

18. The method of claim 16, wherein the cardiac cell is derived from a rat, mouse or human.