Drug screening system

• US 20020115059A1
• Filed: 10/26/2001
• Published: 08/22/2002
• Est. Priority Date: 10/26/2000
• Status: Abandoned Application

• Current Assignee: University of Florida Research Foundation, Incorporated (State University System of Florida)
• Original Assignee: University of Florida Research Foundation, Incorporated (State University System of Florida)
• Inventors: Terada, Naohiro, Hamazaki, Takashi
• Application Number:
  US10/045,721
• Publication Number:
  US 20020115059A1
• Time in Patent Office: Days
• US Class Current: 435/4
• CPC Class Codes: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2501/113 Acidic fibroblast growth factor (aFGF, FGF-1)
• CPC Class Codes: C12N 2501/12 Hepatocyte growth factor [HGF]
• CPC Class Codes: C12N 2501/235 Leukemia inhibitory factor [LIF]
• CPC Class Codes: C12N 2501/237 Oncostatin M [OSM]
• CPC Class Codes: C12N 2501/39 Steroid hormones
• CPC Class Codes: C12N 2503/02 Drug screening
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 5/067 Hepatocytes
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: G01N 33/5008 for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• CPC Class Codes: G01N 33/5023 on expression patterns
• CPC Class Codes: G01N 33/5067 Liver cells
• CPC Class Codes: G01N 33/5073 Stem cells

Abstract

A method for identifying a drug candidate for promoting tissue-specific differentiation of a stem cell includes the steps of: providing a library of test substances and an in vitro culture of stem cells divided into at least two subcultures; contacting one of the subcultures with the first test substance from the library and a second subculture with a second test substance from the library; culturing the subcultures under conditions that would promote tissue-specific differentiation of the stem cells if an agent that promoted tissue-specific differentiation was in contact with the stem cells; and analyzing the cells in the subcultures for increased tissue specific gene expression.

Claims

1. A method for identifying a drug candidate for promoting tissue-specific differentiation of a stem cell, the method comprising the steps of:

(A) providing a library of test substances, the library comprising at least a first test substance and a second test substance, the first and second test substances having different molecular structures;

(B) providing an in vitro culture of stem cells, the culture being divided into at least a first subculture and a second subculture;

(C) contacting the first subculture with the first test substance and the second subculture with the second test substance;

(D) culturing the first and second subcultures respectively contacted with the first and second test substances under conditions that would promote tissue-specific differentiation of the stem cells if an agent that promoted tissue-specific differentiation was in contact with the stem cells; and

(E) analyzing the cells in the first and second subcultures for increased tissue-specific gene expression.

2. The method of claim 1, wherein the stem cells are embryonic stem cells.

3. The method of claim 2, wherein the embryonic stem cells are mammalian embryonic stems cells.

4. The method of claim 3, wherein the mammalian embryonic stem cells are murine embryonic stems cells.

5. The method of claim 4, wherein the murine embryonic stem cells R1 embryonic stems cells.

6. The method of claim 3, wherein the mammalian embryonic stem cells are human embryonic stems cells.

7. The method of claim 1, wherein the conditions that would promote tissue-specific differentiation of the stem cells comprises culturing the first and second subcultures in a differentiating medium.

8. The method of claim 1, wherein the conditions that would promote tissue-specific differentiation of the stem cells comprises culturing the first and second subcultures at about 37°

C.

9. The method of claim 1, wherein the conditions that would promote tissue-specific differentiation of the stem cells comprises culturing the first and second subcultures in a humidified, carbon-dioxide containing incubator.

10. The method of claim 1, wherein the conditions that would promote tissue-specific differentiation of the stem cells comprises culturing the first and second subcultures for a time period of at least five days.

11. The method of claim 10, wherein the time period is at least seven days.

12. The method of claim 11, wherein the time period is between seven and eighteen days.

13. The method of claim 1, wherein the first and second subcultures are cultured in a microtiter plate.

14. The method of claim 1, wherein the step (E) of analyzing the cells in the first and second subcultures for increased tissue-specific gene expression comprises isolating mRNA from the first and second subcultures.

15. The method of claim 14, wherein total cellular RNA is isolated from the first and second subcultures.

16. The method of claim 14, wherein the step (E) further comprises reverse-transcribing the mRNA to create cDNA.

17. The method of claim 1, wherein the step (E) of analyzing the cells in the first and second subcultures for increased tissue-specific gene expression comprises performing a polymerase chain reaction (PCR).

18. The method of claim 14, wherein the isolated mRNA is immobilized on a substrate.

19. The method of claim 18, wherein the substrate is contacted with a probe that specifically hybridizes to the tissue-specific mRNA.

20. The method of claim 1, wherein the step (E) of analyzing the cells in the first and second subcultures for increased tissue-specific gene expression is performing using gene chip technology.