INDUCTION OF PLURIPOTENT STEM CELLS INTO MESODERMAL LINEAGES

• US 20080038820A1
• Filed: 10/22/2007
• Published: 02/14/2008
• Est. Priority Date: 06/22/2004
• Status: Abandoned Application

• Current Assignee: Diane Elizabeth Rudy-Reil
• Original Assignee: Diane Elizabeth Rudy-Reil
• Inventors: Rudy-Reil, Diane
• Application Number:
  US11/876,769
• Publication Number:
  US 20080038820A1
• Time in Patent Office: Days
• US Class Current: 435/377
• CPC Class Codes: A61K 2121/00 Preparations for use in therapy
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/12 Hepatocyte growth factor [HGF]
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2502/1329 Cardiomyocytes
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2533/52 Fibronectin; Laminin
• CPC Class Codes: C12N 5/0657 Cardiomyocytes; Heart cells
• CPC Class Codes: C12N 5/069 Vascular Endothelial cells
• CPC Class Codes: C12N 5/0691 Vascular smooth muscle cells; 3D culture thereof, e.g. models of blood vessels

Abstract

The present invention provides a method of inducing mesoderm derived cells from pluripotent stem cells. In contrast to methods known in the art that are often designed to replicate in vivo events of mesoderm induction, the present invention provides a unique, yet simple, method whereby pluripotent stem cells are mesodermally primed in the presence of factors that concomitantly inhibit the spontaneous differentiation of endoderm and ectoderm during expansion and suspension steps. Exposure and/or adherence of primed aggregates to a extracellular matrix that promotes the commitment and survival of induced mesoderm progenitors, followed by exposure to various mesoderm associated factors, allows for the subsequent induction of such cells into terminally differentiated lineages, such as cardiomyocytes. End products of this induction system will ultimately provide an unlimited source of mesoderm-derived cell types for therapeutic and pharmacological purposes.

Claims

1. A method of inducing mesoderm progenitor cells, the method comprising:

expanding and priming pluripotent cells on a fibroblast layer in the presence of a priming medium;

suspending primed stem cells in the priming medium for a predetermined time period; and

,

exposing the suspended primed stem cells to a substrate.

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

3. The method of claim 1, wherein the priming medium comprises bFGF.

4. The method of claim 3, wherein the priming medium further comprises MEF-CM.

5. The method of claim 1, wherein the predetermined time period between 1 and 4 days.

6. The method of claim 1, wherein the predetermined time period is 3 days.

7. The method of claim 1 wherein the substrate is fibronectin.

8. A method of inducing target mesoderm derived cells from pluripotent stem cells, the method comprising:

inducing mesoderm progenitors from pluripotent stem cells; and

,

exposing the mesoderm progenitor cells to at least one MesA factor known to induce target mesoderm derived cells.

9. The method of claim 8, wherein the target mesoderm derived cells are cardiomyocytes.

10. The method of claim 8, wherein the MesA factor is HGF.

11. The method of claim 8, wherein the mesoderm progenitor cells are exposed to a combination of HGF and bFGF.

12. The method of claim 8 wherein the MesA factor is precardiac mesoderm explant.

13. The method of claim 8, wherein the MesA factor is mesoderm conditioned medium.

14. The method of claim 8, wherein the target mesoderm derived cells are endothelium.

15. The method of claim 14, wherein the MesA factor is BMP-4.

16. A method of inducing target mesoderm derived cells from pluripotent stem cells, the method comprising:

priming the mesoderm of pluripotent stem cells in the presence of a priming medium;

committing the induction of mesodermal progenitor cells from the mesodermally primed stem cells; and

,

exposing the mesodermal progenitor cells to at least one MesA factor known to induce the target mesoderm derived cells.

17. The method of claim 16, wherein the target mesoderm derived calls are cardiomyocytes.

18. The method of claim 16, wherein the target mesoderm derived calls are endothelium.

19. The method of claim 16, wherein the target pluripotent stem cells are human.

20. The method of claim 19, wherein the priming medium includes bFGF+HEF-CM.