Method of forming mesenchymal stem cells from embryonic stem cells

• US 20060008902A1
• Filed: 05/06/2005
• Published: 01/12/2006
• Est. Priority Date: 05/07/2004
• Status: Active Grant

• Current Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Original Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Inventors: Shevde, Nirupama K., Pike, J. Wesley
• Granted Patent: US 7,592,176 B2
• Time in Patent Office: Days
• US Class Current: 435/371
• CPC Class Codes: C12N 2500/38 Vitamins
• CPC Class Codes: C12N 2500/42 Organic phosphate, e.g. beta glycerophosphate
• CPC Class Codes: C12N 2501/30 Hormones derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin C12N2501/85
• CPC Class Codes: C12N 2501/385 of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
• CPC Class Codes: C12N 2501/39 Steroid hormones
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 5/0662 Stem cells

Abstract

This invention relates to methods of producing a substantially homogenous population of mesenchymal stem cells derived from embryonic stem cells. Also, disclosed is a homogenous population of mesenchymal stem cells capable of further differentiating into a variety of specific cell types, characterized by various morphological factors and cell-specific markers. The compositions and methods described in this disclosure are useful for a variety of commercially important diagnostic, drug screening, and therapeutic applications.

Claims

1. A method of producing a substantially homogenous population of mesenchymal stem cells derived from embryonic stem cells, the method comprising the steps of:

a) culturing the embryonic stem cells under conditions conducive to form embryoid bodies;

b) propagating the embryoid bodies in a mesenchyme-specific medium;

c) digesting the embryoid bodies to form mesodermal cells; and

d) culturing the mesodermal cells in the presence of mesenchyme-specific medium to form a substantially homogenous population of mesenchymal stem cells.

2. The method of claim 1 further comprising the steps of:

e) propagating the mesenchymal stem cells in the presence of osteogenic medium to produce a subpopulation of bone precursor cells; and

f) exposing the subpopulation of bone precursor cells to osteoblast differentiation agents in a sequence-specific manner to form a substantially homogenous population of bone precursor cells.

3. The method of claim 1 wherein the mesenchyme-specific medium is MesenCult™

Basal Medium.

4. The method of claim 1 wherein the mesenchymal stem cells exhibit spindle shaped morphology.

5. The method of claim 1 wherein the mesenchyme-specific medium includes 1.25(OH)₂D₃.

6. The method of claim 2 wherein the osteoblast differentiation agents are selected from the group consisting of 1.25(OH)₂D₃, dexamethasone, retinoic acid, ascorbic acid and β

-glycerophosphate.

7. The method of claim 2 wherein the sequence-specific manner of adding osteoblast differentiation agents to the population of bone precursor cells comprises treating the cells with an effective amount of 1.25(OH)₂D₃, dexamethasone, and retinoic acid, or a combination thereof followed by the addition of an effective amount of ascorbic acid and β

-glycerophosphate.

8. The method of claim 1 wherein the embryonic stem cell culture is derived from humans.

9. The method of claim 1 wherein the substantially homogenous population of mesenchymal stem cells include at least 60% mesenchymal stem cells.

10. The method of claim 1 wherein the substantially homogenous population of mesenchymal stem cells includes at least 90% mesenchymal stem cells.

11. The method of claim 2 wherein the bone precursor cells are pre-osteoblast and osteoblast cells.

14. The cell population of claim 11 wherein the mesenchymal stem cells exhibit spindle shaped morphology.

15. The cell population of claim 11 wherein the mesenchymal stem cells are further differentiated into a subpopulation of bone precursors cells having at least 80% pre-osteoblast and osteoblast cells.

16. The cell population of claim 14 wherein the pre-osteoblast and osteoblast cells are characterized by the expression of osteoblast-specific gene markers selected from the group consisting of osteopontin, osteonectin, and osteocalcin.

17. The cell population of claim 14 wherein the cells are characterized by the expression of early transcription factors selected from the group consisting of Cbfa-1, Msx2, and Dlx5.

12. An isolated substantially homogenous cell population comprising at least 60% mesenchymal stem cells.

13. An isolated cell population as claimed in claim 12 which is at least 90% mesenchymal stem cells.

18. A method of producing a substantially homogenous population of mesenchymal stem cells derived from embryonic stem cells, the method comprising the steps of:

(a) inducing human embryonic stem cells to form embryoid bodies;

(b) culturing cells from the embryoid bodies in the presence of 1.25(OH)₂D₃ to make a mesenchyme-specific medium; and

(c) recovering the cells which proliferate in the mesenchyme-specific medium.

19. A method as claimed in claim 18 wherein the time taken by the step of inducing takes 10 to 12 days, and the steps of culturing and recovering together take less than 10 days.

20. A method as claimed in claim 18 wherein the mesenchyme-specific medium further includes serum.

21. A method as claimed in claim 18 further including the step of culturing the cells in a medium including 1.25(OH)₂D₃ to promote differentiation to osteogenic cells.

22. A method as claimed in claim 18 wherein the time period to go from human embryonic stem cells to osteogenic cells is less than 34 days.

23. A method for the formation of human bone forming cells comprising the steps of

(a) culturing human embryonic stem cells into mesenchymal stem cells;

and

(b) inducing the mesenchymal stem cells to differentiate into osteogenic cells.

24. A method as claimed in claim 23 wherein the step of culturing the human embryonic stem cells into mesenchymal stem cells includes the step of culturing the human embryonic stem cells to form embryoid bodies and then culturing cells from the embryoid bodies in the presence of 1.25(OH)₂D₃.

25. A method as claimed in claim 23 wherein the step of culturing human embryonic stem cells into mesenchymal stem cells is performed by culturing the embryonic stem cells so that they form embryoid bodies, propagating the embryoid bodies in a mesenchyme-specific medium, digesting the embryoid bodies, and culturing the resulting cells in the presence of mesenchyme-specific medium to form a culture of mesenchymal stem cells.