Method of forming mesenchymal stem cells from embryonic stem cells

• US 7,592,176 B2
• Filed: 05/06/2005
• Issued: 09/22/2009
• 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
• Primary Examiner(s): Crouch; Deborah
• Assistant Examiner(s): Noble; Marcia S
• Application Number:
  US11/123,794
• Publication Number:
  US 20060008902A1
• Time in Patent Office: 1,600 Days
• Field of Search: None
• US Class Current: 435/373
• 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
• 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 population of human mesenchymal lineage cells comprising mesenchymal stem cells (MSCs) and bone precursor cells (BPCs) from human embryonic stem cells (hESCs), the method comprising the steps of:

a) culturing hESCs under conditions conducive to form embryoid bodies (EBs);

b) propagating the EBs formed in step a) in a mesenchyme-specific medium containing 1.25(OH)₂D₃ to promote formation of EBs having cells of the mesenchymal lineage;

c) digesting the resultant EBs of step b) and culturing the resulting cells in mesenchyme-specific medium to obtain a population of human mesenchymal lineage cells exhibiting spindle shaped morphology, wherein the population is composed of at least 60% human MSCs, and wherein the population exhibits enhanced expression of Cba-1, Msx2, and D1x5 relative to the EBs of step a); and

d) propagating the human MSCs produced by step c).

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

e) culturing the human MSCs in the presence of an effective amount of 1.25(OH)₂D₃, dexamethasone, retinoic acid, or a combination thereof to cause the MSCs to differentiate into a subpopulation of BPCs composed of at least 80% pre-osteoblast and osteoblast cells, wherein the BPCs are characterized by the expression of osteopontin, osteonectin, and, osteocalcin.

3. The method of claim 2 further comprising the step of:

f) exposing the subpopulation of BPCs to an effective amount of ascorbic acid and β

-glycerophosphate to further propagate and differentiate the BPCs into osteoblast cells capable of forming bone nodules.

4. The method of claim 1 wherein the population of human mesenchymal lineage cells includes at least 90% mesenchymal stem cells.

5. A method as claimed in claim 1 wherein the mesenchyme-specific medium further includes serum.

6. A method as claimed in claim 1 further comprising the step of culturing the population of MSCs in a medium having an effective amount of 1.25(OH)₂D₃ to direct differentiation of MSCs into bone precursor cells capable of forming bone nodules.

7. A method as claimed in claim 1 wherein the time period to go from human embryonic stem cells to bone precursor is less than 34 days.

8. A method of producing a population of human bone-precursor cells (BPCs) from human embryonic stem cells (hESCs) comprising the steps of:

a) culturing hESCs under conditions conducive to form embryoid bodies (EBs);

b) culturing the EBs in a mesenchyme-specific medium containing 1,25(OH)₂D₃ to form a population of mesenchymal lineage cells exhibiting spindle shaped morphology, wherein the population is composed of at least 60% human mesenchymal stem cells (MSCs), and wherein the population exhibits enhanced expression of Cba-1, Msx2, and D1x5 relative to the EBs of step a); and

c) culturing the population of MSCs in an effective amount of 1,25(OH)₂D₃, dexamethosone, retinoic acid, or a combination thereof to induce the MSCs to differentiate into a subpopulation of BPCs composed of at least 80% pre-osteoblast and osteoblast cells, wherein the BPCs are characterized by the expression of osteopontin, osteonectin, and osteocalcin.

9. A method of directing the differentiation of human embryonic stem cells (hESCs) into cells of the mesenchymal lineage comprising the steps of:

a) culturing the hESCs under conditions conducive to form embryoid bodies (EBs);

b) propagating the EBs formed in step a) in a mesenchyme-specific medium containing 1,25(OH)₂D₃ to promote formation of EBs having cells of the mesenchymal lineage;

c) digesting the resultant EBs of step b) and culturing the resulting cells in mesenchyme-specific medium to obtain a population of human mesenchymal lineage cells exhibiting spindle shaped morphology, wherein the population is composed of at least 60% human MSCs, and wherein the population exhibits enhances expression of Cba-1, Msx2, and D1x5 relative to the EBs of step a).