Method of forming mesenchymal stem cells from embryonic stem cells
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.
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) 2 D 3 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) 2 D 3 , 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) 2 D 3 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) 2 D 3 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) 2 D 3 , 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) 2 D 3 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).