Mesenchymal-like stem cells derived from human embryonic stem cells, methods and uses thereof
The present invention relates to methods of generating and expanding hitman embryonic stem eel! derived mesenchymal-like stem/siromal cells. These hES-MSCs are characterized at least in part by the low level of expression of IL-6. These cells are useful for the prevention and treatment of T cell related autoimmune disease, especially multiple sclerosis, as well as for delivering agents across the blood-brain barrier and the blood-spinal cord barrier. Also provided is a method of selecting clinical grade hES-MSC and a method of modifying MSC to produced a MSC with specific biomarker profile. The modified MSC are useful for treatment of various diseases.
1. A method for producing human embryonic stem cell-derived mesenchymal stem cells (hES-MSCs) comprising:
a) culturing human embryonic stem cells in a serum free medium comprising at least one GSK3 inhibitor at a concentration ranging from 0.05 μM to 0.2 μM, wherein the human embryonic stem cells are cultured in the absence of feeder cells;
b) culturing the cells resulting from step a) in a serum-free medium comprising vascular endothelial growth factor (VEGF) and bone morphogenic protein 4 (BMP4) in an amount sufficient to induce formation of embryoid bodies comprising human hemangio-colony forming cells;
c) adding at least one growth factor to the culture resulting from step b), wherein the growth factor is in an amount sufficient to expand human hemangio-colony forming cells;
d) disaggregating the hemangio-colony forming cells resulting from step c) into single cells; and
e) culturing the single hemangio-colony forming cells resulting from step d) in mesenchymal stem cell medium containing serum, knockout serum replacement (KOSR) or in a serum-free medium to induce differentiation of the single cells into human mesenchymal stem cells;
wherein at least 90% of the hES-MSCs express CD73; and said hES-MSCs: (i) comprise greater than 95% of cells expressing CD73, CD90, CD105, CD146, CD166, and CD44; (ii) comprise greater than 80% of cells expressing CD13, CD29, CD54, and CD49E; (iii) comprise less than 5% of cells expressing CD45, CD34, CD31 and SSEA4; (iv) express IL-10 and TGFβ; (v) comprise less than 2% of cells expressing IL-6, IL-12 and TNFα; and (vi) comprise less than 0.001% of cells co-expressing OCT4, NANOG, TRA-1-60 and SSEA4.
2. The method of claim 1 , wherein the hES-MSCs do not express IL-6, IL12 and TNFα.
3. The method of claim 1 , wherein the hES-MSCs express TGF-beta1, TGF-beta2 and IL10.
4. The method of claim 1 , wherein the hES-MSCs do not express CCL2, MMP2 and RAGE.
5. The method of claim 1 , wherein the hES-MSCs have lower expression of IFNγR1 and IFNγR2 when compared to IFNγR1 and IFNγR2 expression in bone marrow-derived mesenchymal stem cells.
6. The method of claim 1 , further comprising a step of irradiating the human mesenchymal stem cells.
7. The method of claim 6 wherein the human mesenchymal stem cells are irradiated with gamma-irradiation.
8. The method of claim 1 , wherein the hES-MSCs are further modified by genetic modification, epigenetic regulation, small molecule, nutraceutical, natural compound, or antibody treatment.
9. The method of claim 1 , further comprising co-culturing the hES-MSCs with hematopoietic stem cells.
10. The method of claim 9 , wherein the hematopoietic stem cells comprise bone marrow hematopoietic stem cells, umbilical cord hematopoietic stem cells, or a combination thereof.
11. The method of claim 1 , wherein the GSK3 inhibitor is (2′Z,3′E)-6-Bromoindirubin-3′-oxime (BIO).