Colony forming medium and use thereof
The invention relates to a method for producing a mesenchymal stem cell (MSC), the method comprising culturing a primitive mesoderm cell in a mesenchymal colony forming medium (M-CFM) comprising LiCl and FGF2, but excluding PDGF, under normoxic conditions for sufficient time for a mesenchymal colony to form, and culturing the mesenchymal colony adherently to produce the MSC, wherein the MSC has superior T-cell immunosuppressive properties relative to an MSC not produced in said M-CFM. The invention also relates to an MSC produced by the method, a population of MSCs produced by the method, a therapeutic composition comprising the MSC produced by the method, an M-CFM and an M-CFM in concentrated form, and method and uses of the MSC or population in treating a disease.
1. A method for producing a mesenchymal stem cell (MSC), the method comprising:
(a) culturing a primitive mesodermal cell in a mesenchymal-colony forming medium (M-CFM) comprising LiCl and FGF2, but excluding PDGF, BMP4 and Activin A, under normoxic conditions for sufficient time for a mesenchymal colony to form; and
(b) culturing the mesenchymal colony of (a) adherently to produce the MSC,
wherein the MSC of (b) has superior T-cell immunosuppressive properties relative to an MSC produced by a method comprising:
(a′) culturing a primitive mesodermal cell in a medium comprising FGF2 and PDGF, but excluding LiCl, under normoxic conditions for sufficient time for a mesenchymal colony to form; and
(b′) culturing the mesenchymal colony of (a′) adherently to produce the MSC.
2. The method of claim 1 , wherein the primitive mesodermal cell is a primitive mesodermal cell with mesenchymoangioblast (MCA) potential.
3. The method of claim 2 , wherein the primitive mesodermal cell with MCA potential has a EMH lin − KDR + APLNR + PDGFRalpha + phenotype.
4. The method of claim 1 , wherein sufficient time for the mesenchymal colony to form is about 8 days to about 14 days, about 10 days to about 14 days, about 11 to about 13 days, or about 12 days.
5. The method of claim 1 , wherein the T-cell immunosuppressive properties of the MSC are determined relative to an MSC produced from a primitive mesodermal cell differentiated from a PSC in a differentiation medium comprising FGF2, BMP4, Activin A, and LiCl under hypoxic conditions for about two days.
6. The method of claim 1 , wherein T-cell immunosuppressive properties comprise suppression of proliferation of T helper (CD4 + ) lymphocytes.
7. The method of claim 1 , wherein the M-CFM comprises about 1 mM LiCl.
8. The method of claim 1 , wherein the M-CFM comprises about 5 ng/mL to about 100 ng/mL FGF2, about 10 ng/mL to about 50 ng/mL FGF2, about 10 ng/mL FGF2, or about 20 ng/mL FGF2.
9. The method of claim 1 , wherein the M-CFM comprises about 1 mM LiCl and about 10 ng/mL FGF2, or about 1 mM LiCl and about 20 FGF2.
10. The method of claim 1 , further comprising, prior to culturing the primitive mesodermal cell, differentiating a pluripotent stem cell (PSC) into a primitive mesodermal cell comprising:
culturing; the PSC in a differentiation medium comprising FGF2, BMP4, Activin A, and LiCl under hypoxic conditions for about two days to form the primitive mesoderm; and
replacing the differentiation medium with the M-CFM comprising LiCl and FGF2, but excluding PDGF.
11. The method of claim 1 , wherein the cells are human.
12. The method of claim 10 , wherein the PSC is an induced PSC (iPSC).
13. The method of claim 10 , wherein the differentiation medium comprises:
about 10 ng/mL to about 50 ng/mL FGF2, or about 50 ng/mL FGF2;
about 50 ng/mL to about 250 ng/mL BMP4, or about 50 ng/mL BMP4;
about 1 ng/mL to about 15 ng/mL Activin A, about 10 ng/mL to about 15 ng/mL Activin A, about 12.5 ng/mL Activin A, or about 1.5 ng/mL Activin A; and/or
about 1 mM to about 2 mM LiCl.
14. The method of claim 10 , wherein the differentiation medium comprises:
about 50 ng/mL FGF2;
about 50 ng/mL BMP4;
about 1.5 ng/mL Activin A; and
about 2 mM LiCl.
15. The method of claim 1 , comprising culturing on collagen IV and/or tenascin C.
16. The method of claim 1 , further comprising passaging the mesenchymal colony and/or MSC.
17. The method of claim 16 , wherein passaging comprises:
culturing the mesenchymal colony and/or MSC for about 3 days;
culturing the mesenchymal colony and/or MSC on fibronectin and/or collagen I for
1, 2, 3, 4, 5, or 6 passages.