Human iPSC-derived vascular-related and hematopoetic cells for therapies and toxicology/drug screenings
Described herein are cells, cell culture methods, and cell culture media compositions useful for producing and maintaining iPSC-derived cell lines that are of higher purity and maintain cell type integrity better than current iPSC-derived cell lines. Also disclosed are methods of using the described cells and media, such as therapeutic methods of use for the described cells. The described cells include iPSC-derived mesodermal precursor cells (MPC), which itself may differentiate into at least four different cell types. When cultured under appropriate conditions, the mesodermal precursor cells can be used to produce hematopoietic stem cells (HSC), mesenchymal stem cells (MSC), smooth muscle cells (SMC), or unlimited functional endothelial cells (UFEC). One characteristic that makes the described cells desirable is that they can be maintained in culture for a number of days, or passages, without changing phenotype through differentiation.
1. A method of producing mesodermal precursor cells from induced pluripotent stem cells (iPSC), comprising incubating the iPSCs in a cell culture medium comprising:
Iscove's modified Dulbecco's medium (IMDM),
Ham's F-12 Nutrient Mix, with L-alanyl-L-glutamine additive,
Albumin,
α-monothioglycerol,
protein-free hybridoma mixture II,
L-ascorbic acid 2-phosphate,
L-alanyl-L-glutamine,
Antibiotic,
insulin-transferrin-selenium-ethanolamine supplement,
bone morphogenic protein 4,
vascular endothelial growth factor, and
basic fibroblast growth factor.
2. The method of claim 1 , wherein the cell culture medium comprises cholesterol lipids.
3. The method of claim 1 , wherein the antibiotic is selected from the group consisting of penicillin, streptomycin, and a mixture of penicillin and streptomycin.
4. The method of claim 1 , wherein the concentration of albumin is about 5 mg/ml.
5. The method of claim 1 , wherein the concentration of α-monothioglycerol is from about 350 μM to about 450 μM.
6. The method of claim 1 , wherein the concentration of L-ascorbic acid 2-phosphate is about 50 μg/ml.
7. The method of claim 1 , wherein the concentration of L-alanyl-L-glutamine is about 1 mM to about 2 mM.
8. The method of claim 1 , wherein the concentration of bone morphogenic protein is about 4 ng/ml to about 10 ng/ml.
9. The method of claim 1 , wherein the concentration of vascular endothelial growth factor is about 10 ng/ml.
10. The method of claim 1 , wherein the concentration of basic fibroblast growth factor is about 10 ng/ml.
11. The method of claim 1 , wherein the cell culture medium comprises stem cell factor, Flt-3 ligand, and thrombopoietin.
12. The method of claim 11 , wherein the concentration of stem cell factor is at least 50 ng/ml.
13. The method of claim 11 , wherein the concentration of Flt-3 ligand is at least 50 ng/ml.
14. The method of claim 1 , wherein the iPSCs are incubated in an environment having an attachment surface coated with a basement membrane matrix.
15. A method of producing hematopoietic progenitor cells from induced pluripotent stem cells (iPSCs), comprising incubating the iPSCs in a cell culture medium comprising:
Iscove's modified Dulbecco's medium (IMDM),
Ham's F-12 Nutrient Mix, with L-alanyl-L-glutamine additive,
Albumin,
α-monothioglycerol,
protein-free hybridoma mixture II,
L-ascorbic acid 2-phosphate,
L-alanyl-L-glutamine,
Antibiotic,
insulin-transferrin-selenium-ethanolamine supplement,
bone morphogenic protein 4,
vascular endothelial growth factor, and
basic fibroblast growth factor;
wherein the iPSCS are incubated for about 7 to about 17 days.
16. The method of claim 15 , wherein the cell culture medium comprises cholesterol lipids.
17. The method of claim 15 , wherein the cell culture medium comprises stem cell factor, Flt-3 ligand, and thrombopoietin.
18. The method of claim 15 , wherein the iPSCs are cultured in an environment having an attachment surface coated with a basement membrane matrix.