Analysis of the temporal and concentration-dependent effects of BMP-4, VEGF, and TPO on development of embryonic stem cell-derived mesoderm and blood progenitors in a defined, serum-free media.
OBJECTIVE
To develop a robust serum-free (SF) system for generation of hemogenic mesoderm and blood progenitors from pluripotent cells.


METHODS
Embryonic stem cells (ESCs) maintained in N2B27 supplemented with leukemia inhibitory factor (LIF) and bone morphogenetic protein (BMP)-4 were induced to differentiate into Brachyury/T-expressing cells (measured using a green fluorescent protein reporter) and myeloid-erythroid colony-forming cells (ME-CFCs), by removing LIF, changing the base media formulation, and via the time- and concentration-dependent addition of other factors.


RESULTS
Presence of 10 ng/mL BMP-4 permitted the emergence of cells expressing T and the vascular endothelial growth factor receptor (VEGFR)-2, however, <5% of the cells were double-positive on day 4. Adjusting the SF media formulation allowed only 5 ng/mL BMP-4 to yield 24% +/- 4% Brachyury-green fluorescent protein VEGFR-2(+) cells by day 4. These cells could develop into ME-CFC, producing 4.4 +/- 0.8 CFC per 1000 cells at day 8. We also examined the timing and concentration sensitivity of BMP-4, VEGF, and thrombopoietin (TPO) during differentiation. BMP-4 with 50 ng/mL TPO generated 232 +/- 48 CFC per 5 x 10(4) cells, similar to the serum-control, and this response could be enhanced to 292 +/- 42 CFC per 5 x 10(4) cells by early (between day 0-5), but not late (after day 5) VEGF treatment.


CONCLUSIONS
Moving to SF systems facilitates directed differentiation by eliminating confounding signals. This article describes modifications to the N2B27 media that amplify mesoderm induction and extends earlier work defining blood progenitor cell induction from ESC with BMP-4, VEGF, and TPO.
Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada.
Kelly A
KA
Purpura
Jennifer
J
Morin
Peter W
PW
Zandstra
D000818:Animals; D055415:Bone Morphogenetic Protein 4; D019485:Bone Morphogenetic Proteins / Q000494:pharmacology*; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000187:drug effects; D016895:Culture Media, Serum-Free / Q000494:pharmacology*; D053595:Embryonic Stem Cells / Q000166:cytology / Q000187:drug effects*; D005326:Fetal Proteins / Q000096:biosynthesis; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000187:drug effects*; D006801:Humans; D053605:Leukemia Inhibitory Factor / Q000494:pharmacology; D008648:Mesoderm / Q000166:cytology*; D051379:Mice; D009994:Osmolar Concentration; D020825:T-Box Domain Proteins / Q000096:biosynthesis; D013926:Thrombopoietin / Q000494:pharmacology*; D013997:Time Factors; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000096:biosynthesis; D000098908:Brachyury Protein
D055415:Bone Morphogenetic Protein 4; D019485:Bone Morphogenetic Proteins; D016895:Culture Media, Serum-Free; D005326:Fetal Proteins; D053605:Leukemia Inhibitory Factor; D020825:T-Box Domain Proteins; D013926:Thrombopoietin; D042461:Vascular Endothelial Growth Factor A; D040301:Vascular Endothelial Growth Factor Receptor-2; D000098908:Brachyury Protein; C525809:BMP4 protein, human; C525810:Bmp4 protein, mouse