Purpura Kelly A, Morin Jennifer, Zandstra Peter W
Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada.
Exp Hematol. 2008 Sep;36(9):1186-98. doi: 10.1016/j.exphem.2008.04.003. Epub 2008 Jun 11.
To develop a robust serum-free (SF) system for generation of hemogenic mesoderm and blood progenitors from pluripotent cells.
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.
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.
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.
开发一种强大的无血清（SF）系统，用于从多能细胞生成造血中胚层和血液祖细胞。
将维持在添加白血病抑制因子（LIF）和骨形态发生蛋白（BMP）-4的N2B27培养基中的胚胎干细胞（ESC），通过去除LIF、改变基础培养基配方以及按时间和浓度依赖性添加其他因子，诱导分化为表达Brachyury/T的细胞（使用绿色荧光蛋白报告基因检测）和髓系-红系集落形成细胞（ME-CFC）。
存在10 ng/mL BMP-4时，可出现表达T和血管内皮生长因子受体（VEGFR）-2的细胞，但在第4天，<5%的细胞为双阳性。调整SF培养基配方后，仅5 ng/mL BMP-4在第4天就能产生24%±4%的Brachyury-绿色荧光蛋白VEGFR-2(+)细胞。这些细胞可发育为ME-CFC，在第8天每1000个细胞产生4.4±0.8个集落形成细胞（CFC）。我们还研究了分化过程中BMP-4、血管内皮生长因子（VEGF）和血小板生成素（TPO）的时间和浓度敏感性。50 ng/mL TPO与BMP-4共同作用时，每5×10⁴个细胞产生232±48个CFC，与血清对照组相似，早期（第0 - 5天之间）而非晚期（第5天后）添加VEGF可将该反应增强至每5×10⁴个细胞产生292±42个CFC。
转向SF系统通过消除混杂信号促进定向分化。本文描述了对N2B27培养基的改良，该改良增强了中胚层诱导，并扩展了早期关于用BMP-4、VEGF和TPO从ESC诱导血液祖细胞的研究工作。
