Gutierrez-Agüera Francisco, Rodriguez-Cortez Virginia, Petazzi Paolo, Bueno Clara, Menendez Pablo
Josep Carreras Research Institute, Barcelona, Spain.
Centro de Investigación Biomédica en Red de Cáncer (CIBER-ONC), Instituto de Salud Carlos III (ISCIII), Barcelona, Spain.
Front Cell Dev Biol. 2021 May 21;9:636704. doi: 10.3389/fcell.2021.636704. eCollection 2021.
The generation of transplantable hematopoietic stem cells (HSCs) from human pluripotent stem cells (hPSCs) remains challenging. Current differentiation protocols from hPSCs generate mostly hematopoietic progenitors of the primitive HSC-independent program, and it remains unclear what is the best combination of cytokines and hematopoietic growth factors (HGFs) for obtaining functional hematopoietic cells . Here, we have used the AND1 and H9 hESC lines and the H9:dual-reporter -GFP--Cherry to compare the hematopoietic differentiation based on the treatment of embryoid bodies (EBs) with the ventral mesoderm inducer BMP4 plus HGFs in the absence (protocol 1) or presence (protocol 2) of stage-specific activation of Wnt/β-catenin and inhibition of Activin/Nodal. Despite a slight trend in favor of protocol 1, no statistically significant differences were observed between protocols at any time point analyzed throughout EB development regarding the frequency of hemogenic endothelial (HE) precursors; CD43+ CD45-, CD45+, and CD45 + CD34 + hematopoietic derivatives; or the output of clonogenic progenitors. Similarly, the kinetics of emergence throughout EB development of both + HE and + definitive hematopoiesis was very similar for both protocols. The expression of the early master mesendodermal transcription factors Brachyury, MIXL1, and KDR revealed similar gene expression kinetics prior to the emergence of + definitive hematopoiesis for both protocols. Collectively, the simpler protocol 1 is, at least, as efficient as protocol 2, suggesting that supplementation with additional morphogens/HGFs and modulation of Activin/Nodal and Wnt/β-catenin pathways seem dispensable for hematopoietic differentiation of hPSCs.
从人多能干细胞（hPSC）生成可移植的造血干细胞（HSC）仍然具有挑战性。目前从hPSC进行分化的方案大多产生的是原始的不依赖HSC程序的造血祖细胞，对于获得功能性造血细胞而言，哪种细胞因子和造血生长因子（HGF）的最佳组合仍不清楚。在这里，我们使用了AND1和H9人胚胎干细胞系以及H9:双报告基因-GFP-樱桃红，以比较基于用腹侧中胚层诱导剂BMP4加HGF处理胚状体（EB）的造血分化情况，分别在不存在（方案1）或存在（方案2）Wnt/β-连环蛋白的阶段特异性激活和激活素/节点抑制的情况下进行。尽管有略微倾向于方案1的趋势，但在整个EB发育过程中分析的任何时间点，就造血内皮（HE）前体的频率、CD43 + CD45-、CD45 +和CD45 + CD34 +造血衍生物或克隆形成祖细胞的产量而言，两种方案之间均未观察到统计学上的显著差异。同样，两种方案中+ HE和+确定性造血在整个EB发育过程中的出现动力学非常相似。在+确定性造血出现之前，两种方案中早期主中胚层转录因子Brachyury、MIXL1和KDR的表达显示出相似的基因表达动力学。总体而言，更简单的方案1至少与方案2一样有效，这表明补充额外的形态发生素/HGF以及调节激活素/节点和Wnt/β-连环蛋白途径对于hPSC的造血分化似乎是不必要的。
