造血干细胞能够自我更新并分化成所有血细胞谱系。造血干细胞移植是治疗造血系统恶性肿瘤和低增殖性疾病的主要手段。人多能干细胞(hPSC)的体外造血分化是用于模拟造血发育和细胞替代疗法的有希望的方法。尽管使用hPSC来获得造血祖细胞在过去已经取得了一些成功,从hPSC分化以产生可以提供稳健的长期多谱系移植的所有造血细胞仍然非常困难。这里,我们报道了一种在缺氧条件下具有最佳细胞因子组合的人胚胎干细胞(hESCs)造血分化的新型培养系统。
利用缺氧分化系统从hESCs体外生产T系造血干/祖细胞,通过RT-qPCR验证研究了低氧促进T谱系造血的作用和潜在机制,细胞周期测定和流式细胞术分析。
使用我们的分化系统,hESCs产生的CD45+细胞约80%为造血细胞,特别是在体外可进一步诱导为CD3+TCRαβ+T细胞。我们检测到更多的CD34+CD144+造血内皮祖细胞(HEP)诱导从hESCs比那些在常氧条件下,早期HEPs相关基因DLL4通过潜在的HIF-1α/NOTCH1/DLL4轴增强缺氧信号上调,因此在造血过程中驱动T谱系。引人注目的是,在我们的系统中产生的造血细胞表现出所有多谱系重建的潜力,包括淋巴,通过移植测定体内髓系和红系谱系。
我们的结果表明,缺氧通过激活HIF-1α信号通路促进动脉内皮基因DLL4的表达和NOTCH1的上调,在T谱系造血中起重要作用。这些结果提供了从hESC体外和体内生产全功能造血干/祖细胞的重要方法。
Hematopoietic stem cells are able to self-renew and differentiate into all blood cell lineages.
Hematopoietic stem cell transplantation is a mainstay of life-saving therapy for
hematopoietic malignancies and hypoproliferative disorders. In vitro
hematopoietic differentiation of human pluripotent stem cells (hPSCs) is a promising approach for modeling
hematopoietic development and cell replacement therapies. Although using hPSCs to derive
hematopoietic progenitor cells has achieved some successes in the past, differentiation from hPSCs to produce all hematopoietic cells which can provide robust long-term multilineage engraftment is still very difficult. Here, we reported a novel culture system for hematopoietic differentiation from human embryonic stem cells (hESCs) with optimal cytokines combinations under hypoxia condition.
In vitro production of T lineage hematopoietic stem/progenitor cells from hESCs by using hypoxia differentiation system, the effects and the potential mechanism of hypoxia promoting T lineage hematopoiesis were investigated by RT-qPCR validation, cell cycle assay and flow cytometry analysis.
Using our differentiation system, almost 80% CD45+ cells generated from hESCs were hematopoietic cells and particularly could be further induced into CD3+TCRαβ+ T cells in vitro. We detected more CD34+CD144+ hematopoietic endothelial progenitors (HEPs) induced from hESCs than those in normoxia conditions, and the early HEPs-related gene DLL4 was upregulated by enhancing the hypoxia signaling via potential HIF-1α/NOTCH1/DLL4 axis to enhance arterial feature, thus drove T lineage during the hematopoiesis. Strikingly, hematopoietic cells generated in our system exhibited the potential for all multilineage reconstruction including lymphoid, myeloid and erythroid lineages in vivo by transplantation assay.
Our results demonstrated that hypoxia plays an important role in T lineage hematopoiesis by promoting the expression of arterial endothelial gene DLL4 and upregulation of NOTCH1 through the activation of the HIF-1α signaling pathway. These results provide a significant approach for in vitro and in vivo production of fully functional hematopoietic stem/progenitor cells from hESCs.