Ex vivo development, expansion and in vivo analysis of a novel lineage of dendritic cells from hematopoietic stem cells.

Shuhong Han, Yichen Wang, Bei Wang, Ekta Patel, Starlyn Okada, Li-Jun Yang, Jan S Moreb, Lung-Ji Chang
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引用次数: 7

Abstract

Dendritic cells (DCs) play a key role in innate and adaptive immunity but the access to sufficient amount of DCs for basic and translational research has been limited.We established a novel ex vivo system to develop and expand DCs from hematopoietic stem/progenitor cells (HPCs). Both human and mouse HPCs were expanded first in feeder culture supplemented with c-Kit ligand (KL, stem cell factor, steel factor or CD117 ligand), Flt3 ligand (fms-like tyrosine kinase 3, Flt3L, FL), thrombopoietin (TPO), IL-3, IL-6, and basic fibroblast growth factor (bFGF), and then in a second feeder culture ectopically expressing all above growth factors plus GM-CSF and IL-15.In the dual culture system, CD34+ HPCs differentiated toward DC progenitors (DCPs), which expanded more than five orders of magnitude. The DCPs showed myeloid DC surface phenotype with up-regulation of transcription factors PU.1 and Id2, and DC-related factors homeostatic chemokine ligand 17 (CCL17) and beta-chemokine receptor 6 (CCR6). Multiplex ELISA array and cDNA microarray analyses revealed that the DCPs shared some features of IL-4 and IL-15 DCs but displayed a pronounced proinflammatory phenotype. DCP-derived DCs showed antigen-uptake and immune activation functions analogous to that of the peripheral blood-derived DCs. Furthermore, bone marrow HPC-derived DCP vaccines of tumor-bearing mice suppressed tumor growth in vivo.This novel approach of generating DCP-DCs, which are different from known IL-4 and IL-15 DCs, overcomes both quantitative and qualitative limitations in obtaining functional autologous DCs from a small number of HPCs with great translational potential.

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造血干细胞树突状细胞新谱系的体外发育、扩增和体内分析。
树突状细胞(dc)在先天免疫和适应性免疫中发挥关键作用,但获得足够数量的树突状细胞用于基础和转化研究的途径有限。我们建立了一种新的体外系统,从造血干细胞/祖细胞(HPCs)中培养和扩增DCs。人和小鼠的HPCs首先在添加c-Kit配体(KL,干细胞因子,钢因子或CD117配体),Flt3配体(fms样酪氨酸激酶3,Flt3L, FL),血小板生成素(TPO), IL-3, IL-6和碱性成纤维细胞生长因子(bFGF)的饲养培养基中扩增,然后在第二个饲养培养基中异位表达所有上述生长因子以及GM-CSF和IL-15。在双重培养体系中,CD34+ HPCs分化为DC祖细胞(dcp),扩展超过5个数量级。dcp表现髓样DC表面表型,转录因子PU.1和Id2上调,DC相关因子稳态趋化因子配体17 (CCL17)和趋化因子受体6 (CCR6)上调。多重ELISA阵列和cDNA芯片分析显示,dcp具有IL-4和IL-15 dc的一些特征,但表现出明显的促炎表型。dcp来源的dc具有类似于外周血来源的dc的抗原摄取和免疫激活功能。此外,荷瘤小鼠骨髓hpc来源的DCP疫苗在体内抑制肿瘤生长。这种生成dcp - dc的新方法不同于已知的IL-4和IL-15 dc,克服了从少量具有巨大转化潜力的HPCs中获得功能性自体dc的定量和定性限制。
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