Immunological and ultrastructural characterization of endothelial cell cultures differentiated from human cord blood derived endothelial progenitor cells
Immunological and ultrastructural characterization of endothelial cell cultures differentiated from human cord blood derived endothelial progenitor cells
复制标题
DOI：
10.1007/s00418-006-0201-6
复制
发表时间：
2006-12-01
影响因子：
2.3
通讯作者：
Huber, Johannes
中科院分区：
文献类型：
--
作者：
Neumueller, Josef;Neumueller-Guber, Sylvia-Emanuela;Huber, Johannes
用内皮祖细胞替代内皮细胞以改善缺血器官的血管状态是目前血管研究的热点。本研究的目的是探讨来源于外周血单个核细胞（PBMNCs-衍生的EPCs）或来源于CD 34（+）造血干细胞（HSCs-衍生的EPCs）的EPCs（两者均分离自人脐带血）是否能够在一定的体外条件下分化为早期成熟的内皮细胞（ECs）。我们通过流式细胞术、相差显微镜、荧光显微镜和共聚焦激光扫描显微镜以及透射和扫描电子显微镜的超微结构来表征这两种细胞群。虽然PBMNC在几天后产生梭形样EPC簇，但在体外条件下没有进一步成熟，但成熟的EC只能从分离的HSC的起始群体成功繁殖。PBMNCs和HSC来源的EPCs均能摄取Dil标记的乙酰化低密度脂蛋白（Dil-Ac-LDL），并能在细胞表面对CD 31、CD 105、血管内皮生长因子受体2（VEGFR-2，KDR）和荆豆凝集素1（UEA-1）进行阳性染色。EPC表面表达CD 54和CD 106。然而，只有一小部分HSC来源的EPCs为CD 54阳性，而CD 106为阴性。血管性血友病因子（vWF）的细胞内染色在PBMNC来源的EPCs中提供了均匀的染色，而在HSC来源的EPCs中，在培养2-3周期间，越来越多的与韦伯-帕拉德小体（WPB）相关的典型点状染色模式可见。通过相差显微镜和扫描电镜观察，PBMNCs来源的EPCs呈索状排列。在这些形成中，细胞显示出平行排列，但仅表现出很少的细胞接触。在PBMNCs来源的EPCs中从未发现发育良好的WPB。与此相反，分化的HSC衍生的EPCs发展粘附连接，交错连接以及韧带。在成熟过程中，纺锤形细胞类型出现丰富的WPB以及鹅卵石样细胞类型与这些细胞器的含量较少。WPB，在梭形细胞类型显示出明显的形状，并集中在靠近富含维生素A的区域。HSC来源的EPCs表现出高合成活性的迹象，如发达的粗面内质网（RER）和多个高尔基复合体。在反式高尔基体网络（TGN）中，靠近高尔基体复合体，可以观察到新的WPB形成。这些形态特征与高生长能力相关。虽然这是不可能的，以证明完整的分化线从HSC的早期成熟的EC的免疫标记，因为有限的细胞数量可用于这样的调查，不同的形态成熟阶段可以显示在光镜和电子显微镜水平。总之，这里提出的研究不仅表征了参与早期EPCs分化为成熟EC的不同细胞群体，而且还表征了通过证明WPB的新形成而发生成熟步骤的过渡阶段。在这方面，这些调查提供了新的见解，在体外分化可能有一些在体内的相关性。
The replacement of endothelium by endothelial progenitor cells (EPCs) for therapeutic use in order to ameliorate the vascular status of ischemic organs is now in the focus of vascular research. The aim of our studies was to investigate whether EPCs derived from peripheral blood mononuclear cells (PBMNCs-derived EPCs) or EPCs propagated from CD34(+) hematopoietic stem cells (HSCs-derived EPCs), both isolated from human cord blood, are able to differentiate into early mature endothelial cells (ECs) under certain in vitro conditions. We characterized both cell populations by flow cytometry, phase contrast microscopy, fluorescence microscopy and confocal laser scanning microscopy as well as ultrastructurally using transmission and scanning electron microscopy. While PBMNCs gave rise to clusters of spindle-like EPCs after few days but did not further mature under in vitro conditions, mature ECs could only be successfully propagated from a starting population of isolated HSCs. Both, PBMNCs- and HSCs-derived EPCs, took up Dil-labeled acetylated low density lipoprotein (Dil-Ac-LDL) and could be positively stained for CD31, CD105, the vascular endothelial growth factor receptor 2 (VEGFR-2, KDR) and ulex europaeus agglutinin 1 (UEA-1) at the cell surface. EPC showed surface expression of CD54 and CD106. However, only a small portion of HSCs-derived EPCs was positive for CD54 but negative for CD106. Intracellular staining for von Willebrand factor (vWF) provided a homogenous stain in PBMNC-derived EPCs while in HSCs-derived EPCs, during cultivation for 2-3 weeks, more and more a typical punctuated staining pattern related to Weibel-Palade bodies (WPBs) was visible. By phase contrast and scanning electron microscopy, an arrangement of PBMNCs-derived EPCs in cord-like structures could be demonstrated. In these formations, cells showed parallel alignment but exhibited only few cell contacts. Well-developed WPBs could never be found in PBMNCs-derived EPCs. In contrast, differentiating HSCs-derived EPCs developed adherence junctions, interdigitating junctions as well as syndesmos. During maturation, spindle-like cell types appeared with abundant WPBs as well as cobblestone-like cell types with a fewer content of these organelles. WPBs, in the spindle-like cell types displayed conspicuous shapes and were concentrated in close proximity to mitochondria-rich areas. HSCs-derived EPCs exhibited signs of high synthetic activity such as a well-developed rough endoplasmic reticulum (RER) and multiple Golgi complexes. In the trans-Golgi network (TGN), close to the Golgi complex, a new formation of WPBs could be observed. These morphological features correlated well with a high growing capacity. Although it was not possible to demonstrate the complete differentiation line from HSCs to early matured ECs by immunologic markers because of the limited number of cells available for such investigations, distinct morphologic maturation stages could be shown at light and electron microscopical levels. In conclusion, the study presented here characterizes not only the different cell populations involved in the differentiation of early EPCs into mature ECs but also the transition stage where the maturation step takes place by demonstration of the new formation of WPBs. In this respect, these investigations provide new insights into the in vitro differentiation which could have some in vivo correlation.