Fluorescent-activated cell sorting (FACS) remains a powerful tool to enrich blood-derived progenitor cells for the establishment of highly proliferative endothelial colony-forming cells (ECFC). Further investigation remains necessary to determine whether the retention of progenitor cell phenotypes after expansion can identify ECFC with enhanced proangiogenic and regenerative functions. This study employed FACS purification to segregate umbilical cord blood-derived ECFC using conserved provascular progenitor cell markers CD34 or aldehyde dehydrogenase (ALDH) activity. ECFC FACS purified for high versus low ALDH activity formed single cell-derived colonies and demonstrated tubule formation in Matrigel at comparable rates. Surprisingly, FACS purification of ECFC for CD34 enriched cells with enhanced colony-forming capabilities and tubule formation within the CD34⁻ population. CD34 expression was enriched on early ECFC populations; however, steady-state expression of CD34 rapidly declined and stabilized on expanded ECFC after serial passage. CD34 expression on ECFC was shown to be cell density dependent and coincided with a loss of progenitor cell characteristics in vitro. Silica-bead surface membrane capture followed by proteomic analysis by label-free liquid chromatography tandem mass spectrometry (LC-MS/MS) identified >100 distinctions (P < 0.05) associated with the plasma membrane of CD34⁻ versus CD34⁺ ECFC, including a significant enrichment of CD143 (angiotensinogen converting enzyme) on CD34⁺ cells. Despite an enrichment for traditional endothelial cell markers on the CD34⁺ ECFC in vitro, implantation of both CD34⁺ and CD34⁻ ECFC within Matrigel plugs in immunodeficient NOD.SCID mice promoted the formation of vessel-like structures with equivalent integration of human cells at 7 days post-transplantation. Although positive selection of CD34 enriched ECFC establishment before culture, FACS-purified CD34⁺ ECFC demonstrated reduced colony and tubule formation in vitro, yet demonstrated equivalent vessel formative function in vivo compared to CD34⁻ counterparts. The knowledge will support future studies aiming to identify ECFC subsets with enhanced vessel forming functions for applications of regenerative medicine.

中文翻译：

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脐带血衍生的内皮集落形成细胞体外扩增后 CD34 表达细胞亚群的纯化和功能表征。

荧光激活细胞分选 (FACS) 仍然是丰富血液源性祖细胞以建立高度增殖的内皮集落形成细胞 (ECFC) 的有力工具。还需要进一步研究以确定扩增后祖细胞表型的保留是否可以识别具有增强的促血管生成和再生功能的 ECFC。本研究采用 FACS 纯化，使用保守的促血管祖细胞标记 CD34 或醛脱氢酶 (ALDH) 活性分离脐带血来源的 ECFC。针对高与低 ALDH 活性进行纯化的 ECFC FACS 形成了单细胞衍生的集落，并在 Matrigel 中以可比的速率证明了小管形成。出奇，^-人口。CD34 表达在早期 ECFC 群体中富集；然而，CD34 的稳态表达在连续传代后迅速下降并稳定在扩大的 ECFC 上。ECFC 上的 CD34 表达被证明是细胞密度依赖性的，并且与体外祖细胞特征的丧失一致。二氧化硅珠表面膜捕获，然后通过无标记液相色谱串联质谱 (LC-MS/MS) 进行蛋白质组学分析，确定了 与 CD34 ^-与 CD34 ⁺ ECFC的质膜相关的> 100 个差异 ( P < 0.05) ，包括CD34 ⁺细胞上 CD143（血管紧张素原转化酶）的显着富集。尽管 CD34 上的传统内皮细胞标志物富集⁺ ECFC 体外，将 CD34 ⁺和 CD34 ^- ECFC植入免疫缺陷 NOD.SCID 小鼠的 Matrigel 栓内促进了血管样结构的形成，并在移植后 7 天促进了人体细胞的等效整合。尽管 CD34 的阳性选择在培养前丰富了 ECFC 的建立，但 FACS 纯化的 CD34 ⁺ ECFC 在体外表现出减少的集落和小管形成，但与 CD34 ^-对应物相比，在体内表现出等效的血管形成功能。这些知识将支持未来的研究，旨在识别具有增强血管形成功能的 ECFC 子集，以用于再生医学的应用。