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Abstract：
BACKGROUND: Bone marrow-derived mesenchymal stem cell (BMMSC)-based therapy has become a major focus for treating liver fibrosis/cirrhosis. However, although these cell therapies promote the treatment of this disease, the heterogeneity of BMMSCs, which causes insufficient efficacy during clinical trials, has not been addressed. In this study, we describe a novel Percoll-Plate-Wait procedure (PPWP) for the isolation of an active cell subset from BMMSC cultures that was characterized by the expression of neuroglial antigen 2 (NG2/BMMSCs).
METHODS: By using the key method of PPWP and other classical biological techniques we compared NG2/BMMSCs with parental BMMSCs in biological and functional characteristics within a well-defined diethylnitrosamine (DEN)-induced liver fibrosis/cirrhosis injury male C57BL/6 mouse model also in a culture system. Of note, the pathological alterations in the model is quite similar to humans\'.
RESULTS: The NG2/BMMSCs revealed more advantages compared to parentalBMMSCs. They exhibited greater proliferation potential than parental BMMSCs, as indicated by Ki-67 immunofluorescence (IF) staining. Moreover, higher expression of SSEA-3 (a marker specific for embryonic stem cells) was detected in NG2/BMMSCs than in parental BMMSCs, which suggested that the \"stemness\" of NG2/BMMSCs was greater than that of parental BMMSCs. In vivo studies revealed that an injection of NG2/BMMSCs into mice with ongoing DEN-induced liver fibrotic/cirrhotic injury enhanced repair and functional recovery to a greater extent than in mice treated with parental BMMSCs. These effects were associated with the ability of NG2/BMMSCs to differentiate into bile duct cells (BDCs). In particular, we discovered for the first time that NG2/BMMSCs exhibit unique characteristics that differ from those of parental BMMSCs in terms of producing liver sinusoidal endothelial cells (LSECs) to reconstruct injured blood vessels and sinusoidal structures in the diseased livers, which are important for initiating hepatocyte regeneration. This unique potential may also suggest that NG2/BMMSCs could be an novel off-liver progenitor of LSECs. Ex vivo studies revealed that the NG2/BMMSCs exhibited a similar trend to that of their in vivo in terms of functional differentiation responding to the DEN-diseased injured liver cues. Additionally, the obvious core role of NG2/BMMSCs in supporting the functions of BMMSCs in bile duct repair and BDC-mediated hepatocyte regeneration might also be a novel finding.
CONCLUSIONS: Overall, the PPWP-isolated NG2/BMMSCs could be a novel effective cell subset with increased purity to serve as a new therapeutic tool for enhancing treatment efficacy of BMMSCs and special seed cell source (BDCs, LSECs) also for bioliver engineering.
METHODS: By using the key method of PPWP and other classical biological techniques we compared NG2/BMMSCs with parental BMMSCs in biological and functional characteristics within a well-defined diethylnitrosamine (DEN)-induced liver fibrosis/cirrhosis injury male C57BL/6 mouse model also in a culture system. Of note, the pathological alterations in the model is quite similar to humans\'.
RESULTS: The NG2/BMMSCs revealed more advantages compared to parentalBMMSCs. They exhibited greater proliferation potential than parental BMMSCs, as indicated by Ki-67 immunofluorescence (IF) staining. Moreover, higher expression of SSEA-3 (a marker specific for embryonic stem cells) was detected in NG2/BMMSCs than in parental BMMSCs, which suggested that the \"stemness\" of NG2/BMMSCs was greater than that of parental BMMSCs. In vivo studies revealed that an injection of NG2/BMMSCs into mice with ongoing DEN-induced liver fibrotic/cirrhotic injury enhanced repair and functional recovery to a greater extent than in mice treated with parental BMMSCs. These effects were associated with the ability of NG2/BMMSCs to differentiate into bile duct cells (BDCs). In particular, we discovered for the first time that NG2/BMMSCs exhibit unique characteristics that differ from those of parental BMMSCs in terms of producing liver sinusoidal endothelial cells (LSECs) to reconstruct injured blood vessels and sinusoidal structures in the diseased livers, which are important for initiating hepatocyte regeneration. This unique potential may also suggest that NG2/BMMSCs could be an novel off-liver progenitor of LSECs. Ex vivo studies revealed that the NG2/BMMSCs exhibited a similar trend to that of their in vivo in terms of functional differentiation responding to the DEN-diseased injured liver cues. Additionally, the obvious core role of NG2/BMMSCs in supporting the functions of BMMSCs in bile duct repair and BDC-mediated hepatocyte regeneration might also be a novel finding.
CONCLUSIONS: Overall, the PPWP-isolated NG2/BMMSCs could be a novel effective cell subset with increased purity to serve as a new therapeutic tool for enhancing treatment efficacy of BMMSCs and special seed cell source (BDCs, LSECs) also for bioliver engineering.
摘要：
背景：基于骨髓间充质干细胞（BMMSC）的治疗已成为治疗肝纤维化/肝硬化的主要焦点。然而,尽管这些细胞疗法促进了这种疾病的治疗，BMMSCs的异质性，导致临床试验中疗效不足，尚未解决。在这项研究中,我们描述了一种新的Percoll-Plate-Wait程序（PPWP），用于从BMMSC培养物中分离活性细胞亚群，其特征在于神经胶质抗原2（NG2/BMMSC）的表达。
方法：通过使用PPWP和其他经典生物学技术的关键方法，我们在明确定义的二乙基亚硝胺（DEN）诱导的肝纤维化/肝硬化雄性C57BL/6小鼠模型中也在培养系统中比较了NG2/BMMSCs与亲本BMMSCs的生物学和功能特征。值得注意的是，模型中的病理改变与人类非常相似。
结果：NG2/BMMSCs比肠胃外BMMSCs更有优势。它们表现出比亲本BMMSCs更大的增殖潜力，如Ki-67免疫荧光（IF）染色所示。此外，与亲本BMMSCs相比，在NG2/BMMSCs中检测到更高的SSEA-3（胚胎干细胞特异性标志物）表达，这表明NG2/BMMSCs的“干性”大于亲本BMMSCs。体内研究表明，与用亲本BMMSC治疗的小鼠相比，向正在进行的DEN诱导的肝纤维化/肝硬化损伤的小鼠注射NG2/BMMSC在更大程度上增强了修复和功能恢复。这些作用与NG2/BMMSCs分化为胆管细胞（BDCs）的能力有关。特别是,我们首次发现NG2/BMMSCs在产生肝窦内皮细胞（LSEC）以重建患病肝脏中受损的血管和窦结构方面表现出与亲本BMMSCs不同的独特特征，这对启动肝细胞再生很重要。这种独特的潜力也可能表明NG2/BMMSC可能是LSEC的新型肝外祖细胞。离体研究表明，NG2/BMMSC在响应DEN患病的受损肝脏线索的功能分化方面表现出与其体内相似的趋势。此外,NG2/BMMSCs在支持BMMSCs在胆管修复和BDC介导的肝细胞再生中的重要作用也可能是一个新发现。
结论：总体而言，PPWP分离的NG2/BMMSCs可能是一种新型的有效细胞亚群，纯度更高，可作为一种新的治疗工具，用于增强BMMSCs和特殊种子细胞来源的治疗功效（BDCs，LSEC）也用于生物工程。
方法：通过使用PPWP和其他经典生物学技术的关键方法，我们在明确定义的二乙基亚硝胺（DEN）诱导的肝纤维化/肝硬化雄性C57BL/6小鼠模型中也在培养系统中比较了NG2/BMMSCs与亲本BMMSCs的生物学和功能特征。值得注意的是，模型中的病理改变与人类非常相似。
结果：NG2/BMMSCs比肠胃外BMMSCs更有优势。它们表现出比亲本BMMSCs更大的增殖潜力，如Ki-67免疫荧光（IF）染色所示。此外，与亲本BMMSCs相比，在NG2/BMMSCs中检测到更高的SSEA-3（胚胎干细胞特异性标志物）表达，这表明NG2/BMMSCs的“干性”大于亲本BMMSCs。体内研究表明，与用亲本BMMSC治疗的小鼠相比，向正在进行的DEN诱导的肝纤维化/肝硬化损伤的小鼠注射NG2/BMMSC在更大程度上增强了修复和功能恢复。这些作用与NG2/BMMSCs分化为胆管细胞（BDCs）的能力有关。特别是,我们首次发现NG2/BMMSCs在产生肝窦内皮细胞（LSEC）以重建患病肝脏中受损的血管和窦结构方面表现出与亲本BMMSCs不同的独特特征，这对启动肝细胞再生很重要。这种独特的潜力也可能表明NG2/BMMSC可能是LSEC的新型肝外祖细胞。离体研究表明，NG2/BMMSC在响应DEN患病的受损肝脏线索的功能分化方面表现出与其体内相似的趋势。此外,NG2/BMMSCs在支持BMMSCs在胆管修复和BDC介导的肝细胞再生中的重要作用也可能是一个新发现。
结论：总体而言，PPWP分离的NG2/BMMSCs可能是一种新型的有效细胞亚群，纯度更高，可作为一种新的治疗工具，用于增强BMMSCs和特殊种子细胞来源的治疗功效（BDCs，LSEC）也用于生物工程。
