Impact of Tissue Factor Gene Knockout on Coagulation Properties of Umbilical Cord-Derived Multipotent Mesenchymal Stromal/Stem Cells

IF 2.8 3区 生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY Cell Biochemistry and Function Pub Date : 2024-12-11 DOI:10.1002/cbf.70021
Zahra Heidari, Jafar Fallahi, Mohsen Sisakht, Fatemeh Safari, Kamran Hosseini, Ardeshir Bahmanimehr, Amir Savardashtaki, Sahar Khajeh, Seyed Mohammad Bagher Tabei, Vahid Razban
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Abstract

Multipotent mesenchymal stromal/stem cells (MSCs) refer to a population of stem cells that exhibit distinct progenitor cell characteristics including the potential for differentiation into a wide range of cell types. MSCs have become a promising candidate for cell therapy and tissue regeneration due to their unique properties, such as their ability to differentiate into multiple cell types, their capacity for expansion, self-renewal, and immune-regulatory effects. However, reports have brought attention to thrombosis-related complications associated with MSCs therapy in the last decade. As tissue factor (TF) is a powerful coagulation activator expressed by MSCs that stimulates the extrinsic coagulation pathway, we investigated the thrombotic properties of human umbilical cord MSCs (HUCMSCs) after knocking out the TF gene. MSCs populations that obtained from umbilical cord were cultured and expanded in the appropriate medium cell culture. The identity of the MSCs was verified through flow cytometry, and their ability to differentiate into osteogenic and adipogenic lineages. Two gRNAs for Exons 1 and 2 of the TF gene have been designed and cloned into px458 vector's backbone (pSpCas9 (BB)−2A-GFP). Following transfecting of gRNAs into HUCMSCs and successfully knocking out the TF gene using GAP-PCR, the impact of normal and knockout HUCMSCs on coagulation was assessed through prothrombin time (PT), D-dimer level, clotting time (CT), and turbidity assay. Furthermore, the impact of TF knockout (TFKO) on MMP19 expression was assessed. Our results revealed that the PT was prolonged and D-dimer level was decreased in TFKO group compared to normal HUCMSCs. These findings suggest that TF gene plays a crucial role in regulating coagulation in HUCMSCs. Also, a significant reduction in MMP19 expression was observed within the TFKO group.

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组织因子基因敲除对脐带源性多能间充质基质/干细胞凝血特性的影响
多能间充质基质/干细胞(MSCs)是指具有明显祖细胞特征的干细胞群,包括分化为多种细胞类型的潜力。由于其独特的特性,如分化成多种细胞类型的能力、增殖能力、自我更新能力和免疫调节作用,间充质干细胞已成为细胞治疗和组织再生的一个有希望的候选者。然而,在过去的十年中,有报道引起了人们对与MSCs治疗相关的血栓相关并发症的关注。由于组织因子(TF)是一种强大的凝血激活因子,由MSCs表达,刺激外源性凝血途径,我们在敲除TF基因后研究了人脐带MSCs (HUCMSCs)的凝血特性。从脐带中获得的MSCs群体在适当的培养基细胞培养中培养和扩增。通过流式细胞术验证了MSCs的身份,以及它们分化成成骨和脂肪谱系的能力。设计了TF基因外显子1和2的两个grna,并将其克隆到px458载体的主干(pSpCas9 (BB)-2A-GFP)中。将grna转染到HUCMSCs中并使用GAP-PCR成功敲除TF基因后,通过凝血酶原时间(PT)、d -二聚体水平、凝血时间(CT)和浊度测定来评估正常和敲除的HUCMSCs对凝血的影响。此外,我们还评估了TF敲除(TFKO)对MMP19表达的影响。结果显示,与正常HUCMSCs相比,TFKO组PT时间延长,d -二聚体水平降低。这些发现表明,TF基因在调节HUCMSCs的凝血中起着至关重要的作用。此外,在TFKO组中观察到MMP19表达显著降低。
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来源期刊
Cell Biochemistry and Function
Cell Biochemistry and Function 生物-生化与分子生物学
CiteScore
6.20
自引率
0.00%
发文量
93
审稿时长
6-12 weeks
期刊介绍: Cell Biochemistry and Function publishes original research articles and reviews on the mechanisms whereby molecular and biochemical processes control cellular activity with a particular emphasis on the integration of molecular and cell biology, biochemistry and physiology in the regulation of tissue function in health and disease. The primary remit of the journal is on mammalian biology both in vivo and in vitro but studies of cells in situ are especially encouraged. Observational and pathological studies will be considered providing they include a rational discussion of the possible molecular and biochemical mechanisms behind them and the immediate impact of these observations to our understanding of mammalian biology.
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