Abstract：
BACKGROUND: This study investigates the role of CXXC5 in the self-renewal and differentiation of hematopoietic stem cells (HSCs) within the bone marrow microenvironment, utilizing advanced methodologies such as single-cell RNA sequencing (scRNA-seq), CRISPR-Cas9, and proteomic analysis.
METHODS: We employed flow cytometry to isolate HSCs from bone marrow samples, followed by scRNA-seq analysis using the 10x Genomics platform to examine cell clustering and CXXC5 expression patterns. CRISPR-Cas9 and lentiviral vectors facilitated the knockout and overexpression of CXXC5 in HSCs. The impact on HSCs was assessed through qRT-PCR, Western blot, CCK-8, CFU, and LTC-IC assays, alongside flow cytometry to measure apoptosis and cell proportions. A mouse model was also used to evaluate the effects of CXXC5 manipulation on HSC engraftment and survival rates.
RESULTS: Our findings highlight the diversity of cell clustering and the significant role of CXXC5 in HSC regulation. Knockout experiments showed reduced proliferation and accelerated differentiation, whereas overexpression led to enhanced proliferation and delayed differentiation. Proteomic analysis identified key biological processes influenced by CXXC5, including cell proliferation, differentiation, and apoptosis. In vivo results demonstrated that CXXC5 silencing impaired HSC engraftment in a bone marrow transplantation model.
CONCLUSIONS: CXXC5 is crucial for the regulation of HSC self-renewal and differentiation in the bone marrow microenvironment. Its manipulation presents a novel approach for enhancing HSC function and provides a potential therapeutic target for hematological diseases.
METHODS: We employed flow cytometry to isolate HSCs from bone marrow samples, followed by scRNA-seq analysis using the 10x Genomics platform to examine cell clustering and CXXC5 expression patterns. CRISPR-Cas9 and lentiviral vectors facilitated the knockout and overexpression of CXXC5 in HSCs. The impact on HSCs was assessed through qRT-PCR, Western blot, CCK-8, CFU, and LTC-IC assays, alongside flow cytometry to measure apoptosis and cell proportions. A mouse model was also used to evaluate the effects of CXXC5 manipulation on HSC engraftment and survival rates.
RESULTS: Our findings highlight the diversity of cell clustering and the significant role of CXXC5 in HSC regulation. Knockout experiments showed reduced proliferation and accelerated differentiation, whereas overexpression led to enhanced proliferation and delayed differentiation. Proteomic analysis identified key biological processes influenced by CXXC5, including cell proliferation, differentiation, and apoptosis. In vivo results demonstrated that CXXC5 silencing impaired HSC engraftment in a bone marrow transplantation model.
CONCLUSIONS: CXXC5 is crucial for the regulation of HSC self-renewal and differentiation in the bone marrow microenvironment. Its manipulation presents a novel approach for enhancing HSC function and provides a potential therapeutic target for hematological diseases.
摘要：
背景：这项研究调查了CXXC5在骨髓微环境中造血干细胞（HSC）自我更新和分化中的作用，利用先进的方法，如单细胞RNA测序（scRNA-seq），CRISPR-Cas9和蛋白质组学分析。
方法：我们采用流式细胞术从骨髓样本中分离HSC，然后使用10x基因组学平台进行scRNA-seq分析，以检查细胞聚类和CXXC5表达模式。CRISPR-Cas9和慢病毒载体促进HSC中CXXC5的敲除和过表达。通过qRT-PCR评估对HSC的影响，蛋白质印迹,CCK-8,CFU,和LTC-IC分析，与流式细胞术一起测量细胞凋亡和细胞比例。还使用小鼠模型来评估CXXC5操作对HSC植入和存活率的影响。
结果：我们的发现强调了细胞聚集的多样性以及CXXC5在HSC调节中的重要作用。基因敲除实验显示增殖减少，分化加速，而过度表达导致增殖增强和分化延迟。蛋白质组学分析确定了受CXXC5影响的关键生物过程，包括细胞增殖，分化,和凋亡。体内结果表明，CXXC5沉默会损害骨髓移植模型中的HSC移植。
结论：CXXC5对于调节骨髓微环境中的HSC自我更新和分化至关重要。它的操作提供了一种增强HSC功能的新方法，并为血液病提供了潜在的治疗靶标。
方法：我们采用流式细胞术从骨髓样本中分离HSC，然后使用10x基因组学平台进行scRNA-seq分析，以检查细胞聚类和CXXC5表达模式。CRISPR-Cas9和慢病毒载体促进HSC中CXXC5的敲除和过表达。通过qRT-PCR评估对HSC的影响，蛋白质印迹,CCK-8,CFU,和LTC-IC分析，与流式细胞术一起测量细胞凋亡和细胞比例。还使用小鼠模型来评估CXXC5操作对HSC植入和存活率的影响。
结果：我们的发现强调了细胞聚集的多样性以及CXXC5在HSC调节中的重要作用。基因敲除实验显示增殖减少，分化加速，而过度表达导致增殖增强和分化延迟。蛋白质组学分析确定了受CXXC5影响的关键生物过程，包括细胞增殖，分化,和凋亡。体内结果表明，CXXC5沉默会损害骨髓移植模型中的HSC移植。
结论：CXXC5对于调节骨髓微环境中的HSC自我更新和分化至关重要。它的操作提供了一种增强HSC功能的新方法，并为血液病提供了潜在的治疗靶标。
