Single-Nuclear RNA Sequencing of Endomyocardial Biopsies Identifies Persistence of Donor-Recipient Chimerism With Distinct Signatures in Severe Cardiac Allograft Vasculopathy.
Single-Nuclear RNA Sequencing of Endomyocardial Biopsies Identifies Persistence of Donor-Recipient Chimerism With Distinct Signatures in Severe Cardiac Allograft Vasculopathy.
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心内膜心肌活检的单核 RNA 测序鉴定了严重心脏同种异体移植血管病中具有独特特征的供体-受体嵌合体的持续存在。
DOI：
10.1161/circheartfailure.122.010119
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发表时间：
2023
期刊：
影响因子：
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通讯作者：
Moslehi,Javid
中科院分区：
文献类型：
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作者：
Amancherla,Kaushik;Qin,Juan;Hulke,MichelleL;Pfeiffer,RyanD;Agrawal,Vineet;Sheng,Quanhu;Xu,Yaomin;Schlendorf,KellyH;Lindenfeld,JoAnn;Shah,RaviV;Freedman,JaneE;Tucker,NathanR;Moslehi,Javid
心脏同种异体移植血管病变（CAV）是心脏移植后迟发同种异体移植失败和死亡的主要原因。 1 组织学上，CAV 是一种慢性血管排斥反应，其特征是大血管和微血管的弥漫性内膜增厚。虽然体外细胞模型和体内组织学观察表明内皮细胞、成纤维细胞和平滑肌细胞在 CAV 病理学中具有协调反应，但尚未研究移植的人心脏中这些细胞之间的细胞特异性转录特征。由于目前 CAV 的诊断和治疗标准有很大的局限性，了解细胞特异性反应可能对于开发改进的检测策略和新的治疗方法至关重要。心脏移植受者接受常规心内膜心肌活检 (EMB) 以进行排斥反应监测，这为直接对人类心肌进行深度分子表型分析提供了独特的机会。在这里，我们证明了对常规临床实践中获得的人类 EMB 进行单核 RNA 测序的可行性。我们将 4 名严重 CAV 个体再移植时获得的组织与 3 名无 CAV 移植后个体的 EMB 标本进行了比较（图 [A]）。从右心室获取样本。 4 名严重 CAV 患者中，有 3 名还采集了左心室样本（总共 10 个样本）。将大约 3 至 10 mg 的 EMB 组织和 25 mg 的外植组织安装在低温恒温器上，然后切成 100 μm 的切片。然后用松散和紧密的杵对组织进行几轮杜恩斯均质化，然后顺序过滤碎片（100μm，20μm）。在倾倒过程中对原子核的释放进行监测，以尽量减少核损伤。将细胞核转移至冷重悬缓冲液中并在血细胞计数器上计数，然后装入 10× Genomics 单细胞 3'v3。 1 个平台，预计每个样品可回收 8000 个细胞核。文库根据制造商的说明进行处理，并进行如前所述的修改。 2 Illumina 测序的目标是每个核 25,000 个读数。原始 FASTQ 文件存放在美国国立卫生研究院/国家生物技术中心基因表达综合数据存储库 (GSE203548)。详细的核分离方法和用于分析的代码存放在 https://github 上。 com/learning-MD/CAV。这项研究得到了范德比尔特大学医学中心机构审查委员会的批准。我们成功分离了 62 465 个细胞核，并鉴定了 17 种具有异质分布的主要细胞类型
Cardiac allograft vasculopathy (CAV) is the leading cause of late allograft failure and mortality after heart transplantation. 1 Histologically, CAV is chronic vascular rejection characterized by diffuse intimal thickening of macro-and microvasculature. While in vitro cellular models and in vivo histologic observations suggest coordinated responses of endothelial, fibroblast, and smooth muscle cells in CAV pathology, cell-specific transcriptional signatures among these in the transplanted human heart have not been studied. As current standards of diagnosis and treatment of CAV have significant limitations, understanding cell-specific responses may prove critical for developing improved detection strategies and novel therapeutics. Recipients of heart transplantation undergo routine endomyocardial biopsies (EMBs) for rejection surveillance, offering a unique opportunity to perform deep molecular phenotyping of human myocardium directly. Here, we demonstrate the feasibility of performing single-nuclear RNA sequencing of human EMBs obtained during routine clinical practice. We compared tissue obtained at the time of re-transplantation from 4 individuals with severe CAV to EMB specimens from 3 individuals post-transplant without CAV (Figure [A]). Samples were obtained from the right ventricle. In 3 out of 4 patients with severe CAV, left ventricular samples were also obtained (for a total 10 samples). Approximately 3 to 10 mg of EMB tissue and 25 mg of explanted tissue were mounted onto a Cryostat, followed by sectioning into 100 μm sections. The tissue was then disrupted by several rounds of dounce homogenization with loose and tight pestles before sequential filtration of debris (100 μm, 20 μm). Liberation of nuclei was monitored over the course of douncing to minimize nuclear damage. Nuclei were transferred to a cold resuspension buffer and counted on a hemocytometer before being loaded into a 10× Genomics single cell 3’v3. 1 platform for an estimated recovery of 8000 nuclei per sample. Libraries were processed according to manufacturer’s instructions, with modifications as described previously. 2 Illumina sequencing targeted 25 000 reads per nucleus. Raw FASTQ files are deposited at the National Institutes of Health/National Center for Biotechnology Gene Expression Omnibus data repository (GSE203548). Detailed nuclear isolation methods and code used for analyses are deposited at https://github. com/learning-MD/CAV. This study was approved by the Vanderbilt University Medical Center’s Institutional Review Board. We successfully isolated 62 465 nuclei and identified 17 major cell types with heterogenous distribution