背景：经典的Ph（Philadelphia）染色体阴性骨髓增殖性肿瘤（myeloproliferative neoplasm，MPN）是髓系造血干细胞的克隆性恶性疾病。这一类疾病被分为原发性血小板增多症（essential thrombocythemia，ET）、真性红细胞增多症（polycythemia vera，PV）和原发性骨髓纤维化（primary myelofibrosis，PMF）。骨髓纤维化包括PMF、ET后MF（post-ET MF）、PV后MF（post-PV MF）。骨髓纤维化的临床表现为贫血、肝脾大、血栓、出血等，其发生是由于骨髓网状纤维增加，进一步胶原纤维生成及骨硬化。骨髓微环境包括骨髓间充质基质细胞（mesenchymal stromal cell，MSC）、内皮细胞、血管旁细胞、胶质细胞等，以及分泌的一些细胞因子、血管、神经等，微环境的异常在骨髓纤维化的发生发展中有着重要作用。

目的：本研究旨在描绘MPN患者及小鼠骨髓基质细胞单细胞转录组图谱，分析与骨髓纤维化发生进展有关的细胞及机制。

方法：抽取MPN患者骨髓液，通过磁珠分选、流式分选去除造血细胞富集基质细胞，再进行10xGenomics单细胞转录组测序。取JAK2^V617F转基因鼠或CALR 52-bp deletion敲入鼠子代骨髓进行移植，产生不同疾病表型（ET、PV、MF）或非疾病小鼠。对移植后鼠同样进行磁珠分选（magnetic-activated cell sorting ，MACS）、流式分选（fluorescence-activated cell sorting，FACS）去除造血细胞富集基质细胞，再进行10xGenomics单细胞转录组测序。单细胞转录组数据分析包括原始数据质控、降维、去批次、聚类、差异基因表达分析、细胞发育轨迹分析、基因集评分、差异基因富集分析、细胞通讯分析、群相关性分析。

结果：本研究纳入非骨髓纤维化MPN患者5例（4例ET、1例PV），骨髓纤维化患者3例（post-ET MF、post-PV MF、PMF各1例），捕获骨髓微环境细胞包括MSC、内皮细胞，发现MF患者MSC出现明显成骨分化偏倚、造血支持功能减弱、胶原纤维形成能力增强，而内皮细胞出现动脉化基因表达趋势。小鼠骨髓微环境捕获细胞包括MSC、内皮细胞、血管旁细胞、成纤维细胞，与MF有关的主要是MSC的改变，表现为MF小鼠成纤维MSC亚群、成骨细胞比例明显增加。人与小鼠成纤维MSC亚群具有较强的相关性，干扰素α-2（interferon α-2, IFN-α-2）能够降低小鼠成纤维MSC亚群比例。

结论：本研究描绘了MPN患者及小鼠模型的骨髓基质细胞单细胞转录组图谱，骨髓纤维化患者于小鼠的基质细胞与非纤维化患者/小鼠存在明显不同，通过分析发现，与骨髓纤维化进展最相关的基质细胞是MSC，识别了可能与骨髓纤维化进展有关的MSC亚群，该亚群在人和小鼠中具有较强的相关性。靶向减少MSC成纤维细胞亚群可能为延缓骨髓纤维化进展提供一种策略。

Background: The classic Ph (Philadelphia) chromosome negative myeloproliferative tumor (myeloproliferative neoplasm, MPN) is a clonal malignant disease of myeloid hematopoietic stem cells. This type of disease is divided into essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF). Bone marrow fibrosis includes PMF, post-ET MF and post-PV MF. The clinical manifestations of bone marrow fibrosis are anemia, hepatosplenomegaly, thrombosis, hemorrhage, etc.. The occurrence of bone marrow fibrosis is due to the increase of bone marrow reticular fibers, further collagen fiber production and osteosclerosis. Bone marrow microenvironment includes bone marrow mesenchymal stromal cells (MSC), endothelial cells, perivascular cells, glial cells, as well as some cytokines, blood vessels and nerves. Abnormal microenvironment plays an important role in the occurrence and development of bone marrow fibrosis.

Objective: This study aimed to characterize the single-cell transcriptome profile of bone marrow stromal cells in MPN patients and mice and analyze the cells and mechanisms related to the progression of myelofibrosis.

Methods: The bone marrow aspirate of MPN patients was extracted, and hematopoietic cells were removed to enrich stromal cells through magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS), and then 10 x Genomics single-cell transcriptome sequencing was performed. Bone marrow from the offspring of JAK2V617F transgenic mice or CALR52-bpdeletion knock-in mice were transplanted to produce mice with different disease phenotypes (ET, PV, MF) or non-disease mice. The transplanted mice were also sacrificed to obtain bone marrow cells. MACS and FACS were performed to enrich stromal cells, and then 10xGenomics single-cell transcriptome sequencing was performed. Single-cell transcriptome data analysis includes raw data quality control, reduction of dimensionality, batch removal, clustering, analysis of differential gene expression, cell trajectory, scoring of gene sets, differential gene enrichment, cell communication and clusters correlation.

Results: This study included 5 non-myelofibrosis MPN patients (4 ET, 1 PV) and 3 myelofibrosis patients (1 post-ET MF, 1 post-PV MF, and 1 PMF). Cells in the bone marrow microenvironment were including MSCs and endothelial cells. It was found that MSCs from MF patients showed a significant osteogenic differentiation bias, weakened hematopoietic support function, and enhanced collagen fiber formation ability, while endothelial cells showed an arterial gene expression trend. Cells captured in the mouse bone marrow microenvironment include MSCs, endothelial cells, perivascular cells, and fibroblasts. The main changes related to MF are MSCs, which are manifested in a significant increase in the proportion of fibrogenic MSC subcluster and osteoblasts in MF mice. There is a strong correlation between human and mouse fibrogenic MSC subclusters, and interferon α-2 could reduce the proportion of mouse fibrogenic MSC subcluster.

Conclusion: This study depicts the single-cell transcriptome profile of bone marrow stromal cells in MPN patients and mouse models. The stromal cells in mice from patients with myelofibrosis are significantly different from those in non-fibrosis patients/mouse. The stromal cells most relevant to myelofibrosis progression are MSCs, and a fibrogenic subcluster of MSCs that may be involved in myelofibrosis progression was identified with a strong correlation in humans and mice. Targeted reduction of fibrogenic MSC subcluster may provide a strategy to delay the progression of myelofibrosis.