原发性骨髓纤维化（PMF）是一种以骨髓（BM）纤维化为特征的骨髓增生性肿瘤（MPN）。纤维化前 PMF（pre-PMF）进展为明显 PMF。巨核细胞 (MK) 在 PMF 中起主要作用；然而，MK 亚群和其他造血细胞在 PMF 进展过程中的功能仍不清楚。因此，我们使用单细胞 RNA 测序 (scRNA-seq) 分析了 PMF 前、PMF 和其他 MPN 中的 BM 抽吸物。我们鉴定了 14 种细胞类型及其子集，包括造血干细胞和祖细胞 (HSPC) 以及 MK。明显 PMF 中的 HSPC 偏向 MK，并且富含炎症/纤维化。在 MK 中，上皮间质转化 (EMT) 丰富的子集在明显的 PMF 中突然增加。非纤维化/非 PMF MPN 中的 MK 富含 MK 分化，而纤维化/非 PMF MPN 中的 MK 富含炎症/纤维化。总体而言，HSPC、MK 和 CD14 单核细胞亚群的炎症/纤维化特征从 PMF 前增加到明显的 PMF。 T 细胞和 NK 细胞的细胞毒性和功能障碍评分也有所增加。临床上，外周血原始细胞≥1%的患者中，具有高炎症/纤维化特征的MK和HSPC亚群很常见。 scRNA-seq 预测明显 PMF 中的 MK 分化、炎症/纤维化、免疫效应/功能障碍和肿瘤相关信号传导的细胞通讯高于 PMF 前。然而，在 PMF 进展过程中没有出现决定性的子集。我们的研究表明，HSPC、单核细胞和淋巴细胞有助于 PMF 进展，并且在炎症/纤维化和免疫功能障碍方面存在子集特异性。 PMF 的进展可能取决于多种细胞类型的改变，而富含 EMT 的 MK 可能是诊断和治疗进展的潜在靶点。

Primary myelofibrosis (PMF) is a myeloid proliferative neoplasm (MPN) characterized by bone marrow (BM) fibrosis. Pre-fibrotic PMF (pre-PMF) progresses to overt PMF. Megakaryocytes (MKs) play a primary role in PMF; however, the functions of MK subsets and those of other hematopoietic cells during PMF progression remain unclarified. Therefore, we analyzed BM aspirates in pre-PMFs, overt PMFs, and other MPNs using single-cell RNA sequencing (scRNA-seq). We identified 14 cell types with subsets, including hematopoietic stem and progenitor cells (HSPCs) and MKs. HSPCs in overt PMF were MK-biased and inflammation/fibrosis-enriched. Among MKs, the epithelial-mesenchymal transition (EMT)-enriched subset was abruptly increased in overt PMF. MKs in non-fibrotic/non-PMF MPN were MK differentiation-enriched, whereas those in fibrotic/non-PMF MPN were inflammation/fibrosis-enriched. Overall, the inflammation/fibrosis signatures of the HSPC, MK, and CD14+ monocyte subsets increased from pre-PMF to overt PMF. Cytotoxic and dysfunctional scores also increased in T and NK cells. Clinically, MK and HSPC subsets with high inflammation/fibrosis signatures were frequent in the patients with peripheral blood blasts ≥1%. scRNA-seq predicted higher cellular communications of MK differentiation, inflammation/fibrosis, immunologic effector/dysfunction, and tumor-associated signaling in overt PMF than pre-PMF. However, no decisive subset emerged during PMF progression. Our study demonstrated that HSPCs, monocytes, and lymphoid cells contribute to PMF progression, and subset specificity existed regarding inflammation/fibrosis and immunologic dysfunction. PMF progression may depend on multiple cell types' alterations, and EMTenriched MKs may be potential targets for the diagnosis and therapy of the progression.