Laboratory of Regenerative Hematopoiesis, Institute of Bioengineering and Institute for Experimental Cancer Research, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland.
Animal Imaging and Technology Core, Center for Biomedical Imaging, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland.
Front Endocrinol (Lausanne). 2020 Sep 24;11:480. doi: 10.3389/fendo.2020.00480. eCollection 2020.
The bone marrow (BM) exists heterogeneously as hematopoietic/red or adipocytic/yellow marrow depending on skeletal location, age, and physiological condition. Mouse models and patients undergoing radio/chemotherapy or suffering acute BM failure endure rapid adipocytic conversion of the marrow microenvironment, the so-called "red-to-yellow" transition. Following hematopoietic recovery, such as upon BM transplantation, a "yellow-to-red" transition occurs and functional hematopoiesis is restored. Gold Standards to estimate BM cellular composition are pathologists' assessment of hematopoietic cellularity in hematoxylin and eosin (H&E) stained histological sections as well as volumetric measurements of marrow adiposity with contrast-enhanced micro-computerized tomography (CE-μCT) upon osmium-tetroxide lipid staining. Due to user-dependent variables, reproducibility in longitudinal studies is a challenge for both methods. Here we report the development of a semi-automated image analysis plug-in, , which employs the open-source software QuPath, to systematically quantify multiple bone components in H&E sections in an unbiased manner. discerns and quantifies the areas occupied by bone, adipocyte ghosts, hematopoietic cells, and the interstitial/microvascular compartment. A separate feature, , fragments adipocyte ghosts in H&E-stained sections of extramedullary adipose tissue to render adipocyte area and size distribution. Quantification of BM hematopoietic cellularity with lies within the range of scoring by four independent pathologists, while quantification of the total adipocyte area in whole bone sections compares with volumetric measurements. Employing our tool, we were able to develop a standardized map of BM hematopoietic cellularity and adiposity in mid-sections of murine C57BL/6 bones in homeostatic conditions, including quantification of the highly predictable red-to-yellow transitions in the proximal section of the caudal tail and in the proximal-to-distal tibia. Additionally, we present a comparative skeletal map induced by lethal irradiation, with longitudinal quantification of the "red-to-yellow-to-red" transition over 2 months in C57BL/6 femurs and tibiae. We find that, following BM transplantation, BM adiposity inversely correlates with kinetics of hematopoietic recovery and that a proximal to distal gradient is conserved. Analysis of recovery through magnetic resonance imaging (MRI) reveals comparable kinetics. On human trephine biopsies successfully recognizes the BM compartments, opening avenues for its application in experimental, or clinical contexts that require standardized human BM evaluation.
骨髓（BM）根据骨骼位置、年龄和生理状况呈现出造血/红色或脂肪/黄色骨髓的异质性。在接受放射/化学治疗或患有急性 BM 衰竭的小鼠模型和患者中，骨髓微环境会迅速发生脂肪细胞转化，即所谓的“红转黄”。在造血恢复后，如骨髓移植后，会发生“黄转红”转变，并恢复功能性造血。评估骨髓细胞组成的金标准是病理学家在苏木精和伊红（H&E）染色组织切片中评估造血细胞的细胞密度，以及用锇四氧化物脂质染色后对比增强微计算机断层扫描（CE-μCT）测量骨髓脂肪含量的体积测量。由于用户依赖的变量，这两种方法在纵向研究中的可重复性都是一个挑战。在这里，我们报告了一种半自动图像分析插件的开发，它使用开源软件 QuPath 来系统地、无偏地定量 H&E 切片中的多个骨成分。 可以区分和定量由骨、脂肪细胞幽灵、造血细胞和间质/微血管区占据的区域。一个单独的功能， ，将髓外脂肪组织的 H&E 染色切片中的脂肪细胞幽灵分割成碎片，以呈现脂肪细胞面积和大小分布。 使用 对骨髓造血细胞密度的定量与四位独立病理学家的评分范围一致，而对整个骨切片中总脂肪细胞面积的定量与体积测量一致。使用我们的工具，我们能够在稳态条件下开发出 C57BL/6 小鼠中骨髓造血细胞密度和脂肪含量的标准化图谱，包括在尾骨近端和胫骨近端到远端的高度可预测的“红转黄”转变的定量。此外，我们还展示了一种由致死性照射引起的比较性骨骼图谱，在 C57BL/6 股骨和胫骨中进行了 2 个月的“红转黄转红”转变的纵向定量。我们发现，在骨髓移植后，骨髓脂肪含量与造血恢复的动力学呈负相关，并且存在从近端到远端的梯度。通过磁共振成像（MRI）分析 恢复情况揭示了类似的动力学。 在人类环钻活检中， 成功识别了 BM 隔室，为其在需要标准化人类 BM 评估的实验或临床环境中的应用开辟了道路。
