Osteomacs promote maintenance of murine hematopoiesis through megakaryocyte-induced upregulation of Embigin and CD166

Maintenance of hematopoietic stem cell (HSC) function in the niche is an orchestrated event. Osteomacs (OM), are key cellular components of the niche. Previously, we documented that osteoblasts, OM, and megakaryocytes interact to promote hematopoiesis. Here, we further characterize OM and identify megakaryocyte-induced mediators that augment the role of OM in the niche. Single cell mRNAseq, mass spectrometry, and CyTOF examination of megakaryocyte-stimulated OM suggested that upregulation of CD166 and Embigin on OM augment their hematopoiesis maintenance function. CD166 knockout OM or shRNA-Embigin knockdown OM, confirmed that loss of these molecules significantly reduced OM ability to augment the osteoblast-mediated hematopoietic enhancing activity. Recombinant CD166 and Embigin partially substituted for OM function, characterizing both proteins as critical mediators of OM hematopoietic function. Our data identify Embigin and CD166 as OM-regulated critical components of HSC function in the niche and potential participants in various in vitro manipulations of stem cells. Overall design: 1x105 neonatal calvarial cells (NCC) were cultured for 16hrs with or without 50,000 megakaryocytes were stained with CD45, F4/80, and CD41. Sorted CD45+F4/80+CD41- cells were suspended at 3x105 cells/ml PBS then dispensed into medium sized IFC chips (Fluidigm C1 Single-Cell Auto Prep System)(Zhang et al., 2019). mRNA was isolated followed by cDNA synthesis (Clontech SMART-Se v4 Ultra Low Input RNA Kit). Up to 0.4ng cDNA was used for library preparation and indexing with Nextera XT DNA Library Prep Kit (Illumina, Inc., San Diego, CA). Pooled libraries (4nM, quality checked) were used for 150b paired-end sequencing (NextSeq 500). Cells that passed quality testing were analyzed.

造血干细胞（hematopoietic stem cell, HSC）在干细胞龛中的功能维持是一个协同调控的过程。骨巨噬细胞（Osteomacs, OM）是该干细胞龛的关键细胞组分。此前本团队已证实，成骨细胞、骨巨噬细胞与巨核细胞可相互作用以促进造血过程。本研究进一步对骨巨噬细胞进行了系统表征，并鉴定出巨核细胞诱导产生的、可增强骨巨噬细胞在干细胞龛中功能的介导因子。对巨核细胞刺激后的骨巨噬细胞开展单细胞mRNA测序（single cell mRNAseq）、质谱分析（mass spectrometry）以及质谱流式细胞术（CyTOF）检测后发现，骨巨噬细胞表面CD166与Embigin的上调可增强其维持造血功能的能力。敲除CD166的骨巨噬细胞或通过短发夹RNA（short hairpin RNA, shRNA）下调Embigin表达的骨巨噬细胞实验证实，这两种分子的缺失会显著削弱骨巨噬细胞增强成骨细胞介导的造血促进活性的能力。重组CD166与Embigin蛋白可部分替代骨巨噬细胞的功能，证实这两种蛋白均为骨巨噬细胞造血功能的关键介导因子。本研究结果表明，Embigin与CD166是骨巨噬细胞调控的、干细胞龛中维持造血干细胞功能的关键组分，且可能参与干细胞的各类体外操作。
实验整体设计：将1×10^5个新生颅盖骨细胞（neonatal calvarial cells, NCC）与5×10^4个巨核细胞（或无巨核细胞）共培养16小时，随后用CD45、F4/80及CD41对细胞进行染色。分选得到CD45+F4/80+CD41-的细胞，以3×10^5个/毫升的浓度重悬于磷酸盐缓冲液（phosphate buffered saline, PBS）中，随后接种至中型IFC芯片（Fluidigm C1单细胞自动制备系统）（Zhang等，2019）。提取mRNA后采用Clontech SMART-Seq v4超低输入RNA试剂盒进行cDNA合成。取至多0.4 ng cDNA用于文库构建，并采用Nextera XT DNA文库制备试剂盒（Illumina公司，美国加利福尼亚州圣地亚哥）进行索引标记。将质控合格的混合文库（浓度4 nM）置于NextSeq 500测序平台进行150 bp双端测序。对通过质量检测的细胞进行数据分析。