Gene expression profile in single cells of neonatal murine anemic liver

In the mice fetus or neonate, the liver is the major hematopoietic organ before the bone marrow takes over, and anemic-associated hypoxia may alter the hematopoietic niche in the liver. Using scRNA-seq analysis, we analyzed the cell diversity in the neonatal murine anemic liver. Briefly, C57BL/6 mice from each litter were randomly assigned to two study groups: (a) naive control and (b) phlebotomy-induced anemia. Phlebotomy-induced anemia was performed in mouse pups by facial vein phlebotomy to remove 20µL blood/g body weight on P2, P4, P6, P8, and P10 to reduce the hematocrit (Hct) index from an initial 45-60% range, down to 20-24%. Hematocrits and RBC indices were measured at each phlebotomy; 5 µL blood was diluted 1:20 in Cellpak reagent (Sysmex America, Mundelein, IL; catalog #DCL-310A), and analyzed in the Sysmex XT-2000iV veterinary hematology analyzer. Non-phlebotomized control animals were handled similarly to phlebotomized animals with a daily non-phlebotomizing needle stick. Pups were weighed daily to determine the volume of blood to be drawn. The animals were euthanized 24 h (P11) later, and the liver tissue was processed for scRNA sequencing. Overall design: We used phlebotomy-induced anemia in C57BL6/J neonatal mouse pups and extracted the liver tissue for single cell isolation by using mouse liver dissociation kit (Miltenyi Biotech). Single-cell RNA-sequencing was performed on dissociated liver single cells using the Chromium Next GEM Single-Cell 3' Reagent Kits v3.1 (10× Genomics) at the Single Cell & Transcriptomics Core, Johns Hopkins University. Mice with and without phlebotomy-induced anemia were analyzed by scRNA-Seq and compared as two study groups: (a) naive control and (b) phlebotomy-induced anemia.

在小鼠胎儿及新生阶段，骨髓接管造血功能之前，肝脏是主要的造血器官；贫血相关的缺氧可能会改变肝脏内的造血微生态位（hematopoietic niche）。本研究通过单细胞RNA测序（scRNA-seq）分析，探究了贫血新生小鼠肝脏中的细胞多样性。
简言之，将每窝C57BL/6小鼠随机分为两个研究组：(a) 未放血对照组，(b) 放血诱导贫血组。针对幼鼠的放血诱导贫血造模采用面静脉放血方式：在出生后第2、4、6、8、10天（P2、P4、P6、P8、P10），按每克体重抽取20微升血液，将血细胞比容（hematocrit, Hct）从初始的45%-60%降至20%-24%。每次放血后均检测血细胞比容与红细胞（red blood cell, RBC）相关指标：取5微升血液以1:20的比例在Cellpak试剂（Sysmex America公司，蒙迪莱恩，伊利诺伊州；货号DCL-310A）中稀释后，使用Sysmex XT-2000iV兽用血液分析仪进行检测。未放血对照组小鼠接受与放血组相同的操作流程：每日用穿刺针进行穿刺但不抽取血液。每日称量幼鼠体重，以确定每次放血的血液体积。于最后一次放血后24小时（即出生后第11天，P11）对小鼠实施安乐死，采集肝脏组织用于单细胞RNA测序（scRNA-seq）。
实验整体设计：本研究通过对C57BL/6J新生幼鼠实施放血诱导贫血造模，使用小鼠肝脏解离试剂盒（Miltenyi Biotech公司）分离肝脏单细胞。在约翰斯·霍普金斯大学单细胞与转录组学核心实验室，使用Chromium Next GEM单细胞3'端测序试剂盒v3.1（10× Genomics公司）对解离得到的肝脏单细胞进行单细胞RNA测序。以放血诱导贫血组与未放血对照组为两个研究组，通过单细胞RNA测序（scRNA-seq）对两组小鼠进行分析并比较。