Putative Regulators for the Continuum of Erythroid Differentiation Revealed by Single-cell Transcriptome of Human BM and UCB Cells[scRNA-seq]

Fine-resolution differentiation trajectories of adult human hematopoietic stem cells (HSC) involved in the generation of red cells is critical for understanding dynamic developmental changes that accompany human erythropoiesis. Using Single-cell RNA sequencing (scRNA-seq) of primary human terminal erythroid cells (CD34−CD235a+) isolated directly from adult bone marrow (BM) and umbilical cord blood (UCB), we documented the transcriptome of terminally differentiated human erythroblasts at unprecedented resolution. The insights enabled us to distinguish polychromatic erythroblasts (PolyE) at the early and late stages of development as well as the different development stages of orthochromatic erythroblasts (OrthoE). We further identified a set of putative regulators of terminal erythroid differentiation and functionally validated three of the identified genes, AKAP8L, TERF2IP and RNF10, by monitoring cell differentiation and apoptosis. We documented that knockdown of AKAP8L suppressed the commitment of HSC to erythroid lineage and cell proliferation, and delayed differentiation of colony forming unit-erythroid (CFU-E) to the proerythroblast stage (ProE). In contrast, the knockdown of TERF2IP and RNF10 delayed differentiation of PolyE to OrthoE stage. Taken together, the convergence and divergence of the transcriptional continuums at single-cell resolution underscore the transcriptional regulatory networks that underlie human fetal and adult terminal erythroid differentiation. CD235a+ cells were isolated from 3 adult bone marrow and 3 umbilical cord blood samples and further sequenced by 10x Genomics platform to obtain transcriptomic profiles.UMI Count matrices were generated by cellranger. >>> Submitter states that raw data have been deposited in EGA (EGAS00001003114)<<<

成人造血干细胞（hematopoietic stem cells, HSC）参与红细胞生成的高分辨率分化轨迹，对于解析人类红细胞生成过程中伴随的动态发育变化至关重要。本研究通过对直接取自成人骨髓（bone marrow, BM）与脐带血（umbilical cord blood, UCB）的原代人终末红系细胞（CD34−CD235a+）进行单细胞RNA测序（Single-cell RNA sequencing, scRNA-seq），以前所未有的分辨率解析了终末分化人成红细胞的转录组特征。基于该转录组数据，本研究得以区分发育早晚期的嗜多染性成红细胞（polychromatic erythroblasts, PolyE）以及正染性成红细胞（orthochromatic erythroblasts, OrthoE）的不同发育阶段。本研究进一步筛选得到一批终末红系分化的潜在调控因子，并通过监测细胞分化与凋亡过程，对其中AKAP8L、TERF2IP及RNF10三个基因进行了功能验证。研究发现，敲低AKAP8L会抑制造血干细胞向红系谱系的定向分化与细胞增殖，并延缓红系集落形成单位（CFU-E）向早幼红细胞（proerythroblast, ProE）的分化进程。与之相反，敲低TERF2IP与RNF10则会延缓嗜多染性成红细胞向正染性成红细胞的分化进程。综上，单细胞分辨率下转录连续体的趋同与趋异特征，凸显了支撑人类胎儿与成人终末红系分化的转录调控网络。本研究从3例成人骨髓与3例脐带血样本中分离得到CD235a+细胞，并通过10x Genomics平台进行测序以获取转录组图谱；UMI计数矩阵由Cell Ranger软件生成。>>> 数据提交者声明：原始数据已存储至欧洲基因组表型档案库（EGA），存档编号为EGAS00001003114 <<<