Isolation and Transcriptome Analyses of Human Erythroid Progenitors: BFU-E and CFU-E

Burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E) cells are erythroid progenitors traditionally defined by colony assays. We developed a flow cytometry-based strategy for isolating human BFU-E and CFU-E cells based on the changes in expression of cell surface markers during in vitro erythroid cell culture. BFU-E and CFU-E are characterized by CD45+GPA-IL-3R-CD34+CD36-CD71low and CD45+GPA-IL-3R-CD34-CD36+CD71high phenotypes, respectively. Colony assays validated phenotypic assignment giving rise to BFU-E and CFU-E colonies, both at a purity ~90%. The BFU-E colony forming ability of CD45+GPA-IL-3R-CD34+CD36-CD71low cells required SCF and erythropoietin, while the CFU-E colony forming ability of CD45+GPA-IL-3R-CD34-CD36+CD71high cells required only erythropoietin. Bioinformatic analysis of the RNA-seq data revealed unique transcriptomes in each differentiation stage. The sorting strategy was validated in uncultured primary cells isolated from bone marrow and peripheral blood, indicating that marker expression is not an artifact of in vitro cell culture, but represents an in vivo characteristic of erythroid progenitor populations. The ability to isolate highly pure human BFU-E and CFU-E progenitors will enable detailed cellular and molecular characterization of these distinct progenitor populations and define their contribution to disordered erythropoiesis in inherited and acquired hematological disease. Our data provide important resource for future studies. Transcription profiles of Human erythroid progenitors at distinct developmental stages were generated by deep sequencing, in triplicate, using IlluminaHiSeq 2000. The complete dataset comprises 4 sample types: CD34, BFU, CFU, and Pro (reanalysis of GSM1304777-GSM1304779).

红系爆式集落形成单位（Burst-forming unit-erythroid, BFU-E）与红系集落形成单位（colony-forming unit-erythroid, CFU-E）是传统上通过集落培养检测定义的红系祖细胞。本研究建立了一种基于流式细胞术的分选策略，可在体外红系细胞培养过程中，根据细胞表面标志物的表达变化分离人BFU-E与CFU-E细胞。二者的细胞表型分别为CD45+GPA-IL-3R-CD34+CD36-CD71low与CD45+GPA-IL-3R-CD34-CD36+CD71high。集落培养检测验证了该表型分型，分选得到的BFU-E与CFU-E集落纯度均约为90%。CD45+GPA-IL-3R-CD34+CD36-CD71low细胞形成BFU-E集落的能力依赖于干细胞因子（Stem Cell Factor, SCF）与促红细胞生成素（erythropoietin, EPO），而CD45+GPA-IL-3R-CD34-CD36+CD71high细胞形成CFU-E集落的能力仅需促红细胞生成素即可实现。对RNA测序（RNA-seq）数据的生物信息学分析显示，两个分化阶段的细胞具有独特的转录组特征。该分选策略在从骨髓与外周血分离的未培养原代细胞中得到了验证，表明上述标志物的表达并非体外细胞培养的人为假象，而是红系祖细胞群体在体内的真实特征。分离高纯度人BFU-E与CFU-E祖细胞的能力，将为这两类不同的祖细胞群体开展精细的细胞与分子生物学表征提供可能，并明确它们在遗传性与获得性血液系统疾病中对红系生成紊乱的贡献。本研究数据为后续相关研究提供了重要的资源。本研究通过Illumina HiSeq 2000测序平台开展三次生物学重复的深度测序，构建了不同发育阶段人红系祖细胞的转录组图谱。完整数据集包含4种样本类型：CD34+细胞、BFU-E细胞、CFU-E细胞与Pro细胞（即对GSM1304777至GSM1304779的重新分析）。