Developmental differences between neonatal and adult human erythropoiesis

Studies of human erythropoiesis have relied, for the most part, on the in vitro differentiation of hematopoietic stem and progenitor cells (HSPC) from different sources. Here, we report that despite the common core erythroid program that exists between cord blood- and peripheral blood-HSPC induced towards erythroid differentiation in vitro, significant functional differences exist. We undertook a comparative analysis of human erythropoiesis using these two different sources of HSPC and differentiated them in vitro. We observed that cells derived from cord blood proliferate 4.5 times more than cells derived from peripheral blood. However, these cells present a delay in their differentiation pattern due to increased quantities of progenitors, notably CFU-E. Using our method of immunophenotyping for the study of erythroid progenitors, we document the presence and maintenance of a specific population in peripheral blood-derived erythroid progenitors. This population, defined as IL3R-GPA-CD34+CD36+, has the ability to form both BFU-E and CFU-E colonies in colony-forming assays, reflecting a higher potential. To further understand the differences between cord blood- and peripheral blood- HSPC, we sorted all stages of erythropoiesis from both sources and compared their transcriptome. We document differences at the CD34, BFU-E, poly- and orthochromatic stages. Among the genes presenting the highest differences in expression, many are involved in the regulation of the cell cycle and autophagy. Altogether, our studies provide a qualitative and quantitative comparative analysis of human erythropoiesis and highlight functional differences, critical to our understanding of the impact of the developmental origin of HSPCs on erythroid differentiation. RNA was extracted from FACS-sorted cells at 8 distinct stages of erythropoiesis, derived from CB- and PB-CD34+ cells. cDNA libraries were prepared using the Illumina TruSeq kit and sequenced on the Illumina HiSeq 2500 (Epigenomics Core of Weill Cornell Medical College, New York). This sequencing strategy produced ~15-80 million 50bp single end reads per sample. For each distinct stage of erythropoiesis, 3 biological replicates were obtained from independently cultured and sorted samples from different donations. Quality control of reads was performed and low-quality reads removed. Reads were aligned to the hg19 reference genome using HISAT2. Raw read counts were extracted from the aligned reads using the featureCounts program. Differential expression analysis was done at each stage comparing the two original sources of CD34+ cells using the DESeq2 bioconductor package. Using gene expression data from each stage, the subset of genes differentially expressed as a function of HSPC source was clustered using divisive hierarchical clustering and split into 10 clusters. Gene ontology enrichment analysis of the biological process gene sets was then performed on each cluster using the cluster Profiler R package with the gene background of all genes expressed across all stages in both sources.

关于人类红细胞生成的研究，迄今为止大多依赖于不同来源造血干祖细胞（hematopoietic stem and progenitor cells, HSPC）的体外分化培养模型。本研究发现，尽管体外诱导向红细胞系分化的脐带血来源与外周血来源HSPC拥有共同的核心红细胞生成程序，但二者存在显著的功能差异。
我们针对这两种不同来源的HSPC开展了人类红细胞生成的比较分析，并对其进行体外诱导分化。实验观察到，脐带血来源的细胞增殖能力为外周血来源细胞的4.5倍。然而，由于祖细胞（尤其是红细胞集落形成单位（CFU-E））数量增多，脐带血细胞的分化进程出现延迟。
借助我们用于红细胞祖细胞研究的免疫分型方法，我们证实外周血来源的红细胞祖细胞中存在并维持着一类独特细胞群。该细胞群的表型为IL3R-GPA-CD34+CD36+，在集落形成实验中可同时生成爆式红细胞集落形成单位（BFU-E）与CFU-E集落，提示其具有更高的分化潜能。
为进一步解析脐带血与外周血来源HSPC的差异，我们分别从两种来源中分离出红细胞生成过程的各个阶段细胞，并对其转录组进行比较分析。我们在CD34+细胞、BFU-E、多染性红细胞以及正染性红细胞阶段均检测到表达差异。在表达差异最为显著的基因中，许多参与细胞周期调控与自噬过程。
综上，本研究对人类红细胞生成开展了定性与定量的比较分析，明确了二者的功能差异，这对于解析HSPC的发育起源对红细胞系分化的影响具有关键意义。
本研究的样本为脐带血（CB）与外周血（PB）来源的CD34+细胞，我们从红细胞生成8个不同阶段的流式分选（FACS）细胞中提取总RNA。
采用Illumina TruSeq试剂盒构建cDNA文库，并在Illumina HiSeq 2500测序平台完成测序（实验由纽约威尔康奈尔医学院表观基因组学中心执行）。
该测序策略每个样本可产生约1500万至8000万条50bp单端读段。
针对红细胞生成的每个阶段，我们均设置3个生物学重复，样本来自不同供体的独立培养与分选体系。
我们对原始读段进行质量控制并移除低质量读段，随后通过HISAT2将读段比对至hg19参考基因组。
使用featureCounts程序从比对后的读段中提取原始读段计数。
利用DESeq2生物信息学R包，在每个发育阶段对比两种来源CD34+细胞的基因表达差异。
基于各阶段的基因表达数据，我们将随HSPC来源不同而呈现表达差异的基因子集采用分裂式层级聚类法进行聚类，划分为10个基因簇。
随后，使用clusterProfiler R包，以两种来源所有发育阶段中均表达的基因作为基因背景，对每个基因簇的生物过程基因集开展基因本体（GO）富集分析。