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. Overall design: 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.