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Putative regulators for the continuum of erythroid differentiation revealed by single-cell transcriptome of human BM and UCB cells

Significance Using scRNA-seq, we identified a dynamic gene expression profile during terminal erythroid differentiation. Recent efforts revealed the expression features at various developmental stages during hematopoietic differentiation of human stem cells derived from human fetal cord blood and adult bone marrow. However, the transcription dynamics for erythropoiesis remain elusive. Here, we dissected the gene expression dynamics from ProE to OrthoE by carrying out scRNA-seq of erythroblasts isolated from human cord blood and bone marrow cells. We subdivided the human erythropoiesis PolyE and OrthoE into early-/late-PolyE and early-/late-OrthoE, respectively. We also predicted a list of regulators during terminal erythroid differentiation and tested in vitro differentiation experiments. We provide a foundational human scRNA-seq dataset and candidate master regulators of erythropoiesis for further study. Fine-resolution differentiation trajectories of adult human hematopoietic stem cells (HSCs) 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 (PolyEs) at the early and late stages of development as well as the different development stages of orthochromatic erythroblasts (OrthoEs). 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 HSCs 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.