# Decoding human in vitro terminal erythropoiesis originating from umbilical cord blood mononuclear cells and pluripotent stem cells

## TL;DR

This study compares red blood cell development from cord blood and stem cells, identifying factors that hinder adult globin expression and maturation in stem cell-derived cells.

## Contribution

The study provides a single-cell atlas of terminal erythropoiesis and identifies CD99high progenitors and regulators affecting globin expression and enucleation.

## Key findings

- PSCE showed absent BCL11A and ZBTB7A, limiting adult globin expression.
- CD99high cells are a proliferative subpopulation in erythroid progenitors.
- TRIB3 agonists enhance adult globin expression in PSCE.

## Abstract

Ex vivo red blood cell (RBC) production generates unsatisfactory erythroid cells. A deep exploration into terminally differentiated cells is required to understand the impairments for RBC generation and the underlying mechanisms. Here, we mapped an atlas of terminally differentiated cells from umbilical cord blood mononuclear cells (UCBMN) and pluripotent stem cells (PSC) and observed their dynamic regulation of erythropoiesis at single‐cell resolution. Interestingly, we detected a few progenitor cells and non‐erythroid cells from both origins. In PSC‐derived erythropoiesis (PSCE), the expression of haemoglobin switch regulators (BCL11A and ZBTB7A) were significantly absent, which could be the restraint for its adult globin expression. We also found that PSCE were less active in stress erythropoiesis than in UCBMN‐derived erythropoiesis (UCBE), and explored an agonist of stress erythropoiesis gene, TRIB3, could enhance the expression of adult globin in PSCE. Compared with UCBE, there was a lower expression of epigenetic‐related proteins (e.g., CASPASE 3 and UBE2O) and transcription factors (e.g., FOXO3 and TAL1) in PSCE, which might restrict PSCE's enucleation. Moreover, we characterized a subpopulation with high proliferation capacity marked by CD99high in colony‐forming unit‐erythroid cells. Inhibition of CD99 reduced the proliferation of PSC‐derived cells and facilitated erythroid maturation. Furthermore, CD99–CD99 mediated the interaction between macrophages and erythroid cells, illustrating a mechanism by which macrophages participate in erythropoiesis. This study provided a reference for improving ex vivo RBC generation.

scRNA‐seq and cell typing of late‐stage umbilical cord blood mononuclear cells‐ and pluripotent stem cell (PSC)‐derived cells were conducted. Stress erythropoiesis was more active in UCBMN‐derived erythropoiesis (UCB‐E) than in PSC‐derived erythropoiesis (PSC‐E). Regulators that impair adult globin expression and enucleation between UCB‐E and PSC‐E were compared at single cell level. CD99high progenitor cells were a proliferating colony forming unit erythroid subpopulation.