TGF-β1 Reverses Hemoglobin Switching in Erythroid Progenitor Cells before Erythropoietin Induction

Objective: This study aims to elucidate the pattern and mechanism by which Transforming Growth Factor-β1 (TGF-β1) regulates human hemoglobin switching, providing foundational evidence for subsequent clinical translational research.

Methods: Human umbilical cord blood CD34+ hematopoietic stem and progenitor cells (HSPCs) were enriched using magnetic beads and subjected to a biphasic erythroid in vitro culture. The effect of TGF-β1 on globin switching was assessed at various stages of erythroid differentiation: the EPO-independent erythroid progenitor cell stage, the EPO-dependent erythroid precursor cell stage, and under different EPO concentrations. Flow cytometry (FCM) was used to analyze the expression of erythroid maturation markers including CD71 and CD235 to evaluate the differentiation stage. Real-time quantitative PCR (RT-qPCR) was employed to verify the mRNA expression of target genes, and Western Blot was used to confirm the expression of target proteins. Moreover, HSPCs were maintained in culture for 7 days, followed by RNA transcriptome sequencing (RNA-seq) and single-cell RNA-seq, both in control and TGF-β1-treated groups. Bioinformatics analysis was conducted to elucidate potential downstream mechanisms. Genes with at least a two-fold change in expression (Log2 Fold Change > 1) in RNA sequencing were selected for Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and Gene Set Enrichment Analysis to preliminarily identify involved functional pathways. The activation of downstream TGF-β1 signaling pathways was further examined, and endogenous and exogenous small molecule inhibitors of the predicted signaling pathways were used for phenotype validation.

Results: At the erythroid precursor cell stage induced by effective concentrations of EPO, TGF-β1 significantly promoted all total globin expression and erythroid differentiation without altering globin gene expression patterns. However, TGF-β1 upregulated ε-globin and γ-globin expression while inhibiting β-globin expression at the EPO-independent HSPC stage. With increasing EPO concentrations, the effect of TGF-β1 on globin switching in HSPCs gradually diminished, though removing TGF-β1 during differentiation partially maintained this effect. Single-cell RNA sequencing revealed that TGF-β1 remodels the development of various hematopoietic lineages in progenitor cells, confirming that TGF-β1 can reverse globin switching in three subpopulations of progenitor cells with erythroid differentiation potential: CD34+ GATA2+ progenitor cells, megakaryocyte-erythroid progenitors (MEPs), and erythroid progenitors (EryPs). RNA transcriptome sequencing indicated that TGF-β1 regulates the expression of NF-kB signaling pathway genes in HSPCs. The non-canonical NF-kB signaling pathway downstream of TGF-β1 was activated in HSPCs, and both the endogenous small molecule all-trans retinoic acid (ATRA) and the exogenous small molecule celastrol could rescue the reversal effect of TGF-β1 on globin switching in CD34+ HSPCs. The canonical SMAD signaling pathway downstream of TGF-β1 was activated in HSPCs but did not participate in globin switching regulation.

Conclusion: This study identified a cell stage-specific pattern of TGF-β1 on the retardation of hemoglobin switching, which begins at the progenitor cell stage before EPO intervention and is diminished during EPO-induced erythroid differentiation. The NF-kB signaling pathway is involved in this regulatory process. These findings offer new insights into the regulation of globin switching by the hematopoietic microenvironment and provide a foundation for translational research on HSPC transplantation for β-hemoglobinopathies.