Stage-Specific Multiomic Analysis of Human Erythroid Differentiation

Significance: Reduced red blood cell (RBC) production resulting in anemia is a common symptom of many diseases. RBC transfusion and erythropoiesis-stimulating agents (ESAs), primarily erythropoietin (Epo) and its analogs, are major treatments for anemia, but often lead to organ damage and/or ESA resistance. Understanding the mechanisms of Epo-dependent erythroid differentiation is critical for identifying targets for the future development of therapeutics that increase RBC production and improve patient outcomes. Background: Erythropoiesis, the process of RBC (erythrocyte) production from hematopoietic stem cells (HSCs), occurs through four phases: erythroid progenitor development (EPD), early erythropoiesis (EE), terminal erythroid differentiation (TED), and erythrocyte maturation (EM). The current paradigm of EE describes HSC progression through two erythroid-committed progenitor cells: burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E). Recent studies have demonstrated heterogeneity within EE, but Epo-dependence within heterogeneous EE populations remains poorly understood. In preliminary investigations, we observed an Epo-mediated increase in high expression of CD71 (transferrin receptor) and CD105 (endoglin), leading to our hypothesis that Epo-dependence in heterogeneous EE populations can be resolved based on changes in the erythroid markers CD71 and CD105, which are dynamically expressed in BFU-Es and CFU-Es. Methods: To define Epo-dependent transitions within EE, we utilized human bone marrow (BM)-derived CD34⁺ hematopoietic stem and progenitor cells (HSPCs) cultured ex vivo in the presence or absence of recombinant Epo. To determine the Epo-dependent EE stage, we developed a multiparametric flow cytometry strategy for identification of heterogeneous populations. This strategy also facilitated isolation of individual EE sub-populations via fluorescent activated cell sorting (FACS) which were then plated for re-culture to assess differentiation potential. Untargeted LC-MS/MS–based lipidomic analyses were performed to determine changes in EE lipid metabolite profiles dependent on Epo. Finally, intracellular flow cytometry was used to validate expression of the enzymes involved in lipid metabolism. Results: Based on the expression of CD34, CD71, and CD105 in cultured BM-derived cells, we identified five sub-populations that temporally emerged during EE: two CD34+ BFU-Es, early (earlyB; CD71loCD105lo) and late (lateB; CD71hiCD105lo), and three CD34– CFU-Es, early (earlyC; CD71loCD105lo ), mid (midC; CD71hiCD105lo), and late (lateC; CD71hiCD105hi). These cell populations were present in prospectively analyzed BM, providing evidence that human erythroid differentiation may occur through these five EE stages. Isolated EE populations cultured with Epo progressed into TED via two alternate routes from earlyB: one through earlyC and another through lateB, both converging at midCs that transition into lateCs. These results indicate more than one path of EE cell differentiation. Additional assessment of these subpopulations by flow cytometry revealed that transition from midC to lateC requires Epo, suggesting that acquisition of the CD71hiCD105hi phenotype is Epo-dependent. Subsequent scRNA-seq analyses confirmed a differentiation arrest preceding cells highest in expression of TFRC (CD71) and ENG (CD105) in the absence of Epo, supporting our immunophenotypic findings. Moreover, scRNA-seq identified a unique transcriptional program associated with Epo signaling within EE, which included transient upregulation of genes involved in lipid and cholesterol metabolism, implicating these pathways in Epo-dependent differentiation. Untargeted lipidomic analyses uncovered changes in glycerophospholipid (GPL) and lysophospholipid species, consistent with differential expression of GPL metabolism genes observed in the scRNA-seq dataset. These included MBOAT2, LPCAT3, CHPT1, and PEMT, whose Epo-mediated changes were validated at the protein level. Together, our findings underscore an Epo-dependent reprogramming of GPL metabolism in EE. Discussion: Overall, our work has identified the Epo-dependent transition in EE that is defined by high expression of CD71 and CD105, which was observed both immunophenotypically and transcriptionally, and uncovered GPL metabolism reprogramming as a potentially crucial process underlying this stage. These findings have revealed novel mechanisms for EE differentiation, including alternative routes of differentiation, and established a framework for studying Epo-dependent human erythropoiesis—integrating immunophenotypic, transcriptomic, and lipidomic analyses. Together these results not only expand our understanding of erythroid development but identify MBOAT2, LPCAT3, CHPT1, and PEMT as candidates for additional investigation, which may be used in the future development of therapies against anemia.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

doctoral

Discipline thesis:degree_discipline

Graduate College

Grantor dc:publisher

The University of Arizona.

Year dc:date.issued

2025

Author dc:creator

• Schippel, Natascha Nichole

Advisor dc:contributor.advisor

• Sharma, Shalini

Committee members dc:contributor.committeemember

• Kim, Suwon
• Gustin, Kurt
• Miranti, Cynthia

• Erythropoiesis
• Erythropoietin
• Flow Cytometry
• Lipidomics
• Red Blood Cells
• scRNA-seq

Statement dc:rights

• Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction, presentation (such as public display or performance) of protected items is prohibited except with permission of the author.

Licence dc:rights.uri

• http://rightsstatements.org/vocab/InC/1.0/

Language dc:language.iso

en

Handle dc:identifier.uri

http://hdl.handle.net/10150/679323

OAI identifier oai:identifier

oai:repository.arizona.edu:10150/679323