Comprehensive Characterization and Transcriptome Analysis of Terminal Erythropoiesis in Human Fetal Liver

Background and Research Question

Erythropoiesis is the process of red blood cell production. Initially, “primitive” erythropoiesis occurs in the yolk sac, gradually replaced by “terminal” erythropoiesis in the fetal liver (FL) and postnatal bone marrow (BM). During human fetal development, FL is a crucial organ for erythropoiesis, but our understanding of human FL erythropoiesis is currently very limited. The aim of this study is to comprehensively characterize and analyze the global transcriptome of terminal erythropoiesis in human fetal liver, explore gene expression patterns during erythropoiesis, and further understand the impact of culture systems on gene expression.

Research Source

This study was jointly conducted by Yongshuai Han, Shihui Wang, Yaomei Wang, Yumin Huang, Chengjie Gao, Xinhua Guo, Lixiang Chen, Huizhi Zhao, and Xiuli An. The institutions involved include the School of Life Sciences at Zhengzhou University, the Laboratory of Membrane Biology at New York Blood Center, the Department of Hematology at Zhengzhou University People’s Hospital, the Department of Hematology at Zhengzhou University Affiliated Cancer Hospital, and the Department of Hematology at the First Affiliated Hospital of Zhengzhou University. This article was published on August 30, 2023, in the journal Genomics, Proteomics & Bioinformatics.

Detailed Research Process

1. Experimental Subjects and Methods:
   • Main experimental subjects: Human fetal liver cells.
   • Erythrocyte isolation and RNA sequencing: Primary erythrocytes at different developmental stages were isolated using surface markers and analyzed by RNA sequencing (RNA-seq).
   • Erythropoiesis in culture systems: FL CD34+ cells were cultured in vitro, and RNA-seq was used to compare transcriptomes of primary and cultured erythrocytes.
   • Establishment of immortalized erythroid cell lines: Immortalized erythroid cell lines were established from FL and cord blood (CB) CD34+ cells, followed by phenotypic detection and transcriptome analysis.
2. Isolation of Human Fetal Liver Erythrocytes:
   • A three-phase erythrocyte culture system was used, and a flow cytometry isolation method using glycophorin A, band 3, and α4 integrin as surface markers was developed to successfully isolate erythrocyte populations at various developmental stages.
3. RNA Sequencing Analysis and Gene Expression Patterns:
   • Whole transcriptome analysis of isolated FL erythrocytes revealed a gradual decrease in gene expression from proerythroblasts to orthochromatic erythroblasts, with significant changes.
   • Cluster analysis and Gene Ontology (GO) enrichment analysis identified genes with increased expression in processes such as protein degradation and autophagy, suggesting these pathways play important roles in erythrocyte enucleation.
4. Characteristics of In Vitro Culture Systems and Their Optimization:
   • FL CD34+ cells were cultured in a three-phase system, observing significant cell expansion within 15 days.
   • Expression profile analysis revealed that FL in vitro cultured erythropoiesis patterns were more similar to CB erythropoiesis, with relatively lower enucleation capacity.
5. Establishment and Analysis of Immortalized Erythroid Cell Lines:
   • FL and CB CD34+ cells were immortalized using the HPV-E6/E7 system, resulting in FL-iERY and CB-iERY cell lines. Phenotypic analysis and RNA-seq showed that cell immortalization mainly occurred at the proerythroblast stage.
   • Immortalized erythroid cell lines could undergo terminal erythroid differentiation but had poor enucleation capacity. Transcriptome data comparing enucleation abilities of different erythroid lines indicated significantly reduced expression of genes related to chromosome organization and mitophagy in erythrocytes unable to enucleate.
6. Result Analysis and Key Findings:
   • Compared to primary erythrocytes, in vitro cultured erythrocytes showed upregulation of lipid metabolism-related genes, indicating higher energy requirements in culture systems.
   • Immortalized erythroid cell lines have important research value in studying terminal erythropoiesis. Analysis of differentially expressed hemoglobin and its regulatory factors showed potential applications of immortalized erythroid lines in studying α-type hemoglobin switching.

Results and Conclusions

This study revealed new mechanisms of human fetal liver erythropoiesis through comprehensive transcriptome analysis. Compared to in vitro culture, primary erythrocytes showed more differences in gene expression during erythropoiesis. Through erythrocyte isolation and dual-cell flow cytometry sorting techniques, this study was able to distinguish gene expression at different developmental stages of erythropoiesis, thus revealing key biological pathways in terminal erythropoiesis.

The established immortalized erythroid cell lines have important potential applications in studying terminal erythropoiesis, especially in aspects such as hemoglobin switching. By comparing gene expression in erythrocyte enucleation from different sources, it was found that defects in the regulation of chromosome organization and mitophagy-related gene expression are the main mechanisms leading to erythrocytes’ inability to enucleate.

Research Value and Highlights

The highlights of this study include proposing key gene expression patterns during human fetal liver erythropoiesis, demonstrating the impact of culture systems on erythropoiesis, and establishing the first immortalized erythroid cell lines derived from fetal liver, providing important tools for further research on terminal erythropoiesis. By revealing the mechanism behind the poor enucleation ability of immortalized erythrocytes, this study suggested directions for potential improvement methods. These findings provide important insights into understanding the molecular mechanisms during erythropoiesis, while laying the foundation for future cell and gene therapy research.

Conclusion

This study integrated the different characteristics of erythropoiesis in human fetal liver and culture systems, providing valuable resources for further research on fetal liver erythropoiesis through the establishment of immortalized erythroid cell lines. The RNA-seq data and established cell lines will provide important references for future erythropoiesis research and development of therapeutic approaches.