Deciphering enucleation: Analysis of RNA sequencing data for investigating differences in differentiation behavior between HSCs and erythroblast cell line "imBMEP"

Background

An erythroid progenitor cell line capable of efficient in vitro production of mature red blood cells (RBCs) represents a promising alternative for transfusion medicine. Since an erythroblast line previously established in our lab (imBMEP) showed limitations in efficient maturation, knockout (K.O.) of enucleation inhibitor miR-30a-5p was performed. Although this modification led to a significant promotion of terminal erythropoiesis, insufficient enucleation remains a major challenge.

Methods

To systematically investigate possible causes of the inhibited final maturation a transcriptome analysis was performed. Samples for RNA Sequencing were obtained from imBMEP cells and hematopoietic stem cells (HSCs), acting as control. Extracted RNA was derived at different time points during erythroid differentiation in biological triplicates. RNA quality and concentration were determined. The following enrichment of mRNA was performed via ribosomal RNA depletion with RNAse H. After reverse transcription barcoded cDNA libraries were prepared. The following next generation sequencing was executed paired-ended. After read alignment to a human reference genome and quality control, differential gene expression (DGE) analysis was carried out.

Results

DGE analysis revealed high variance between imBMEP cells and HSC controls. Comparison of raw counts of the different RNA biotypes showed a significant elevation of lncRNAs during differentiation compared to corresponding HSC controls (8,4±0,5% vs. 14,4±1,2%). A notable number of cancer related lncRNAs are among the RNAs with the highest differences in expression between imBMEPs and HSCs. Furthermore, dysregulation of previously described factors involved in terminal erythropoiesis and enucleation, like HDAC5, FOXO3 or GATA2, was observed in imBMEP cells.

Conclusion

RNA Sequencing is a powerful tool for analysing differential gene expression. Comparison of sequencing data between imBMEP cells and HSCs gave first insights in dysregulated processes and possible candidates causing differences in differentiation behaviour. Planned ingenuity pathway analysis (IPA) and pathway enrichment analysis should now help to infer the underlying reasons of observed differences, predict downstream effects and find key candidates for further modifications of the cell line.

Offenlegung Interessenkonflikt:

There are no conflicts of interest to be disclosed.