The donor-independent in vitro production of erythrocytes could be realized by using erythroid progenitor cells as an unlimited cell source. These entail numerous favorable properties for efficient blood pharming, but are currently limited by insufficient terminal maturation. In order to systematically investigate possible causes for the inhibited enucleation, we performed a comparative transcriptome analysis of the erythroblast cell line "imBMEP" and hematopoietic stem cells (HSCs).

RNA Sequencing was performed with RNA samples obtained at different time points during erythroid differentiation from imBMEP cells and HSCs, acting as control. After mapping the sequencing data to a human reference genome, differential gene expression (DGE) analysis was performed. Significant differences in gene expression between imBMEPs and HSCs were assigned to specific signaling pathways and biological functions using Ingenuity Pathway Analysis (IPA). Based on the results, the functionality of the cytoskeleton was examined. Protein expression was quantified by Western Blot, while the distribution of the three cytoskeletal components within the cells was visualized by immunofluorescence staining.

Comparison of the gene expression data between HSCs and imBMEPs using DGE analysis revealed a high degree of variation between both groups. Using IPA, these differences in gene expression were assigned to numerous significantly altered pathways regulating cytoskeletal elements. In particular the actin network is predicted to be affected at several regulatory layers. On protein level there were no considerable differences in the expression of the main cytoskeletal elements actin, tubulin and vimentin but immunofluorescence staining revealed distinct structural changes within the cells. Particularly the actin network is affected, with formation of unstructured actin accumulations in imBMEPs, while a homogenous network is present in the HSCs.

The comparative transcriptome analysis and further examination provided consistent evidence of a dysfunctional cytoskeletal network in imBMEPs. Especially, actin polymerization and reorganization appear to be affected, which is of particular interest in the context of insufficient enucleation, as actin network remodeling plays an essential role in terminal erythropoiesis. This sets the future focus on restoring actin network dynamics in order to achieve efficient enucleation of imBMEPs.

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