Identification of Potential Chemical Compounds Able to Trigger Enucleation of Immortalized Human Erythroid Cell Lines

Ex vivo production of functional red blood cells (RBCs) is a potential method to provide abundant RBCs which therefore is expected as a solution to improve the current shortage of blood supply in donor-dependent transfusion therapies. Immortalized human erythroid cell lines are expected to be an alternative source for ex vivo production of RBCs, as these cells are already committed to the erythroid lineage and still keep a limitless growth capacity. We have previously established immortalized erythroid cell lines derived from human UCB-derived CD34⁺ cells (HUDEP) and human iPS cells (HiDEP) by ectopically overexpressing human papilloma virus E6/E7 gene (Kurita et al., PLoS ONE, 2013). HUDEP/HiDEP sustain the infinite growth capacity, express erythroid specific cell surface markers (e.g. Glycophorin-A) and produce functional hemoglobin. However, in similar to erythroid cells directly differentiated from ES/iPS cells, these cells do not efficiently enucleate and easily cause cell death upon the induction of differentiation.

In this study, we employed an imaging-based high throughput screening system (Cellomics ArrayScan) combined with two distinct DNA dyes (SYTO16 and SYTOXRed) to capture morphological changes of the immortalized erythroid cell lines. Among >1,200 chemical compounds, we identified multiple histone deacetylase (HDAC) inhibitors (HDACi) that largely increased the enucleation of HiDEP. In particular, Fluoro-SAHA and M344 achieved 9.0 fold increase in the enucleation rate. The exert of enucleation was confirmed by morphological examinations using cytospin and motional observations by time-lapse imaging. These assay finally observed that aprrox. 20 % of HiDEP cells enucleated upon Fluoro-SAHA treatment. However, we noticed that a large proportion of enucleated cells were fragile, and a similar number of enucleated cells with damaged membrane were also found. Addition of pan-caspase inhibitor, QVD-OPH, improved the viability of enucleated cells but also blocked enucleation, suggesting that HDACi-induced enucleation is also a caspase-dependent process.

Gene expression profiling revealed that the Fluoro-SAHA and M344 treatment commonly induced cytoskeletal genes including kinesins, e.g. KIF3A, indicating cytoskeletal rearrangement occurred upon Fluoro-SAHA/M344 treatment. Of note, one of main erythroid-membrane components, SPTA1, was significantly down-regulated, whereas non-erythrocytic spectrin, SPTAN1, was abnormally induced. This α-spectrin switching was considered to result in the high fragility of enucleated cells. We therefore generated HiDEP cells with enhanced expression of SPTA1 using CRISPR-activation system. Activation of SPTA1 expression significantly improved viability of the enucleated cells and as the consequence 30 % of cells successfully enucleated while damaged cells were decreased to less than 10 %. Our data propose a potential method to induce enucleation of immortalized erythroid cell lines which would lead to a future large-scale ex vivo RBC production.