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. Disclosures No relevant conflicts of interest to declare.

Inhibition of HDAC5-Mediated p65 Deacetylation Enhances Human Cord Blood Hematopoietic Stem Cell Homing and Engraftment in NSG Mice

Stromal cell derived factor-1α (SDF-1α)/chemokine C-X-C receptor 4 (CXCR4) interactions play a crucial role in hematopoietic stem cells (HSC) trafficking and homing to the bone marrow (BM) environment. To identify new epigenetic regulators of CXCR4 receptor, we screened a chemical compound library of epigenetic enzyme inhibitors to evaluate their effects on membrane CXCR4 expression in CB CD34+ cells. We found that treatment with a couple of histone deacetylase (HDAC) inhibitors, including M344, strongly upregulated membrane CXCR4 expression. We also investigated the effect of M344 on membranal CXCR4 expression in a rigorously defined more primitive HSC cell population (CD34+CD38-CD45RA-CD49f+CD90+) and found that there was a 2.5 fold increase in the M344 treated group compared with vehicle control treated cells. Quantitative RTPCR also showed increased CXCR4 mRNA levels in M344-treated CD34+ cells compared with vehicle control (3.1 fold), indicating that this regulation occurs at the transcriptional level. We next evaluated the effect of M344 treatment on HSC chemotaxis in in vitro transwell migration assays. Both vehicle and M344 treated CB CD34+ cells showed significant migration to 50ng/mL SDF-1α, however, chemotaxis was 2.1 fold higher in M344 treated group. Enhanced migration to SDF-1α by M344 was also observed in the more primitive HSC population. Chemotaxis of CB CD34+ cells to SDF-1α was blocked by CXCR4 antagonist AMD3100, suggesting that the effect was mediated through the CXCR4 receptor. To directly evaluate in vivo homing, vehicle and M344 treated CB CD34+ cells were injected into sublethally irradiated NSG mice, and human cells homing to mouse BM were analyzed 24 hours after transplantation. Consistently, M344 treatment enhanced CB CD34+ cells homing by 2.3 fold in NSG mice. Next, we performed a limiting dilution assay to compare the frequency of SCID-repopulating cells (SRCs) in vehicle and M344 treated CB CD34+ cells. Poisson distribution analysis revealed an SRC frequency of 1/3216 in vehicle control treated group and 1/746 in M344 treatment. We calculated the respective presence of 310.9 SRCs and 1340.5 SRCs in 1×106 cells from vehicle control and M344-treated cultures, so M344 treatment resulted in a 4.3 fold increase in the number of functionally detectable SRCs compared with vehicle control. Eighteen HDACs have been identified in humans and they are divided into four classes. The mechanisms regarding HDAC regulation of HSC homing and engraftment are largely unknown. Using shRNA to knockdown expression of individual HDACs in CB CD34+ cells, we surprisingly found that HDAC5 shRNA transfection resulted in upregulation of membrane CXCR4 expression. LMK235, a selective inhibitor of HDAC5, also increased membrane CXCR4 expression in CB CD34+ cells. In contrast, inhibition of other HDACs did not show any effect on membrane CXCR4 expression. Similar to M344, LMK235 treatment resulted in significantly higher CXCR4 mRNA, membrane CXCR4 expression in CB HSCs, enhanced migration to SDF-1α in chemotaxis assay, and higher number of cells homed to the BM in NSG mice. We next performed chromatin immunoprecipitation (ChIP) assays to examine the chromatin status at the CXCR4 promoter region. H3K9 acetylation levels were significant higher in LMK235-treated CB CD34+ cells compared with vehicle control, suggesting increased H3K9 levels at the CXCR4 promoter region contributes to increased CXCR4 transcription. To further explore the mechanisms underlying HDAC5 regulation of HSC homing, we tested a couple of signaling pathways and found that inhibition of the NF-κB signaling pathway by Andrographolide and Pyrrolidinedithiocarbamate Ammonium, suppressed M344 and LMK235-mediated CXCR4 upregulation on CB CD34+ cells. It has been reported that acetylation of NF-κB p65 subunit enhances its transcriptional activity. We examined the acetylation levels of p65 and found that LMK235 treatment resulted in increased levels of p65 acetylation in CB CD34+ cells, indicating p65 could be a downstream target of HDAC5. Consistently, using ChIP assay we detected increased levels of acetylated p65 binding to the CXCR4 promoter region in the LMK235-treated group. Taken together, our results reveal previously unknown specific epigenetic regulation of HSC homing and engraftment by HDAC5, which suggests a new translational strategy to enhance HSC transplantation. Disclosures Broxmeyer: CordUse: Other: SAB Member.