Direct Generation of Immortalized Erythroid Progenitor Cell Lines from Peripheral Blood Mononuclear Cells

Reliable human erythroid progenitor cell (EPC) lines that can differentiate to the later stages of erythropoiesis are important cellular models for studying molecular mechanisms of human erythropoiesis in normal and pathological conditions. Two immortalized erythroid progenitor cells (iEPCs), HUDEP-2 and BEL-A, generated from CD34+ hematopoietic progenitors by the doxycycline (dox) inducible expression of human papillomavirus E6 and E7 (HEE) genes, are currently being used extensively to study transcriptional regulation of human erythropoiesis and identify novel therapeutic targets for red cell diseases. However, the generation of iEPCs from patients with red cell diseases is challenging as obtaining a sufficient number of CD34+ cells require bone marrow aspiration or their mobilization to peripheral blood using drugs. This study established a protocol for culturing early-stage EPCs from peripheral blood (PB) and their immortalization by expressing HEE genes. We generated two iEPCs, PBiEPC-1 and PBiEPC-2, from the peripheral blood mononuclear cells (PBMNCs) of two healthy donors. These cell lines showed stable doubling times with the properties of erythroid progenitors. PBiEPC-1 showed robust terminal differentiation with high enucleation efficiency, and it could be successfully gene manipulated by gene knockdown and knockout strategies with high efficiencies without affecting its differentiation. This protocol is suitable for generating a bank of iEPCs from patients with rare red cell genetic disorders for studying disease mechanisms and drug discovery.

Generation of an Immortalized Erythroid Progenitor Cell Line Directly from Peripheral Blood Mononuclear Cells without Using Mobilized CD34+ Cells

A reliable stable human erythroid progenitor cell line that can differentiate to the later stages of erythropoiesis is an important cellular model for studying molecular mechanisms of human erythropoiesis in physiological and pathological situations. An erythroid progenitor cell line (HUDEP) was derived from cord blood haematopoietic stem cells (HSCs) by doxycycline inducible expression of HPV E6/E7 gene (Kurita et al., 2013). This cell line could be differentiated further to terminally differentiated red cells, and it has been extensively used for studying transcriptional regulation of human erythropoiesis. Using the same strategy, immortalized erythroid progenitors could also be generated from adult HSCs (Trakarnsanga et al, 2017). However, generation of immortalized erythroid cells from patients using this protocol is challenging as obtaining sufficient number of adult HSCs requires mobilization of HSCs using GCSF. Peripheral blood mononuclear cells (PBMNCs) contain a small number of erythroid progenitors, which can be expanded and differentiated in culture. Till date, there are no reports on the generation of immortalized erythroid progenitors directly from PBMNCs. In this study, we established a protocol for the generation of immortalized erythroid progenitors from PBMNCs of a normal donor. The PBMNCs isolated from 10ml of blood from a normal donor were cultured for 24 hours in the primary erythroid expansion medium as described earlier (Trakarnsanga et al, 2017). These cells were then transduced with lentiviruses to express HPV E6/E7 gene and a fluorescent protein hKO1. After 3 days, the cells were cultured in a serum free medium containing the cytokines (stem cell factor and erythropoietin) and dexamethasone in the presence of doxycycline for 15 days. The cells that expressed hKO1 were sorted by FACS, and they were cultured in the same medium till the immortalization was complete. We continuously monitored the cells for the kinetics in the expression of erythroid cell surface markers, CD36, CD71 and CD235a, till >95% of the cells expressed all these markers. On day 50, all the cells expressed high levels of the erythroid markers and the cell morphology analysis using Giemsa staining showed that 65% of the cells were pronormoblasts, 22% were basophilic normoblasts and the rest of the cells were at the later stages of differentiation. To evaluate the differentiation potential of these cells, the cells were cultured using the media and conditions described by Hawksworth et al, 2018. After the removal of doxycycline from the culture medium, the cells showed haemoglobinization and the morphology analysis showed that 10% of the cells were in the polychromatic stage and 88% of the cells were in the orthochromatic stage, suggesting robust erythroid differentiation of the immortalized erythroid progenitors with the suitable cell culture conditions. These data showed that immortalized erythroid progenitors with differentiation potential could be generated directly from peripheral blood without using mobilized haematopoietic stem cells. This protocol is suitable for the generation of immortalized erythroid cells from the patients with rare red cell genetic disorders for studying disease mechanisms. Disclosures No relevant conflicts of interest to declare.

"C1ORF150", a Novel Mediator of EPO/EPOR/JAK2 Dependent Human Erythroid Progenitor Cell Formation

An understanding of cellular events that are propagated within erythroid progenitor cells upon HGF-R / JAK complex activation is of basic importance for generating new insight into regulated red cell formation, anemia and myeloproliferative disease. Using the EPO/EPOR/JAK2 system as a paradigm, our group is successfully applying post-translational modification-based proteomics to uncover important new mediators of EPO-dependent human erythropoiesis (certain of which may also relate to EPO's untoward effects on hypertension and cancer progression). Here, we report on the discovery of a novel ORF, "C1ORF150", that is strongly tyrosine phosphorylated in response to EPO, possesses several unique features, and modulates EPO- dependent erythroid progenitor cell formation. In human erythroid progenitor UT7epo cells, EPOR ligation leads to C1ORF150 phosphorylation at tandem tyrosine p-Y69, p-Y89 and p-Y110, p-Y129 sites (up to 10-fold within 15 minutes). For each PTM site, EPOR/JAK2 mediated- phosphorylation was validated in independent LC-MS/MS experiments using Hematide as an EPOR agonist. p-Y69 and p-Y89 are predicted SFK sites, while p-Y110 and p-Y129 are predicted RTK sites (including KIT). Notably, C1ORF150 is conserved in H. sapiens and primates, but is not represented in mouse, rat or lower vertebrates. In addition, C1ORF150 has no obvious orthologues, but within EPO-regulated pY regions exhibits sequence homology with HGAL, an important factor for B cell receptor signaling. To assess C1ORF150's functional effects, we used a lentiviral shRNA loss-of-function approach (80% knockdown efficiency). At physiological EPO levels, the knockdown of C1ORF150 substantially compromised UT7epo erythroid progenitor cell (EPC) survival, including 200% increases in apoptosis observed relative to control sh-NT transduced EPCs (p < 0.01). The ectopic expression of C1ORF150, in contrast, heightened baseline JAK2 activation, and potentiated STAT5 activation following EPO challenge. C1ORF150's subcellular localization proved to be predominantly membrane associated. With regards to expression profiles, C1ORF150 levels were markedly elevated in bone marrow (among 30 human tissues), and during erythroid development were maximal at a CFUe stage. Furthermore, transcriptome profiles of myelodysplastic syndrome (MDS) CD34pos hematopoietic progenitor cells revealed elevated C1ORF150 expression in MDS refractory anemia (p=0.005) and refractory anemia-blast patients (p=0.05) compared to normal controls. In summary, via PTM-proteomics we have discovered "C1ORF150" as a major new pY- regulated EPOR/JAK2 target and membrane associated phosphoprotein that is proposed to have evolved in human erythroid progenitor cells to support EPO's cytoprotective effects, and red cell formation, in part by reinforcing JAK2 and STAT5 activation. In anemia and pre-leukemic contexts, attention also is brought to possible roles for C1ORF150 in the onset and progression of MDS. Disclosures No relevant conflicts of interest to declare.