Establishment of an erythroid progenitor cell line capable of enucleation achieved with an inducible c-Myc vector

Abstract

Background A robust scalable method for producing enucleated red blood cells (RBCs) is not only a process to produce packed RBC units for transfusion but a potential platform to produce modified RBCs with applications in advanced cellular therapy. Current strategies for producing RBCs have shortcomings in the limited self-renewal capacity of progenitor cells, or difficulties in effectively enucleating erythroid cell lines. We explored a new method to produce RBCs by inducibly expressing c-Myc in primary erythroid progenitor cells and evaluated the proliferative and maturation potential of these modified cells. Results Primary erythroid progenitor cells were genetically modified with an inducible gene transfer vector expressing a single transcription factor, c-Myc, and all the gene elements required to achieve dox-inducible expression. Genetically modified cells had enhanced proliferative potential compared to control cells, resulting in exponential growth for at least 6 weeks. Inducibly proliferating erythroid (IPE) cells were isolated with surface receptors similar to colony forming unit-erythroid (CFU-Es), and after removal of ectopic c-Myc expression cells hemoglobinized, decreased in cell size to that of native RBCs, and enucleated achieving cultures with 17% enucleated cells. Experiments with IPE cells at various levels of ectopic c-Myc expression provided insight into differentiation dynamics of the modified cells, and an optimized two-stage differentiation strategy was shown to promote greater expansion and maturation. Conclusions Genetic engineering of adult erythroid progenitor cells with an inducible c-Myc vector established an erythroid progenitor cell line that could produce RBCs, demonstrating the potential of this approach to produce large quantities of RBCs and modified RBC products. Electronic supplementary material The online version of this article (10.1186/s12896-019-0515-9) contains supplementary material, which is available to authorized users.

Single-cell multi-omics of human clonal hematopoiesis reveals that DNMT3A R882 mutations perturb early progenitor states through selective hypomethylation

“…Notably, we observed the enrichment of two independent MYC target gene sets, including a MYC signature that was downregulated with monocytic differentiation in an HSPC differentiation cell line model 97,98 . Consistently, MYC has been demonstrated to be a critical factor specifically for erythropoiesis [99][100][101] , and may thus contribute to the observed ME bias (Fig. 2c,d,g).…”

Selective LXR agonist DMHCA corrects retinal and bone marrow dysfunction in type 2 diabetes

“…DMHCA-treated AP1-high stem cells showed increased AP1 complex genes, as well as increased expression of KLF2 and -6, two genes required for normal erythrocyte development (66,67). Of note is the increase in MYC expression, which has recently been shown to induce proliferation of erythroid progenitor cells (68). DGE analysis also showed significantly increased hemoglobin gene expression, suggesting that DMHCA not only increases erythrocyte progenitor flux, but also stimulates the generation of more robust erythrocyte progenitors.…”

BMI1 enables extensive expansion of functional erythroblasts from human peripheral blood mononuclear cells

“… 6 , 21 A study with mouse early erythroid progenitors also reported their immortalization by a c- myc vector, although it is unclear whether it is applicable to human counterparts. 22 Although human erythroblasts can be immortalized by defined genetic factors, full maturation and enucleation of these genetically engineered erythroblasts to produce functional RBCs are usually blocked by the presence of potent pro-growth genes or induced genetic or epigenetic alterations. 21 , 23 , 24 For example, we previously used a lentiviral vector expressing a set of reprogramming factors and immortalized postnatal human umbilical cord blood (UCB)-derived erythroblasts.…”