Establishment of Immortalized Human Erythroid Progenitor Cell Lines Able to Produce Enucleated Red Blood Cells

Abstract

Transfusion of red blood cells (RBCs) is a standard and indispensable therapy in current clinical practice. In vitro production of RBCs offers a potential means to overcome a shortage of transfusable RBCs in some clinical situations and also to provide a source of cells free from possible infection or contamination by microorganisms. Thus, in vitro production of RBCs may become a standard procedure in the future. We previously reported the successful establishment of immortalized mouse erythroid progenitor cell lines that were able to produce mature RBCs very efficiently. Here, we have developed a reliable protocol for establishing immortalized human erythroid progenitor cell lines that are able to produce enucleated RBCs. These immortalized cell lines produce functional hemoglobin and express erythroid-specific markers, and these markers are upregulated following induction of differentiation in vitro. Most importantly, these immortalized cell lines all produce enucleated RBCs after induction of differentiation in vitro, although the efficiency of producing enucleated RBCs remains to be improved further. To the best of our knowledge, this is the first demonstration of the feasibility of using immortalized human erythroid progenitor cell lines as an ex vivo source for production of enucleated RBCs.

An erythroid-specific ATP2B4 enhancer mediates red blood cell hydration and malaria susceptibility

“…Unlike K562 cells that were originally isolated from a naturally occurring human malignancy (chronic myeloid leukemia), HUDEP-2 cells were prospectively isolated from primary hematopoietic stem and progenitor cells transduced by an inducible viral oncogene and continuously cultured under conditions permissive to expansion of erythroid precursors. HUDEP-2 cells are an in vitro model of human erythropoiesis that mimics erythroid development from the proerythroblast to reticulocyte stages (21,35). We have previously established robust methods to perform CRISPR-Cas9 genome editing in HUDEP-2 cells (34,36).…”

“…To test this hypothesis, we deleted this region in a human erythroid precursor cell line (HUDEP-2) using the CRISPR-Cas9 system (21). We introduced 2 independent pairs of guide RNAs, one of which results in a 927-bp deletion and the other in a 889-bp deletion (Supplemental Table 6).…”

An Oncogenic Protein Golgi Phosphoprotein 3 Up-regulates Cell Migration via Sialylation

“…The resultant cDNAs in entry vectors were confirmed by DNA sequencing. The CSIV-TRE-RfA-CMV-KT lentiviral vector was constructed by replacement of the EF promoter with CMV in CSIV-TRE-RfA-EF-KT vector (31,32) The expression vectors pEF puro-RfA and pcDNA3.1-RfA were constructed using the Gateway conversion kit (Invitrogen). Using LR clonase (Invitrogen), the subcloned cDNAs in entry vectors were transferred into pcDNA3.1-RfA for transient expression, CSIV-TRERfA-CMV-KT for the tetracycline inducible overexpression, and pBABE hygro-RfA (33) or pEF puro-RfA for the stable expression.…”

Flicking the switch: adult hemoglobin expression in erythroid cells derived from cord blood and human induced pluripotent stem cells

“…Lu nally differentiate in vitro, 9 and hiPS cells have also been shown to possess hematopoietic potential, using methods similar to those for generating erythroid cells from hESCs. 10 Between the two sources, hiPS cells present two major advantages: they pose no ethical barriers regarding the origin of the cells, and they can be selected for phenotypes of interest. Even though in recent years we have seen great advances in the production of RBCs starting from hESCs and hiPS cells, the procedure needs to be further optimized before clinical applications can even be considered.…”