Generation of an Immortalized Erythroid Progenitor Cell Line Directly from Peripheral Blood Mononuclear Cells without Using Mobilized CD34+ Cells
Authors: Bagchi Abhirup , Nath Aneesha , Rajendiran Vignesh , Maddali Madhavi , Jajodia Ekta , Murugesan Mohankumar Kumarasamypet , Nakamura Yukio , Srivastava Alok , Velayudhan Shaji Ramachandran 
Published: October 2019

Abstract
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.