Large-Sacle ExVivo Generation of Human Red Blood Cells from Cord Blood CD34+ Cells
Authors: Zhang Yu , Wang Chen , Wang Lan , Tian Jing , Shen Bin , Guan Xin , Ren Zhihua , Ding Xinxin , Ma Yupo , Dai Wei , Jiang Yongping 
Published: October 2019

Abstract
Blood transfusion is widely used for various clinical therapies. Ex vivo production of red blood cells (RBCs) in a large-scale from hematopoietic stem cells (HSCs) has been considered as a potential way to overcome the shortage of blood supply. Here, we report that functional human RBCs can be efficiently produced by using a bottle-turning device culture system from cord blood (CB) CD34+cells.
A procedure of four-stage ex vivo expansion and differentiation was developed in a modified IMDM basal medium supplemented with transferrin, insulin, folic acid, fetal bovine serum, and some other nutrients with selected cytokine combinations that contained stem cell factor (SCF), Flt-3 ligand (FL), and thrombopoietin (TPO) in stage I (days 0~5); SCF, FL, erythropoietin(EPO), interleukin 3 (IL-3), and GM-CSF in stage II (days 6~12); SCF, FL, IL-3, and EPO in stage III (days 13~18); SCF and EPO in stage IV (days 19~21). Enriched CD34+ cells were firstly cultured and expanded in 25-T flasks. After 5 day-culture, the cells were transferred to a 2-L bottle with 600 ml of medium in the bottle-turning device system. During the differentiation process, erythroid markers (CD71 and CD235a) and enucleation efficiency (LDS- percentage) of cultured cells were evaluated by flow cytometry. Erythroid progenitor cells were confirmed by clonogenic capacity by colony-forming unit (CFU) assay. Hemoglobin (HGB) content of the cells were determined quantitatively, and RT-qPCR analysis was performed to examine the expression of erythroid-specific genes and the status of proto-oncogenes. Furthermore, generated RBCs were CFSE-labeled and injected into irradiated NOD/SCID mice to monitor the viability and maturation in vivo.
For stage I, the proliferation folds of CD34+ cells reached 20 ± 2.4 while CD34+ rate was maintained at 80% ± 4.3%. Subsequently, CFU assay on Day9 and Day12 showed that over 90% of the total colonies were erythroid burst-forming units (BFU-E) or erythroid colony-forming units (CFU-E), suggesting that these expanded cells were induced toward the erythroid lineage. For 21-day induction, approximate 2×108erythrocytes were produced from one CD34+cell with a purity of CD235a+ and CD71+ cells at 90% ± 6.2% and 54 % ± 7.2%, respectively. Furthermore, the results from flow cytometry of LDS-stained cells showed that 50% ± 5.7% of induced erythrocytes were enucleated. At various time points of the cell culture process, RT-qPCR analysis showed that expressions of erythroid-specific genes were normal and the proto-oncogenes (c-myc, c-myb, Bmi-1, k-ras, cyclinB, and HETRT) were not activated. From days 9 to 21, HGB contents of the cultured cells increased from 12.3 ± 1.5 pg/cell to 31.5 ± 2.4 pg/cell, which was similar to the contents of normal human RBCs (range: 27 - 33 pg/cell). In addition, the induced RBCs, after storing at 4℃ for more than 4 weeks, had normal HGB content, and showed normal expression of both CD235a and CD71. In mouse studies, the CD71+ marker on the 21-day cultured cells was diminished or undetectable in CFSE+ cells 3-day after transplantation. In contrast, LDS- cells among the CFSE+ population increased to 97.1% ± 2.3% 3-day post injection, indicating that the 21-day cultured RBCs could be further enucleated and matured in vivo.
Taken together, we have established a pilot-scale culture system to produce functional human RBCs ex vivo. Considering that one blood transfusion unit contains 0.8×1012RBCs, the CD34+ cells from one CB unit (80 ml with 2×106 CD34+ cells) would generate 4×1014 RBCs, which are equivalent to 500 blood transfusion units in the clinical application.

Disclosures
Ren: Biopharmagen Corp: Employment. Jiang:Biopharmagen Corp: Consultancy.