Generation Of Mature Erythroid Cells From Human Embryonic Stem Cell Derived Sacs and Optimization Of Erythroid Cell Generation
Authors: Fujita Atsushi , Winkler Thomas , Tisdale John F. , Uchida Naoya 
Published: October 2019

Abstract
Human embryonic stem cells (hESCs) represent a potential alternative source for red blood cells (RBCs) for transfusion therapy. However, in vitro production of erythroid cells from hESCs has two critical limitations: low-efficiency of erythroid cell generation and absent production of adult type β globin. Previously, hESC-derived erythroid cells were generated by co-culture with feeder cells through embryoid body formation, and these erythroid cells produced embryonic type ε globin or fetal type γ globin. Recently, embryonic stem cell-derived sacs (ES sacs) were generated with hemangioblast markers and generated functional platelets (Takayama, Blood. 2008). We hypothesized that hESC-derived β globin producing erythroid cells could be efficiently produced through the hemangioblast-like ES sac and developed new strategies to generate mature erythroid cells in vitro from hESCs using the ES sac.
ES sacs were generated from H1 hESCs using ES sac media containing 15% fetal bovine serum supplemented with 20ng/ml VEGF for 14-16 days. Spherical cells in suspension in these ES sacs were harvested, and erythroid cells were generated using a modified human erythroid massive amplification culture for 2 weeks (Migliaccio, Blood cells Mol Dis. 2002). We evaluated total cell counts, hematopoietic stem/progenitor cell marker (CD34) expression, and erythroid cell marker (CD235a) expression. In addition, we analyzed globin types in erythroid cells by hemoglobin electrophoresis.
After erythroid differentiation from suspension cells within ES sacs (containing 12.39% CD34+ cells), we generated CD235a+ erythroid cells (55.47%). Additionally, we confirmed morphology of erythroid cells by cytospin examination and erythroid colony formation by CFU assays using harvested suspension cells within the ES sac. To improve upon erythroid cell differentiation during ES sac production, we added SCF, IL-3, Flt-3L, TPO, and EPO to the ES sac media from day 9-15. The number of CD34+ cells increased 2.7 fold (19x10e3 cells vs. 7x10e3 cells) by supplementation with SCF, IL-3 and EPO while the number of CD235a+ cells increased 6.6 fold (239x10e3 cells vs. 36x10e3 cells) after erythroid differentiation. These data suggest that cytokine stimulation can induce erythroid differentiation during ES sac production.
We then hypothesized that efficient erythroid cell generation might be further promoted by shifting the balance between hematopoietic cell differentiation and endothelial cell differentiation in the hemangioblast-like ES sac. Thus, we added BMP4 to ES sac media from day 6-15 to improve hematopoietic cell differentiation, and we compared no VEGF with normal concentration VEGF (20ng/ml) from day 0-15 to reduce endothelial cell differentiation. After supplementation with BMP4, CD34+ cell numbers increased 3.1 fold (125x10e3 cells vs. 40x10e3 cells) among suspension cells in the ES sac, while the number of CD235a+ cells increase 2.6 fold (646x10e3 cells vs. 250x10e3 cells) after erythroid differentiation. In the no VEGF condition, the number of CD34+ cells increased 3.0 fold (118x10e3 cells vs. 40x10e3 cells) among suspension cells in ES sac, while the number of CD235a+ cells increased 2.8 fold (710x10e3 cells vs. 250x10e3 cells). These data suggest that erythroid differentiation during ES sac production was induced by not only stimulating hematopoietic cell differentiation but also suppressing endothelial cell differentiation.
Finally, we evaluated hemoglobin types among hESC-derived erythroid cells differentiated from ES sacs without cytokine supplementation. We detected embryonic type hemoglobin (27.9%), fetal type hemoglobin (61.3%), and adult type hemoglobin (10.8%) by hemoglobin electrophoresis. These data suggests that ES sac-derived erythroid cells produce more adult type hemoglobin as compared to previous strategies.
In conclusion, we demonstrated for the first time that erythroid cells can be efficiently generated from hESCs derived ES sacs and further improved by cytokine supplementation. Our data suggest that ES sacs have fractions of definitive hematopoiesis which can induce generation of mature red blood cells producing adult type hemoglobin.

Disclosures:
No relevant conflicts of interest to declare.