Erythroid Differentiation of Induced Pluripotent Stem Cells Co-cultured with OP9 Cells for Diagnostic Purposes

Abstract

Background: Reagent red blood cells (RBCs) are prepared from donated whole blood, resulting in various combinations of blood group antigens. This inconsistency can be resolved by producing RBCs with uniform antigen expression. Induced pluripotent stem cells (iPSCs) generated directly from mature cells constitute an unlimited source for RBC production. We aimed to produce erythroid cells from iPSCs for diagnostic purposes. We hypothesized that cultured erythroid cells express surface antigens that can be recognized by blood group antibodies.Methods: iPSCs were co-cultured with OP9 stromal cells to stimulate differentiation into the erythroid lineage. Cell differentiation was examined using microscopy and flow cytometry. Hemoglobin electrophoresis and oxygen-binding capacity testing were performed to verify that the cultured erythroid cells functioned normally. The agglutination reactions of the cultured erythroid cells to antibodies were investigated to confirm that the cells expressed blood group antigens. Results:The generated iPSCs showed stemness characteristics and could differentiate into the erythroid lineage. As differentiation progressed, the proportion of nucleated RBCs increased. Hemoglobin electrophoresis revealed a sharp peak in the hemoglobin F region. The oxygen-binding capacity test results were similar between normal RBCs and cultured nucleated RBCs. ABO and Rh-Hr blood grouping confirmed similar antigen expression between the donor RBCs and cultured nucleated RBCs. Conclusions:We generated blood group antigen-expressing nucleated RBCs from iPSCs co-cultured with OP9 cells that can be used for diagnostic purposes. iPSCs from rare blood group donors could serve as an unlimited source for reagent production.

In vitro erythrocyte production using human-induced pluripotent stem cells: determining the best hematopoietic stem cell sources

“…On the second and fifth days after transfection, each well was supplemented with 1 mL erythroid expansion medium and 1 mL ReproTeSR (Stem Cell Technologies), respectively. Beginning on post-transfection day 7, complete medium changes were performed with 2 mL ReproTeSR every day, and the morphology of colonies was closely monitored until the hiPSC-like colonies appeared [ 26 , 27 ].…”

“…Cells (1 × 10 5 per slide) were centrifuged and immobilized onto a glass microscope slide using a Cytospin 5 cytocentrifuge (Thermo Fisher Scientific) at 700 rpm for 7 min. The slides were stained with Wright–Giemsa dye (Sigma-Aldrich), observed under a BX53 light microscope (Olympus, Tokyo, Japan), and imaged with a DP70 camera (Olympus) [ 26 , 27 ].…”

“…The vial was loaded into the cuvette, and the analysis was performed as per the manufacturer’s instructions. The data obtained from hiPSC-differentiated erythroid cells were compared to those obtained from freshly drawn normal PB [ 21 , 27 , 32 ].…”

Recent updates of stem cell-based erythropoiesis

“…To generate RBCs or erythrocytes, progenitor blood cells are typically differentiated by exposing them to growth factors, such as SCF, IL-3, and EPO, in suspension [ 4 , 61 , 86 , 87 ]. However, current methods are still hindered by low efficiency, with current research attempting to increase RBC differentiation efficiency [ 62 , 76 , 88 ].…”

“… SCF, EPO, etc 15–17 13 RBC [ 62 , 89 ] iPSC from healthy donor PBMC BMP4, WNT3a, Activin A, b-FGF, VEGF, etc. SCF, IL-3, EPO, etc 11 21 RBC [ 87 ] 2D culture (attachment) hiPSC from MUSIi001-A, MUSIi011-A, PBiPSC1 CHIR99021, VEGF, FGF2, SB431542, etc. SCF, IL-3, EPO, transferrin, etc 8–12 20 RBC [ 75 ] Advantages • Feeder-free and serum-free protocols are available • More simple and less demanding culture method compared to 3D and co-culture methods hiPSC derived from Down syndrome or β-thalassemia human fibroblasts BMP4, VEGF, WNT3a, FGF2, SCF, FLT3L, TPO, IL-6, EPO, FICZ, etc.…”