Characterization of Rh(D) and ABO genotype of hESC lines by PCR. (A) Genotyping of RhD locus: Specific primers were designed for the Rh locus that when Rh(D) positive DNA was used, 1200-bp (weak) and 600-bp PCR products were amplified; whereas DNA from RhD-negative cells generated only the 1200-bp fragment. (B,C) Genotyping of the ABO locus: 2 pairs of primers were designed to amplify 2 regions of the ABO locus. The PCR products were digested with restriction enzymes to distinguish ABO types. Vertical white lines have been inserted in panels B and C to indicate repositioned gel lanes. ABO and Rh(D) genotypes are as follows: WA01, O( + ); MA99, B(−); MA133, A(−); WA07 and MA09, B( + ); and WA09 and MA01, A( + ). (D) RhD antigen expression analysis on erythroid cells derived from MA01 and MA99 hESCs by FACS. Erythroid cells generated from MA01 and MA99 hESCs were stained with phycoerythrin-labeled monoclonal anti-RhD antibody and analyzed by FACS. (E) ABO type characterization of hESC-derived erythroid cells. (i) Cells stained with monoclonal antibody against A-antigen (original magnification ×400). (ii) Cells stained with monoclonal antibody against B-antigen (original magnification ×400).