Analysis of erythroid-specific blood group genes using un-mobilized peripheral stem cells cultured in vitro

Abstract

To analyze specific expression of blood group genes using nucleated erythroid cells cultured from un-mobilized peripheral stem cells in vitro. Hematopoietic stem cells(HSC) bearing the CD34 antigen were isolated from peripheral blood by centrifugation and magnetic beads sorting, followed by suspension culture in vitro. Cells were collected from medium on various stages and analyzed by immunofluorescence. The RNA transcription of RH and ABO blood group genes was analyzed using culture cells on day 12. A total of(3.19±0.13) ×10 (4) CD34+cells were isolated from about 50 mL peripheral blood with a recovery rate of 67.3%±2.7%. The cells amount in erythroid-lineage culture system on day 9 reached a plateau of a 237.1±15.5-fold amplification of the initial cell input. The stem cell-specific CD34 antigen was dropped off, while the erythroid-specific CD235a and CD240D antigens were increased in culture period. RHD/CE and ABO genes can be amplified using RNA extracted from culture cells on day 12, and genotypes of Rh and ABO systems by DNA sequencing were consistent with their serologic phenotypes. A method was established to analyze the gene expression of erythroid blood group derived from un-mobilized peripheral stem cells cultured in vitro. It can be used to study the expression of various erythroid-specific genes.