| Objectives:1) to characterize the expression of all CYP genes in CD34(+) human cord blood hematopoietic stem and early progenitor cells (CBHSPCs), as a first step toward assessment of their biological functions in these cells;2) to evaluate mRNA expression levels of CYPIB1and CYP2R1in both CD34(+) CBHSPCs and mononuclear cells(MNCs) using quantitative RT-PCR.Methods:CD34(+)CBHSPCs were purified from umbilical cord blood via monoclonal mouse anti-human CD34antibodies labeled with MACS microbeads. Purity of CD34(+)CBHSPCs was assessed via Fluorescence-Activated Cell Sorting. RNA was extracted from the purified CD34(+)CBHSPCs for RNA-PCR analysis, using primers specific for each of the57CYPs, respectively. Additional sequencing analysis was carried out to identify the detected CYPs. Furthermore, the RNA expression levels of CYP1B1and CYP2R1were evaluated both in CD34(+)CBHSPCs and MNCs, using real time PCR. All experiments were repeated at least3times.Results:Of the57human CYPs, only14were specifically detected by reverse transcription PCR in CD34(+)CBHSPCs, including CYP1A1, CYP1B1, CYP2E1, CYP2C18, CYP2C19, CYP2J2, CYP2R1, CYP2S1, CYP2U1, CYP4F2, CYP4V2, CYP20A1, CYP27A1, and CYP51A1. Further real time PCR analysis of detected CYP transcripts yielded evidence for preferential expression of CYP2R1in CD34(+)CBHSPCs, relative to MNCs, and for greater expression of CYPIB1in MNCs, relative to CD34(+)CBHSPCs.Conclusion:This study provides the first unequivocal evidence of P450expression in human hematopoietic stem and progenitor cells at the early stages of differentiation. Fourteen of the57human CYPs were specifically detected by real time PCR in CD34(+)CBHSPCs. Real time PCR results demonstrated that CYP2R1is expressed at much higher levels in CD34(+)CBHSPCs than in MNCs. In contrast, CYPIB1expression level is much lower in CD34(+)CBHSPCs than in MNCs. These data suggest that, besides their known biological functions, P450enzymes may have novel roles in cell differentiation and maturation of hematopoiesis. |
