Experimental approach to identify genes differentially expressed in FB-HSPCs compared with adult MPB-HSPCs. (A) Development of cDNA libraries for identifying differentially expressed genes in FB-HSPCs. Isolation of Lin^-CD34⁺CD38^- cells from fetal blood (10 samples) and mobilized peripheral blood (6 samples) followed by performing SSH as explained in “Materials and methods.” SSH was performed 2 more times to ensure the quality and reproducibility of the cDNA library. Screening of the colonies in the subtracted library will detect differentially regulated transcripts in FB-HSPCs and BLAST analysis will identify the closest transcripts in the human genome. We further narrowed our analysis toward key regulatory genes including transcription factors, cell cycle regulators, and signal transduction genes presumably responsible for fate determination of HSPCs. (B) Verification and quantification of candidate transcripts identified in the SSH reactions. Lin^-CD34⁺CD38^- cells from fetal blood (5 samples) and mobilized peripheral blood cells (5 samples) and also Lin^-CD34⁺CD38⁺ from several fetal blood (12 samples) were isolated and used for real-time Q-PCR. The Q-PCR experiments were done in 3 different concentrations and each concentration in duplicate. This analysis confirmed and validated our experimental approach to identify genes that are consistently differentially expressed in FB-HSPCs versus adult counterparts from M-PB-HSPCs.