Expression of hematopoiesis-regulating factors by HSCs sorted from 12- to 29-week fetal liver (FL) and bone marrow (FBM). (A) CD34/CD38 two-color stainings of mononucleated cells from 12-week FL and 20-week FBM. The percentages of cells that fall within each of the sorting gates are indicated. The purity of recovered populations was ascertained by PCR-amplification of CD38 cDNA, as shown in (B). (B) Semiquantitative RT-PCR analysis of hematopoiesis-specific genes in the selected CD34⁺CD38⁺ and CD34⁺CD38⁻ cell subsets. Each track is representative of at least four experiments performed on subpopulations sorted from different organs of various stages (see Materials and Methods). Negative control with no cDNA added was included in each PCR experiment and the product size was checked by running molecular weight markers. The amplified products were transferred to nylon membranes and hybridized with internal specific ³²P-labeled cDNA probes. Autoradiography was prolonged for 18 hours for flk-1/KDR PCR products but did not exceed 2 hours for the other gene products. Signals obtained for β-actin amplification were used as reference to normalize quantitative differences between cDNA samples.