ANALYSIS OF GENE-EXPRESSION IN SMALL NUMBERS OF PURIFIED HEMATOPOIETIC PROGENITOR CELLS BY RT-PCR

Primitive hemopoietic stem cells represent the most probable targets f or genetic alterations due to exposure to ionizing irradiation or chem ical carcinogens. We have applied a two-step protocol for the purifica tion of CD34(+)HLA-DR(-/low) hemopoietic progenitor cells from cord bl ood (CB). CD31(+) cells were isolated by monoclonal antibody (mAb) aga inst CD34 (My10) and immunomagnetic beads. Beads were cleaved off the CD34(+) cells by enzymatic treatment with chymopapain. Due to chymopap ain-resistance of epitopes recognized by the used mAbs purity control of CD34(+) cells and separation into CD34(+)HLA-DR(-/low) and CD34(+)H LA-DR(+) subsets could be performed by using flow cytometry, Two minia turized procedures were applied to isolate poly(A)(+) mRNA for the rev erse transcription polymerase chain reaction (RT-PCR) from small numbe rs of CD34(+)HLA-DR(-/low) cells. In five experiments, the mean purity of immunomagnetically isolated CD34(+) cells was 93.8% +/- 3.9. Flow cytometry sorting of CD34(+) cells resulted in pure CD34(+)HLA-DR(-/lo w) populations (purity > 98.8%; range 98.8% to 99.9%; viability > 96%) with an average yield of 2600 +/- 800 cells/5 x 10(7) low density CB cells. By RT-PCR using both poly(A)(+) mRNA isolation procedures, sequ ences corresponding to CD34 and beta 2-microglobulin were amplified fr om as few as 20 cells. Furthermore, a sequence-independent RT-PCR (SIP -RT-PCR) was applied to amplify the cDNA derived from five erythroblas ts isolated from a burst-forming unit-erythroid (BPU-E). Upon hybridiz ation, full-length c-fos message was detected in the SIP-RT-PCR amplif ied material. Our data demonstrate that gene expression can be detecte d at the transcriptional level in small numbers of hemopoietic progeni tor cells. In addition, the SIP-RT-PCR may allow the amplification of unique mRNA species when subtractive hybridization procedures are perf ormed. The presented data should be useful to analyze gene expression in rare subsets of radiation-exposed immature hemopoietic stem/progeni tor cells.