Reverse Transcriptase-PCR Analysis of Gene Expression in Hematopoietic Stem Cells
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The events that determine whether hematopoietic stem cells (HSC) divide in the course of self-renewal or differentiate and become committed progenitor cells are regulated by specific gene expression. Although little is known of the molecular controls for these diverse events, the activation of a single gene may determine which developmental event will occur in an individual HSC. New and more precise information on the controls for gene expression in HSC may provide relevant clues to the regulation of hematopoiesis. Yet investigations of gene expression in HSC have been difficult to perform, primarily because it has been difficult to purify the large numbers of HSC needed to obtain sufficient RNA for Northern analysis or RNase protection assays. These rare cells occur in a ratio of approx 1:10,000 to 1:100,000 bone-marrow cells. Their enrichment is accomplished by coupling several procedures that include the use of lineage-specific monoclonal antibodies (MAbs) and immunomagnetic bead depletion of unfractionated bone marrow followed by fluorescence-activated cell sorting (FACS). The HSC in lineage-negative cell populations from mouse bone marrow are then sorted for HSC using MAbs specific for surface markers such as Sca-1 or c-kit (Fig. 1 ). Human HSC are lineage negative, CD34-positive and CD38-negative.
Fig. 1. This scheme shows the method we use for the purification of mouse and human HSC from elutriated bone marrow. The cells in the elutriated cell fractions (FR = flow rate in mL/min) are labeled with MAbs specific for each hematopoietic lineage. These cell populations are then depleted of lineage-positive cells using immunomagnetic beads. Finally, the lineage-negative cells from mouse bone marrow are incubated with anti c-kit MAb and the lineage-negative cells from human bone marrow are incubated with anti-CD34 and anti-CD38 MAb. With a starting population of 2–4 � 10e8 unfractionated mouse bone-marrow cells, a typical isolation procedure yields approx 2 � 10e4 FR25 lin - c-kit HI cells. This is the most highly enriched HSC population we have been able to obtain to date. As few as 50– 100 of these cells can completely repopulate the hematopoietic tissue of an adult W/W v mouse. However, 2 � 10e4 cells from this highly enriched HSC population do not provide sufficient mRNA for Northern blots or RNase protection assays, but do provide sufficient mRNA for the RT-PCR assay.
