Our recent study demonstrated that the cyclin-dependent kinase inhibitor (CKI) p18Ink4c (p18), also an INK4 family protein acting at early G1-phase, exerts its inhibitory role during the self-renewing division of murine hematopoietic stem cells (HSC) in vivo (Nature Cell Biology 2004). Down-modulating p18 may permit enhanced stem cell expansion in vitro, a hypothesis that is now being testing in our laboratory. To provide the proof-of-the concept, we first took advantage of the murine system by testing the in vivo reconstituting ability of cells that had been cultured under the Dexter culture condition for 19 weeks. 2–20x105 cells with non-adherent and adherent populations were transplanted into lethally irradiated hosts. 3 of 7 mice revealed long-term engraftment in the p18−/− transplanted group (0.5–33% engraftment levels) while there was no engraftment in the p18+/+ group (n=7). Moreover, a substantial level (38.6% on average) of long-term engraftments (7 months) in multilineage was achieved in secondary recipients transplanted with the p18−/− cells (n=3), demonstrating the self-renewal potential of the expanded HSCs after the extended period of long-term culture. These data strongly indicate that p18 absence is able to substantially mitigate the differentiating effect of the ex vivo culture conditions on HSCs and therefore offer a strong rationale for targeting p18 in human HSC expansion. P18 mRNA was detected by RT PCR in human CD34+ cells with a higher expression level in the more primitive subset: CD34+CD38−. To explore the possibility of targeting p18 for expanding human HSCs, we have employed the RNA interference (RNAi) technology in CD34+ cord blood cells. We screened a pool of small interfering RNA (siRNA) oligos and three of them were able to effectively reduce p18 expression by 60–80% in 48 hours as assessed by both RNA and protein analyses in human cells. Further, we tested both transient and permanent delivery methods for introducing the RNAi effect in the CD34+CD38− cells. To demonstrate whether the RNAi method would be sufficient to impact the outcome of cell division after a single or limited cell cycle(s), we chose the nucleofector technology and were able to achieve 48.30±11.66% of transduction efficiency with good viability (50.63±9.38%, n=3) in human CD34+ cells. After a single electroporation pulse, we were able to increase by 2-fold the CD34+CD38− cells associated with the same magnitude of increased colony forming activity under culture condition supplemented with SCF, TPO and Flt3. To observe the long-term effect of p18 downregulation in human HSCs, we constructed a p18 short hairpin (shRNA)-expressing lentiviral vector that was engineered to have the mouse U6 promoter upstream of a CMV-EGFP expression cassette. A transduction efficiency of 30–60% was achieved after overnight infection of the human CD34+ cells with the p18 shRNA or with control lentiviral vectors pseudotyped with the VSV-g envelope. 72–96 hours after the transduction, human p18 protein can be knocked down by the p18 siRNA lentivector at near 100% in the HeLa cell line as determined on the western blot, and at more than 50% in human primary CD34+ cells as determined by real time RT PCR. We are currently undertaking further study aimed at assessing the repopulating ability of the transduced human HSCs with lentivirus-mediated p18 shRNA in NOD/SCID mice. Together, these findings suggest that down-modulating p18 might be a feasible approach for manipulating human HSCs ex vivo.