Control of the HSC Niche Signaling for the Efficient Long-Term Engraftment of Hematopoietic Stem Cells without Irradiation or High-Dose Chemotherapy.

Irradiation and high-dose chemotherapy followed by bone marrow transplantation (BMT) have been a routine treatment for hematological malignancies and solid tumors. These conditioning, however, should be avoided in the cases of BMT for non-malignant diseases since the toxicity extends to all organs generating serious side-effects for both short-term and long-term. Then, we investigated the conditioning methods to obtain the efficient engraftment of hematopoietic stem cells (HSCs) without irradiation before BMT. In the adult BM, HSCs interact with its microenvironment which supports the activity of HSCs to maintain quiescence or self-renew to provide blood cells throughout the lifetime of an individual. This particular microenvironment, called stem cell niche, is critical for the maintenance of HSCs, and osteoblastic cells are considered as a key component for sustaining slow-cycling or quiescence of HSCs (Arai et al., 2007). Then we hypothesized that manipulating the niche may provide solutions for efficient engraftment avoiding redundant side-effects of conditionings. We previously reported that thrombopoietin (THPO)/Mpl signaling pathway is one of the niche factor which maintains HSCs quiescence in the steady state (Yoshihara et al., 2007). Enhancement of THPO/Mpl signal induces quiescence of HSCs in vivo, whereas inhibition of this signaling pathway by the administration of anti-Mpl neutralizing antibody (AMM2) induces cell cycling. We have previously shown that inhibiting THPO/Mpl signal enables HSCs to engraft without lethal irradiation. Administration of AMM2 and 5-FU prior to BMT showed donor chimerism of 5.8 ± 0.7 % (Yoshihara et al., 2007). Recently, Czechowicz et al. (2007) showed strikingly efficient transplantation by administration of c-Kit neutralizing antibody (ACK2) to Rag2-deficient mice. These results imply that inhibition of SCF/c-Kit signaling pathway is a key treatment for endogenous HSCs to deplete and exogenous donor cells to engraft. Then, we performed series of transplantation with various conditions to achieve more efficient engraftment using c-Kit-deficient W/Wv mice as recipient mice. W/Wv mice were conditioned with AMM2 (1 mg/kg body weight, iv) or PBS six days, and 5-FU (150 mg/kg body weight, iv) or PBS two days prior to BMT. Lineage⁻Sca-1⁺c-Kit⁺ (LSK) cells were sorted from BM of donor mice and 10⁴ LSK cells were transplanted intravenously to each recipient. Although the use of the W/Wv mice as the recipient allowed the engraftment of donor cells with the solo administration of AMM2 (6.7%) or 5-FU (9.3 %), we did not find the any reconstitution in W/Wv mice pretreated with PBS alone. Combinational administration of AMM2 and 5-FU increased the engraftment of donor cells (11.3 %) in W/Wv mice, and the engraftment rate in W/Wv mice was higher than that of wild type recipients. These data indicate that inhibition of SCF/c-Kit signaling in endogenous HSCs had synergistic effect on the engraftment of exogenous HSCs without irradiation. In addition, interestingly, increased dose of AMM2 showed the possibility of the efficient long-term engraftment of donor cells. Among high dose AMM2-treated mice (12.5mg/kg body weight iv), maximum donor chimerism was 53.8 %. These data suggest that antibody-mediated BMT is promising by the stem cell regulation in the niche. We are now focusing on the manipulation for the HSC-niche interaction in wild-type recipients. Achievement of non-irradiated BMT method will provide advancement in stem cell therapy for non-malignant diseases such as metabolic disorders or autoimmune disease overcoming serious side-effects.