Cytokine-induced expansion of human CD34+ stem/progenitor and CD34+CD41+ early megakaryocytic marrow cells cultured on normal osteoblasts.

Thrombocytopenia remains a significant cause of morbidity in cancer patients undergoing allogeneic bone marrow transplantation (BMT), which consumes millions each year for frequent platelet transfusions. Using a novel culture system containing appropriate cytokine(s) on a layer of normal human osteoblasts, we investigated the expansion of early megakaryocytic progenitor cells while maintaining the number of CD34+ stem/progenitor marrow cells in an attempt to provide an effective solution for the problem of post-transplant thrombocytopenia. After seven days of culture, normal human osteoblasts alone without cytokines significantly increased the number of CD34+ and CD34+CD41+ marrow cells. Among the various cytokine combinations tested, both stem cell factor (SCF), interleukin 3 (IL-3)+IL-11 and SCF+IL-3+IL-11+thrombopoietin (TPO) emerged as the most effective in expanding early CD34+CD41+ megakaryocytic cells. Early CD34+CD41+ megakaryocytic cells have increased by 3.1- and 4.7-fold compared with day 7 control cultures, and by 62- and 94-fold, respectively, compared with day 0 input, respectively. Also, late CD41+ megakaryocytic cells have increased by 15.4- and 27.5-fold compared with day 7 control cultures in the presence of the same two combinations. In addition, the same cytokine combinations achieved 17.6- and 13.3-fold increases in the number of CD34+ marrow cells after the same seven days of culture on a layer of human osteoblasts. The combination (SCF+IL-3+IL-11+TPO) achieved the highest expansion of CD34+CD41+ early megakaryocytic cells from human marrow CD34+ cells reported so far in the literature. Recently, transplantation of SCF+IL-1+IL-3+TPO ex vivo expanded megakaryocytic progenitor cells as a supplement has been shown to accelerate platelet recovery by three to five days in mice. Therefore, the clinical use of the combination (SCF+IL-3+IL-11+TPO) for ex vivo expansion of CD34+ and megakaryocytic progenitor cells from a portion of the donor's marrow harvest is warranted in allogeneic BMT. Such a protocol would accelerate platelet recovery and shorten the period of hospitalization after allogeneic BMT. The present study has confirmed the role of human osteoblasts in supporting the proliferation and maintenance of human CD34+ stem/progenitor marrow cells. Given the facilitating role of osteoblasts shown previously in several allogeneic BMT studies in mice, it is possible to envisage a future role for donor osteoblasts in clinical BMT. Transplantation of the cultured donor osteoblasts together with the ex vivo expanded CD34+ marrow cells as a supplement might not only accelerate platelet recovery but also prevent acute graft-versus-host disease in allogeneic BMT. The present novel culture system should have useful clinical application in allogeneic BMT.