Stromal factors support the expansion of the whole hemopoietic spectrum from bone marrow CD34⁺DR⁻ cells and of some hemopoietic subsets from CD34⁺ and CD34⁺DR⁺ cells

Abstract

Ex vivo expanded bone marrow CD34⁺DR⁻ cells could offer a graft devoid of malignant cells able to promptly reconstitute hemopoiesis after transplant. We investigated the specific expansion requirements of this subpopulation compared to the more mature CD34⁺ and CD34⁺DR⁺ populations. The role of stromal factors was assessed by comparing the expansion obtained when the cells were cultured in (1) long-term bone marrow culture (LTBMC) medium conditioned by an irradiated human BM stroma (CM), (2) medium supplemented with 15% FBS (FBSM) and (3) non-conditioned LTBMC medium (LTM) for 21 days. The effect of the addition of G-CSF (G) and/or of MIP-1α (M) to a combination of IL-3, SCF, IL-6 and IL-11 (3, S, 6, 11) was analyzed. Compared to CD34⁺DR⁻ cells, CD34⁺ and CD34⁺DR⁺ cells gave rise to a similar number of viable cells and to a lower progenitor expansion. The expansion potential of CD34⁺ and CD34⁺DR⁺ cells was equivalent in CM and in FBSM except for both the emergence of CD61⁺ megakaryocytic cells and LTC-IC maintenance which were improved by culture in CM. In contrast, expansion from CD34⁺DR⁻ cells was enhanced by CM for all the parameters tested. Compared to FBSM, CM induced a higher level of CFU-GM and BFU-E expansion and allowed the emergence of CD61⁺ cells. HPP-CFC were maintained or expanded in CM but decreased in FBSM. Compared to input, the number of LTC-IC remaining after 21 days of CD34⁺DR⁻ expansion culture was strongly decreased in FBSM and variably maintained or expanded in CM. Comparison with LTM indicated that stroma conditioning is responsible for this effect. G-CSF significantly improved CFU-GM and HPP-CFC expansion from CD34⁺DR⁻ cells without being detrimental to the LTC-IC pool. The growth of CD61⁺ cells was significantly enhanced by G-CSF in CM. Addition of MIP-1α had no significant effect either on progenitor expansion or on LTC-IC, regardless of culture medium. We conclude that factors present in stroma- conditioned medium are necessary to support the expansion of the whole spectrum of hematopoietic cells from CD34⁺DR⁻ cells and to support the expansion of cell subsets from CD34⁺ and CD34⁺DR⁺.