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.
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-1alpha (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-1alpha 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+.
Herman
P
P
Hematology Department, Université Catholique de Louvain, Brussels, Belgium.
Ferrant
A
A
De Bruyère
M
M
Straetmans
N
N
D015703:Antigens, CD / Q000096:biosynthesis / Q000502:physiology; D018952:Antigens, CD34 / Q000096:biosynthesis / Q000502:physiology*; D001854:Bone Marrow Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D054405:Chemokine CCL3; D054407:Chemokine CCL4; D002999:Clone Cells; D017077:Culture Media, Conditioned; D016179:Granulocyte Colony-Stimulating Factor / Q000494:pharmacology; D006133:Growth Substances / Q000494:pharmacology*; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D039661:Integrin beta3; D017370:Interleukin-11 / Q000494:pharmacology; D019402:Macrophage Inflammatory Proteins / Q000494:pharmacology; D008533:Megakaryocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D010980:Platelet Membrane Glycoproteins / Q000096:biosynthesis / Q000502:physiology; D019089:Stem Cell Factor / Q000494:pharmacology; D017154:Stromal Cells / Q000166:cytology / Q000378:metabolism; D013997:Time Factors
D015703:Antigens, CD; D018952:Antigens, CD34; D054405:Chemokine CCL3; D054407:Chemokine CCL4; D017077:Culture Media, Conditioned; D006133:Growth Substances; D039661:Integrin beta3; D017370:Interleukin-11; D019402:Macrophage Inflammatory Proteins; D010980:Platelet Membrane Glycoproteins; D019089:Stem Cell Factor; D016179:Granulocyte Colony-Stimulating Factor