title: A clinically suitable ex vivo expansion culture system for LTC-IC and CFC using stroma-conditioned medium.
abstract: FACS-selected CD34+ HLA-DR- cells (DR- cells) may provide a source of benign stem cells suitable for autografting in chronic myelogenous leukemia (CML) and other hematological malignancies. However, DR- cell selection depletes the majority of committed hematopoietic progenitors, which may be important for early engraftment. Furthermore, only a small number of DR- cells may be selectable in certain patients. These impediments to the use of DR- cells for autografting may be overcome through the development of ex vivo culture systems that support expansion and initial differentiation of primitive progenitors. Because 2-week culture of DR- cells in a stroma "noncontact" system supplemented with interleukin-3 (IL-3) and macrophage inflammatory protein 1-alpha (MIP-1alpha) expands both long-term culture-initiating cells (LTC-ICs) and colony-forming cells (CFCs), we adapted this system to a clinically applicable method for expanding LTC-ICs and CFCs ex vivo. In initial small-scale studies, DR cells were grown in stroma conditioned medium (SCM) supplemented with IL-3 with or without additional growth-promoting cytokines and the chemokines PF-4 and BB10010, all approved for clinical use. An IL-3 dose-dependent expansion of committed progenitors and LTC-ICs was observed when DR- cells were cultured in tissue culture plates in SCM+IL-3 for 2 weeks. Similar CFC expansion along with increased (5-fold) LTC-IC expansion was observed following addition of PF-4 to SCM+IL-3 cultures. The addition of stem cell factor (SCF), but not of IL-6, IL-11, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage (GM)-CSF, IL-1, and IL-7, increased CFC and LTC-IC expansion beyond the levels observed with SCM+IL-3 alone. We next evaluated the suitability of this culture system for scale-up. Culture of 2-6 x 10(5) DR- cells in gas-permeable bags with SCM+IL-3 resulted in similar CFC and LTC-IC expansion as seen in small-scale cultures. In addition, we observed that progenitors capable of differentiating to natural killer (NK)-cells were maintained under these conditions. Finally, we found that BCR/ABL mRNA-negative CFCs and LTC-ICs present in DR- cells selected from steady-state CML marrow could be expanded in large-scale SCM+IL-3 cultures. We conclude that culture of DR- cells for 2 weeks in SCM+IL-3 culture, with or without PF-4 or SCF, results in significant CFC and LTC-IC expansion and lymphoid NK progenitor maintenance. This culture system is readily adaptable to the expansion of primitive progenitors for autotransplantation.
authors.affiliation: Department of Medicine, University of Minnesota, Minneapolis 55455, USA.
authors.forename: R
authors.initials: R
authors.lastname: Bhatia
authors.forename: P B
authors.initials: PB
authors.lastname: McGlave
authors.forename: J S
authors.initials: JS
authors.lastname: Miller
authors.forename: S
authors.initials: S
authors.lastname: Wissink
authors.forename: W N
authors.initials: WN
authors.lastname: Lin
authors.forename: C M
authors.initials: CM
authors.lastname: Verfaillie
mesh_terms: D001854:Bone Marrow Cells; D002478:Cells, Cultured; D054405:Chemokine CCL3; D054407:Chemokine CCL4; D017077:Culture Media, Conditioned; D006412:Hematopoietic Stem Cells; D006801:Humans; D007377:Interleukin-3 / Q000494:pharmacology; D007694:Killer Cells, Natural; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive; D019402:Macrophage Inflammatory Proteins / Q000494:pharmacology; D010978:Platelet Factor 4 / Q000494:pharmacology; D017154:Stromal Cells / Q000378:metabolism*
chemical_list: D054405:Chemokine CCL3; D054407:Chemokine CCL4; D017077:Culture Media, Conditioned; D007377:Interleukin-3; D019402:Macrophage Inflammatory Proteins; D010978:Platelet Factor 4