A Clinically Suitable Ex-Vivo Expansion Culture System for LTC-IC and CFC Using Stroma-Conditioned Medium
R. Bhatia,P.B. McGlave, J.S. Miller, S. Wissink, W. Lin, C.M. Verfaillie
Experimental Hematology. 1997. 25:980-991
Hematopoietic progenitors; Stem celIs; Ex vivo expansion; Platelet factor-4; Chronic myelogenous leukemia
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 l-« (MIP-Ia) 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-3cultures. 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 2-6x105 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.