0041-1337/99/6710-1348/0 TRANSPLANTATION Vol. 67, 1348 –1357, No. 10, May 27, 1999 Copyright © 1999 by Lippincott Williams & Wilkins, Inc. Printed in U.S.A. PRESERVED LONG-TERM REPOPULATION AND DIFFERENTIATION PROPERTIES OF HEMATOPOIETIC GRAFTS SUBJECTED TO EX VIVO EXPANSION WITH STEM CELL FACTOR AND INTERLEUKIN 11 1 BEATRIZ ALBELLA, 2 JOSE ´ C. SEGOVIA, 2 GUILLERMO GU ¨ ENECHEA, 2 IAN B. PRAGNELL, 3 AND JUAN A. BUEREN 4,2 Molecular and Cell Biology Unit, Centro de Investigaciones Energe ´ticas, Medioambientales y Tecnolo ´gicas (CIEMAT) Avenida Complutense 22, 28040 Madrid, Spain, and Beatson Institute for Cancer Research, Glasgow G61 1BD, United Kingdom. Background. The ex vivo expansion of hematopoi- etic grafts has been proposed as an efficient procedure for improving the hematological recovery of recipi- ents. The fate of the long-term repopulating cells dur- ing the ex vivo manipulation of the graft is, however, a critical issue in ex vivo expansion protocols and ulti- mately will define the applicability of this new tech- nology in hematopoietic transplants. Methods. The repopulating ability of mouse hemato- poietic samples was determined by means of bone marrow (BM*) competition assays, using congenic strains that express the pan-leukocyte Ly-5.1 and Ly- 5.2 antigens. The generation of potential changes in the repopulating properties of human hematopoietic samples subjected to ex vivo expansion was deter- mined by comparing the engraftment of fresh and ex vivo-manipulated CD34  cord blood cells in irradiated nonobese diabetic/severe-combined immunodeficient (NOD/SCID) mice. Results. Under our optimized conditions of mouse BM incubation (stem cell factor plus interleukin-11, either with or without macrophage inflammatory pro- tein-1 or Flt3 ligand), both the short-term and the mid-term repopulating ability of the ex vivo-expanded samples were significantly improved when compared with fresh samples. In the long-term, no changes in the repopulation and differentiation properties of the graft were observed as a result of the ex vivo expan- sion process. As deduced from the analysis of NOD/ SCID mice transplanted with fresh and ex vivo ex- panded human CD34 cord blood cells, the in vitro stimulation mediated by SCF/IL-11/FLT3L was capa- ble of preserving the ability of the grafts to repopulate the lympho-hematopoiesis of recipents for at least 3 months. Conclusion. These results indicate that under our optimized conditions of ex vivo expansion, the ampli- fication of the hematopoietic progenitors responsible for the short- and mid-term repopulating properties of the graft can take place without compromising the long-term lympho-hematopoietic repopulating prop- erties. A number of studies have demonstrated the feasibility of amplifying the short-term repopulating progenitors in vitro and have shown the relevance of ex vivo expansion ap- proaches in hematopoietic transplants (1–6) and gene ther- apy protocols (7–10). Despite the beneficial effects associated with the ex vivo expansion of the short-term repopulating progenitors, the fate of the true hematopoietic stem cells during the ex vivo expansion process is still a matter of discussion. In the mouse model, a number of studies have observed an impairment in the long-term repopulating capacity of ex vivo-expanded grafts (11–13). We have recently described how a modest restriction in the repopulating properties of 5-fluorouracil (5FU)-preactivated bone marrow (BM*) takes place during ex vivo expansion protocols involving the use of either interleu- kin (IL)-3 plus IL-6 or IL-3 plus stem cell factor (SCF) (14). In some instances, the administration of 5FU was considered to be the cause of this effect (14). However, the ex vivo expan- sion process per se has also been considered the main cause of the repopulation defect, either because of changes in the grafting properties of this population (11, 13) or because of a differentiation stress on the true stem cells (14). To prevent the prompt differentiation of the stem cells during the ex vivo expansion process, new combinations of early acting cytokines have been used to facilitate the self- renewal divisions in the hematopoietic stem cell compart- ment. In particular, it has been shown that the ex vivo expansion of mouse BM with IL-11 plus SCF improved the short-term repopulating ability of the samples and enhanced the capacity of the graft to sustain hematopoiesis over serial transplants (3). Trevisan et al. (15) and Yonemura et al. (16) 1 Supported by grants of Comisio ´n Interministerial de Ciencia y Tecnologı ´a (SAF 95–1548-C02– 01 and 98 – 0008-C04 – 01) and Euro- pean Commission RTD actions, CT-96 –3784). 2 Molecular and Cell Biology Unit, Centro de Investigaciones En- erge ´ticas, Medioambientales y Tecnolo ´gicas (CIEMAT). 3 Beatson Institute for Cancer Research. 4 Address correspondence to: Juan A. Bueren, Unidad de Biologı ´a Molecular y Celular. CIEMAT. Avenida Complutense 22. 28040. Madrid, Spain. E-mail: bueren@ciemat.es. * Abbreviations used: MEM, alpha medium; BM, bone marrow; CB, cord blood; CFU, colony forming unit; CFU-GM, granulocyte- macrophage colony-forming unit; CRA, competitive repopulating abil- ity; FACS, fluorescent activated cell sorting; FBS, foetal bovine serum; FITC, fluorescein isothiocyanate; 5FU, 5-fluorouracil; Flt3L, Flt-3 li- gand; G-CSF, granulocyte-colony stimulating factor; GM-CSF, granu- locyte-macrophage-colony stimulating factor; HGFs, hematopoietic growth factors; HrIL, human recombinant interleukin; HS, horse se- rum; IL, interleukin; IMDM, Iscove’s modified Dulbecco’s medium; MN, mononuclear cells; NOD/SCID mice, non-obese diabetic/severe-com- bined immunodeficient mice; PB, peripheral blood; PBA; PI, propidium iodide; RU, repopulating units; SCF, stem cell factor. 1348