Bone Marrow Transplantation (2002) 29, 443–448  2002 Nature Publishing Group All rights reserved 0268–3369/02 $25.00 www.nature.com/bmt Technical report Role of different medium and growth factors on placental blood stem cell expansion: an in vitro and in vivo study M Berger 1 , F Fagioli 1 , W Piacibello 2 , F Sanavio 2 , K Mareschi 1 , E Biasin 1 , S Bruno 2 , L Gammaitoni 2 , M Gunetti 2 , F Nesi 1 , E Madon 1 and M Aglietta 2 1 Department of Paediatrics, University of Turin Medical School, Turin, Italy; and 2 Institute for Cancer Research and Treatment, IRCC, Candiolo, University of Turin Medical School, Turin, Italy Summary: Expansion of haemopoietic stem cells from placental blood has been obtained with a combination of flt3 ligand (FL), thrombopoietin (TPO), kit-ligand (KL) with or without interleukin-6 (IL6) in serum-replete medium. For clinical use, cell expansion in the absence of serum is a clear advantage. Therefore, stem cell expansion in serum-free (SF) medium with a combi- nation of three (FL, TPO, KL) or four (FL, TPO, KL, IL6) growth factors was compared with the results obtained using fetal calf serum (FCS) or human serum (HS). Human CD34 + placental blood cells were cultured in the presence of FL, TPO, KL  IL6 with SF medium, HS and FCS for up to 8 weeks. CD34 + , CFC, LTC-IC content was measured at intervals. To determine the in vivo repopulating capacity of expanded cells, CD34 + expanded cells were transplanted in sublethally irradiated NOD/SCID mice. With the three growth fac- tor combination the CD34 + cell number increased stead- ily up to the 8 weeks of culture. CD34 + cells were expanded 67.5-fold with SF, 11.7 with HS and 49.2 with FCS. However, when CFCs and LTC-ICs were con- sidered, a continuous expansion was observed only with HS and FCS, whereas in SF medium after 6 weeks their number started to decline. The addition of IL-6 did not change the expansion significantly. Cells grown ex vivo for 14 days were transplanted into NOD/SCID mice. The engraftment of human cells in mice was higher for serum-replete than for SF expanded cells. Nevertheless, SF cultured cells were also able to engraft both marrow and spleen in all animals. In addition, engrafted human cells still maintained clonogenic ability. With KL, FL, TPO  IL6 it is possible to expand haemopoietic pro- genitor cells in a SF medium. Compared with serum- replete cultures, the absolute number of clonogenic cells and in vivo repopulating cells is lower. Although the degree of expansion remains significant, a clinical trial still needs to be carried out to address the question of Correspondence: Dr F Fagioli, Dipartimento di Scienze Pediatriche e dell’Adolescenza, Ospedale Infantile Regina Margherita, Piazza Polonia 94, 10126 Torino, Italy Received 6 September 2001; accepted 2 January 2002 whether this expansion might be useful in reducing post-transplant aplasia. Bone Marrow Transplantation (2002) 29, 443–448. DOI: 10.1038/sj/bmt/1703390 Keywords: placental blood; long-term culture; serum- free expansion; NOD/SCID mice Human placental blood is a source of haemopoietic stem cells both for transplantation 1–4 and gene therapy appli- cations. 5 However, considerable interest in developing methods for expanding cord blood stem cell numbers in vitro 6–14 has been stimulated by the fact that a single cord blood collection may not be sufficient to guarantee engraftment of adult allogeneic recipients. The cultures are traditionally performed in a medium containing fetal calf serum (FCS) but to utilise expanded cells in clinical prac- tice, ‘good manufacturing practice’ (GMP) standards and serum-free (SF) conditions are required. In particular, FCS must be excluded from the medium because it may contain allergenic substances and transmissible infections. Further- more, pooled human serum (HS) should be excluded to eliminate variables linked to individual donors. 15,16 In this study we investigated how CD34 + placental blood cells grow in SF conditions in the presence of haemopoietic growth factors (flt3 ligand (FL), thrombopoietin (TPO), kit ligand (KL)  interleukin 6 (IL6) ) but in the absence of stroma. At different times, total cell, CD34 + , CFC and LTC-IC content was measured. Moreover, after 14 days expansion the in vivo repopulating capacity of cells was determined. The results were compared with those obtained in the presence of HS or FCS. Materials and methods CD34 + cell purification Twenty-four placental blood samples were obtained, with the informed consent of the mother, from full-term deliver- ies. CD34 + cells were isolated from the placental blood mononuclear fraction using a MiniMACS CD34 isolation kit (Miltenyi Biotech, Bisley, UK) according to the manu-