Systematic delineation of optimal cytokine concentrations to expand hematopoietic stem/progenitor cells in co-culture with mesenchymal stem cells Pedro Z. Andrade, a Francisco dos Santos, a Grac¸a Almeida-Porada, b Cla´udia Lobato da Silva a and Joaquim M. S. Cabral a Received 28th October 2009, Accepted 12th March 2010 First published as an Advance Article on the web 27th April 2010 DOI: 10.1039/b922637k The major obstacle to the widespread use of umbilical cord blood (UCB) in hematopoietic stem/progenitor (HSC) cell therapy is the low cell dose available. A cytokine cocktail for the ex vivo expansion of UCB HSC, in co-culture with a bone marrow (BM) mesenchymal stem cells (MSC)-derived stromal layer was optimized using an experimental design approach. Proliferation of total cells (TNC), stem/progenitor cells (CD34 + ) and colony-forming units (CFU) was assessed after 7 days in culture, while sole and interactive effects of each cytokine on HSC expansion were statistically determined using a two-level Face-Centered Cube Design. The optimal cytokine cocktail obtained for HSC-MSC co-cultures was composed by SCF, Flt-3L and TPO (60, 55 and 50 ng mL 1 , respectively), resulting in 33-fold expansion in TNC, 17-fold in CD34 + cells, 3-fold in CD34 + CD90 + cells and 21-fold in CFU-MIX. More importantly, these short-term expanded cells preserved their telomere length and extensively generated cobblestone area-forming cells (CAFCs) in vitro. The statistical tools used herein contributed for the rational delineation of the cytokine concentration range, in a cost-effective way, while systematically addressing complex cytokine-to-cytokine interactions, for the efficient HSC expansion towards the generation of clinically significant cell numbers for transplantation. Introduction The efficient ex vivo expansion of hematopoietic stem/progenitor cells (HSC), especially those from the umbilical cord blood (UCB), which contains a limited numbers of bone marrow repopulating cells per unit, would clearly widespread their clinical applications to adult patients. 1 The generation of clinical significant cell numbers for a HSC transplant, while maintaining their multilineage engraftment capability, would potentially reduce the hematopoietic reconstitution time and/or increase the engraftment success rate. 2 The delineation of protocols for expanding long-term engrafting CD34 + cells would be of direct clinical utility, since most of the transplants performed today with enriched hematopoietic progenitor cells use CD34-enriched cells. 3 In fact, a very recent study demonstrated that after a 10-day ex vivo expansion, UCB CD34 + cells showed multilineage repopulation after long-term and secondary engraftment in sublethally irradiated NOD/SCID/IL-2R / mouse, comparable to non-expanded HSC. 4 Within the CD34 + cell fraction, CD34 + CD90 + cells, in particular, demonstrated to be efficient in achieving rapid and sustained cell engraftment; 5 indeed, CD90 (Thy-1) expression has been recognized as superior to CD38 (i.e. CD34 + CD38  ) and other markers as predictor of the repopulating activity of CD34 + cells. 6 Several ex vivo culture systems have been used with different rates of success namely by testing different cytokine cocktails in stroma-containing or stroma-free cultures with serum or in serum-free conditions. 7–11 Indeed, most cytokine combinations tested include SCF, Flt-3L and TPO presumed to promote extensive cell self-renewal and to limit levels of apoptosis. 9,12 Nevertheless, numerous other molecules have been tested in ex vivo HSC cultures: Zhang and co-workers obtained a 20-fold increase in SCID repopulating cells when IGF-binding protein 2 and angiopoietin-like 5 were added to a cytokine cocktail composed by SCF, FGF and TPO; 13 Araki and collaborators tested the chromatin modifying agents 5-aza- 2 0 -deoxycytidine D (5-AzaD) and trichostatin (TSA) with encouraging HSC expansion levels, 14 whereas Peled and co-workers reported significant enhancement of HSC proliferation with lower levels of differentiation using the copper-chelator agent, TEPA. 15 In addition, in combination with other cytokines, ex vivo expanded UCB CD133 + cells using TEPA have been used in a phase I/II clinical trial, with high engraftment rates. 16 In our laboratory, we have previously established a serum- free culture system using human BM MSC-derived feeder layers, supplemented with SCF, Flt-3, bFGF and LIF, which allowed an efficient expansion/maintenance of HSC from BM and UCB. 3,17 In particular for UCB, HSC-MSC cellular interactions were shown to be crucial for the extensive expansion of CD34 + , CD34 + CD38  cells and CFU-MIX in vitro. 18 a IBB-Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Instituto Superior Te ´cnico, Lisboa, Portugal. E-mail: joaquim.cabral@ist.utl.pt; Fax: +351 218419062; Tel: +351 218419063 b Department of Animal Biotechnology, University of Nevada, Reno, NV, USA This journal is  c The Royal Society of Chemistry 2010 Mol. BioSyst., 2010, 6, 1207–1215 | 1207 PAPER www.rsc.org/molecularbiosystems | Molecular BioSystems Downloaded on 03 February 2011 Published on 27 April 2010 on http://pubs.rsc.org | doi:10.1039/B922637K View Online