Journal of Biotechnology 144 (2009) 127–134 Contents lists available at ScienceDirect Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec The potential of human peripheral blood derived CD34+ cells for ex vivo red blood cell production Daniela Boehm a , William G. Murphy b,c , Mohamed Al-Rubeai a,∗ a School of Chemical and Bioprocess Engineering, and Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin, Ireland b School of Medicine and Medical Science, University College Dublin, Ireland c Irish Blood Transfusion Service, Dublin, Ireland article info Article history: Received 8 April 2009 Received in revised form 16 July 2009 Accepted 31 August 2009 Keywords: Ex vivo erythropoiesis Peripheral blood CD34+ cells Erythroid differentiation abstract The potential of peripheral blood derived CD34+ cells for ex vivo erythropoiesis was investigated in a stroma-free culture system using a novel strategy of daily passaging. By expanding PB-derived CD34+ cells up to 1.5 × 10 6 -fold this method achieved expansion factors previously only reported for CD34+ cells derived from more potent stem cell sources such as cord blood, bone marrow and mobilized peripheral blood. Analysis of cell surface markers showed differentiation of immature CD34+ cells to populations with 80% CD71-/GpA+ cells and up to 45% enucleated cells, indicating a significant amount of terminal maturation. Cell crowdedness was found to have decisive effects on in vitro erythropoiesis. Cell density per surface area rather than cell concentration per media volume determined cell expansion during exponen- tial growth where more crowded cells showed reduced overall expansion. In late stage erythropoiesis, however, when cells no longer proliferating, increased cell density was seen to enhance cell viability. These results indicate that peripheral blood derived haematopoietic stem cells can be an alternative to cells sourced from bone marrow, cord blood or leukapheresis in terms of expansion potential. This pro- vides distinct advantages in terms of availability for studies of conditions for scale-up and maturation, and may have particular clinical applications in the future. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Erythropoiesis is the body’s most productive cell production process, yielding approximately 2 × 10 11 new red cells produced from haematopoietic stem cells (HSCs) of the bone marrow every day. HSCs are a rare population comprising only 0.01% of nucleated bone marrow cells (Rizo et al., 2006) but they possess the poten- tial for both self-renewal and differentiation into all lineages of blood cells (Nielsen, 1999; Rizo et al., 2006; Suda et al., 2005). In the multi-step process of erythropoiesis, HSCs give rise to committed progenitor cells that terminally differentiate to mature erythro- cytes with nuclear condensation and extrusion marking key events of late stage erythropoiesis (Arcasoy and Jiang, 2004; Testa, 2004). Research aimed at finding alternative means to conventional blood transfusion relying on donor blood has focused on either the development of cell-free blood substitute products (reviewed by Kjellstrom (2003)) or establishing in vitro production methods for red blood cells (reviewed by Douay and Andreu (2007)). How- ∗ Corresponding author at: School of Chemical and Bioprocess Engineering, Uni- versity College Dublin, Engineering and Materials Science Centre, Belfield, Dublin 4, Ireland. Tel.: +353 17161862; fax: +353 17161177. E-mail address: m.al-rubeai@ucd.ie (M. Al-Rubeai). ever, significant advances in ex vivo erythropoiesis have recently been achieved with both high expansion factors and high enucle- ation efficiency, making this approach, at least methodologically, a promising option (Fujimi et al., 2008; Giarratana et al., 2005; Miharada et al., 2006; Vlaski et al., 2009). The expansion of HSCs obtained from cord blood, bone marrow, leukapheresis or peripheral blood has been reported in liquid cul- ture (Collins et al., 1998) using a variety of recombinant human cytokine combinations of which stem cell factor (SCF) and erythro- poietin (EPO) seem to play a non-redundant role in erythropoiesis (Munugalavadla et al., 2005). For full terminal maturation co- culture on human (Baek et al., 2008) or murine stromal feeder cells (Giarratana et al., 2005; Vlaski et al., 2009) or co-culture with macrophages (Fujimi et al., 2008) have been employed but suc- cessful enucleation in the absence of stromal support has also been reported (Miharada et al., 2006). Best results have been achieved with cord blood derived CD34+ cells and most research has focused on this source of haematopoietic stem cells due to its higher expan- sion potential (Fujimi et al., 2008; Miharada et al., 2006). Peripheral blood has received less attention as stem cell source in attempts to achieve transfusable yields of red blood cells through in vitro erythropoiesis due to generally lower expansion poten- tial and lower yields of CD34+ cells in comparison to cord blood, bone marrow or leukapheresis. We show an expansion potential 0168-1656/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2009.08.017