Vox Sanguinis (2009) 96, 256–265 © 2008 The Author(s) ORIGINAL PAPER Journal compilation © 2008 International Society of Blood Transfusion DOI: 10.1111/j.1423-0410.2008.01138.x CharacterizationBlackwell Publishing Ltd and comparison of bone marrow and peripheral blood mononuclear cells used for cellular therapy in critical leg ischaemia: towards a new cellular product J.-C. Capiod,1 C. Tournois,2,3 F. Vitry,4 M.-A. Sevestre,5 S. Daliphard,2 T. Reix,5 P. Nguyen,2,3 J.-J. Lefrère1 & B. Pignon3,6 1Laboratory of Haematology, University Hospital of Amiens, Amiens, France 2Laboratory of Haematology, University Hospital of Reims, Reims, France 3EA3801, Institut Fédératif de Recherche 53, Laboratoire d’Hématologie, Centre Hospitalier Universitaire Reims, Hôpital Robert-Debré, Reims Cedex, France 4Department of Methodology, University Hospital of Reims, Reims, France 5Department of Vascular Surgery, University Hospital Amiens, Amiens, France 6Department of Cell Therapy, University Hospital Reims, Reims, France Background and Objectives Autologous transplantation of either bone marrow (BM) or peripheral blood (PB) mononuclear cells (MNC) induces therapeutic angiogenesis in patients with peripheral arterial occlusive disease. Yet, the precise nature of the cellular product obtained from BM or PB and used in these therapeutic strategies remains unclear. Materials and Methods We have analysed the characteristics of BM-MNC and PB-MNC collected without mobilization and implanted in patients with critical limb ischaemia in a clinical trial of cellular therapy including 16 individuals treated by BM-MNC and eight by PB-MNC. These MNCs were characterized by cell counts, viability assessment and enumeration of leucocyte subsets, CD34 stem and endothelial progenitor cells (EPCs) (CD34+/CD133+/VEGF-R2+) by flow cytometry. Mean fluorescence intensity ratios were determined for CD34, CD133 and VEGF-R2 markers. All analyses were simultaneously performed in two laboratories. Results Accuracy and reliability between both laboratories were achieved. BM-MNCs and PB-MNCs were quantitatively and qualitatively heterogeneous and quite different from each other. Stem cells and EPCs were significantly more present in BM- compared to PB-cell products, but with similar mean fluorescence intensity ratios. A weakly positive correlation was observed between CD34+ cell counts and EPCs levels, confirming the specificity of cell identification. Conclusion A great variability was observed in cell product characteristics according to their origin and also between individuals. These data stress the necessity of optimal Received: 11 August 2008, characterization of cell products especially in multicentric clinical trials. revised 28 October 2008, accepted 12 November 2008, Key words: cellular therapy, cellular product, critical leg ischaemia, endothelial published online 15 December 2008 progenitor cells, therapeutic angiogenesis. Correspondence: Claire Tournois, EA3801, Institut Fédératif de Recherche 53, Laboratoire d’Hématologie, Centre Hospitalier Universitaire Reims, Hôpital Robert-Debré, 51092 Reims Cedex, France E-mail: [email protected] Statement of equal author contribution: Jean-Claude Capiod and Claire Tournois contributed equally to this study. Conflict of interest: all authors disclose conflict of interest of any kind related to this manuscript. Authors had full access to the data. Abbreviations: 7-AAD, 7-aminoactinomycin; BM, bone marrow; BMI, body mass index; CLI, critical limb ischaemia; ECD, phycoerythrin-texas red; ECs, endothelial cells; EPCs, endothelial progenitor cells; FC, flow cytometry; GFR, glomerular filtration rate; MDRD, modification of diet in renal disease; Med, median; MGG, May Grünwald Giemsa; PB, peripheral blood; PCy5, phycocyanin 5; PCy7, phycocyanin7; VEGF-R2, vascular endothelial growth factor receptor 2. 256 Cell therapy in critical limb ischaemia 257 Table 1 Patient’s characteristics Introduction Several studies have shown that autologous transplantation BM cell products PB cell products of either bone marrow (BM) or peripheral blood (PB) mono- group (n = 16) group (n = 8) nuclear cells (MNC) could be effective in inducing angiogenesis Age (years) 71 [45–84] 60 [37–84] in patients with peripheral arterial occlusive disease [1–6]. Gender Experimental studies have shown that circulating BM- Male 14 (87·5%) 6 (75%) derived endothelial progenitor cells (EPCs) can be mobilized Female 2 (12·5%) 2 (25%) and incorporated into sites of active ischaemia to increase BMI (kg/m2) 24·8 [20·8–30·8] 22·7 [18·5–31·0] postnatal neovascularization [7,8], and that circulating BM- Smoking one current, 12 past, two current, 4 past, derived EPCs are able to differentiate into mature endothelial 3 never 2 never cells (ECs) [9]. The mechanisms regulating the differentiation Disorders of BM-derived EPCs, their mobilization and their homing to Arterial hypertension 11 (69%) 6 (75%) ischaemic sites remain partially unknown [10], but the Hypercholesterolemia 12 (75%) 3 (37·5%) implantation of BM-MNC [11] and of PB-MNC [12] into Diabetes mellitus 6 (37·5%) 1 (12·5%) 2 a ischaemic tissues (through the intramuscular local transplan- GFR (ml/min/1·73 m ) 59 [30–86] 81 [59–172] Haemoglobin (g/l) 132 [104–154] 124 [100–141] tation of these cells) induces collateral vessel formation in White blood cells (109/l) 7·2 [5·0–10·4] 7·4 [5·6–8·9] animals, suggesting that this cell therapy could constitute a new strategy for therapeutic angiogenesis. Quantitative variables were expressed as median and range [min–max]. The questions of which cells and which optimal effective aGFR, Glomerular filtration rate (MDRD or modification of diet in renal quantity to implant are crucial as long as the precise neovas- disease). cularization mechanisms are not completely elucidated. However, in most clinical trials, the exact composition of the cellular product used remains elusive and is obviously highly unilateral CLI and were not suitable candidates for surgery, variable. In fact, several parameters such as the origin of the non-surgical acts or revascularization. Patients with poorly MNC (BM or PB), the health status of the donor and the controlled diabetes mellitus or with a history of malignant preparation procedures [13] may lead to major differences in disorder were excluded. The ethical committee of each this cellular product, which is perhaps destined to become a participating centre approved the protocol. Written informed new ‘transfusion product’. For this reason, it is important to consent was obtained from all patients. Twenty-four patients try to characterize most precisely such a product before the were included: 16 treated with BM-MNC and eight with implementation of future multicentre clinical trials aiming to PB-MNC (Table 1). Patients were consecutively included as fully demonstrate the efficacy and safety of this cellular soon as they presented with appropriate criteria and were not therapy in critical limb ischaemia (CLI). Such an approach selected to receive one or another type of cells. The first 16 would allow choosing the ‘best’ cell product in terms of selected patients were treated with BM-MNC; the following efficacy, to standardize this product by cell characterization ones were treated with PB-MNC. Cells were implanted 1–3 h methods easy to perform, and to contribute to the best knowledge after preparation by 30 intramuscular injections into the of involved mechanisms. We report here our results in gastrocnemius of the ischaemic leg. Total injection volume characterizing BM-MNC and PB-MNC implanted in patients was 30 ml. In this article, we focalized on the two types with CLI in a French clinical trial. This trial was an open cellular products used. bicentric prospective study performed in two academic centres of vascular surgery and tested the safety and efficacy Bone marrow and peripheral blood mononuclear of BM-MNC or non-mobilized PB-MNC implanted in individuals cell samples with CLI. The clinical results will be presented as soon as all patients will have achieved sufficient follow-up. However, an For the preparation of BM-MNC, 500 ml of bone marrow was interim analysis was performed that confirmed the safety and collected under general anaesthesia through multiple was in favour of similar efficacy for both cell products. punctures of the posterior iliac crest using a Jamshidi needle. MNC were isolated using a blood cell separator (Cobe Spectra, version 4, Bone Marrow Processing Program, Gambro BCT, Materials and methods Lakewood, CO, USA). PB-MNC were collected by cytapheresis A prospective clinical trial was initiated in two French academic of one blood mass (5·1 ± 1·1 l) during 90 min with the hospitals in order to establish whether intramuscular same blood cell separator (Cobe Spectra, version 6, auto- implantation of BM-MNC or PB-MNC can induce clinical PBSC program, Gambro BCT). In both cases, the blood cell improvement in patients with CLI. Enrolled patients had separator was programmed to obtain a final volume of 40 ml © 2008 The Author(s) Journal compilation © 2008 International Society of Blood Transfusion, Vox Sanguinis (2009) 96, 256–265 258 J.-C. Capiod et al. MNC concentrate (30 ml for autologous re-injection and CD34+ cell enumeration was also ensured during the first 10 ml for controls and analyses). There was no previous step of EPCs quantification with phycocyanin 5 (PCy5)- mobilization by any haematopoietic growth factor. Blood cell CD34. separator was centralized in one centre (Reims Hospital). Samples of the end-product were collected and