David Chalmers

Direct selection of human bone marrow mesenchymal stem cells using an anti-CD49a antibody reveals their CD45med,low phenotype.

Summary. Human bone marrow mesenchymal stem cells (MSC) generate, via a fibroblast colony‐forming unit (CFU‐F), osteo‐chondroblastic cells as well as adipocytes and stromacytes. To date, these stem cells are isolated indirectly using a cell culture method and phenotyped as CD45 negative while the in vivo counterparts are undetermined. Our aim was to develop a direct selection method and to determine the phenotype of the MSC isolated in this way. Mesenchymal cells were selected with anti‐CD49a and/or anti‐CD45 antibodies using either flow cytometry or a magnetic beads method. All CFU‐F were always detected in the small population of CD49a‐positive cells. These CFU retained their differentiation potential and gave rise to osteo‐chondroblastic cells, adipocytes and stromacytes. Phenotypic studies on uncultured cells revealed a CD45med,low, CD34low, HLA‐II– cell population. Flow cytometry cell sorting showed that MSC with CFU‐F potential were obtained only from a CD49a+/CD45med,low population. In addition, when cultured, they clearly became CD45–, CD34–, HLA‐II–, CD49a+. These results confirmed that MSC can be directly selected easily from human bone marrow using magnetic beads without altering their differentiation potential. These cells expressed mildly the haematopoietic marker CD45, which was dramatically downregulated by in vitro culture. The expression of CD45 coupled to CD49a thus enabled direct selection of the MSC.

Development of a Competitive PCR Method for In Vitro and In Vivo Quantification of Herpes Simplex Virus Thymidine Kinase and Neomycin Resistance-Expressing Cells Used in a Clinical Trial

The aim of this study was to set up a sensitive and specific method to quantify the number of gene-modified cells in a gene therapy clinical trial currently underway at our institution. This trial involves the use of retrovirally transduced allogeneic T cells expressing the herpes simplex-1 thymidine kinase (HSV-TK) and neomycin-phosphotransferase (NeoR) resistance gene. Quantification by competitive PCR was performed, with two homologous internal standards (deltaTK, deltaNeoR), 30 bp shorter than the target sequences (TK, NeoR), coupled to fluorescent laser-based detection. Assessment of the amplification systems procedures was carried out for each sequence. The 30-bp deletion did not affect the amplification efficiency significantly. Determination of the plateau phase of both amplified sequences demonstrated that each sample must be quantified during the predetermined exponential phase. Finally, a blinded study of a transduced cell dilutions panel validated the overall methodology. The competitive PCR was applied to quantification of the retroviral transduction process by quantifying the NeoR gene in transduced PBMC samples (prior to G418 selection) from 18 donors in our clinical trial. A mean transduction efficiency of 9.78% +/- 1.37% was observed. We also quantified TK-expressing donor transgenic T cells in a murine GvHD model. Results demonstrated on initial expansion of donor HSV-TK- expression T cells as well as a significant ganciclovir (GCV)-induced decrease correlated with the number of circulating gene-modified T cells. Therefore, we have developed an efficient gene quantification tool that should be useful for in vivo monitoring of gene-modified cells.

248 Presence of endothelial colony-forming cells is associated with reduction of microvascular obstruction limiting infarct size and left ventricular remodelling: MRI study in acute myocardial infarction

Background Endothelial colony-forming cells (ECFCs) have proliferative and vasculogenic capacities and can be detected in patients (pts) with myocardial infarction (MI). High ECFC levels reportedly lead to positive left ventricular (LV) remodelling after acute MI, but the mechanism of this improvement has never been assessed. We evaluated the relationship between ECFC levels and microvascular obstruction (MVO), and the impact of this relation on infarct size and LV remodelling at 6 months as assessed by magnetic resonance imaging (MRI). Methods 109 pts Results ECFC colonies were detected in 51/109 pts (47.2%) at admission (ECFC pos pts). At 5 days, MVO was more frequently observed (63% vs 33%; p = 0.003) and of greater magnitude (7 ± 6% vs 3 ± 5%, p = 0.0004) in ECFC neg patients versus ECFC pos pts respectively. At 6 months, there was a significantly greater reduction in infarct size in ECFC pos pts (−33.7 ± 33.2% vs −15.1 ± 24.6%, ECFC pos vs ECFC neg respectively; p = 0.003). This reduction in infarct size was associated with a significant improvement in LV ejection fraction and a significant reduction in LV end diastolic and systolic volumes in ECFC pos pts. A significant positive correlation was observed among ECFC pos pts between MVO at day 5 and infarct size at 6 months (r 2 =0.58, p 2 =0.33, p Conclusion The presence of ECFC colonies is associated with a reduced degree of microvascular obstruction early after myocardial infarction, leading to reduced infarct size and positive LV remodelling at 6 months and can be considered as a marker of microvascular integrity in acute MI pts.