Ex-vivo expansion of cord blood hematopoietic stem and progenitor cells for transplantation using an antioxydant-supplemented medium and a cytokine cocktail inducing hypoxic-like cellular response

We recently developed a clinical grade ex vivo cord blood (CB) expansion procedure enabling a massive amplification of hematopoietic progenitors without any loss of stem cell potential as revealed on the basis of serial engraftment of Nod/SCID mice (Ivanovic et al, Cell Transplant. 2011). This procedure, in line with our concept “Oxygen Stem Cell Paradigm” (Ivanovic, J Cell Physiol 2009), is based on Day 14 liquid cultures of CB CD34+ cells, in medium Macopharma HP01 (containing several antioxidant molecules and nicotinamide) and in presence of Stem Cell Factor - SCF (100 ng/ml), fms-Related Tyrosine Kinase 3 – Ligand - FLT3-ligand (100ng/ml), Megakaryocyte Growth and Developmental Factor - MGDF (100 Ng/ml) (these two cytokines acting in favor of HIF-1alpha transcript stabilization) and Granulocyte – Colony Stimulating Factor -G-CSF (10 Ng/ml). This cocktail had to be modified due to the commercially unavailability of clinical grade MGDF molecule. So, MGDF was replaced by Thrombopoietin - TPO in five-fold lower dose (20 ng/ml) and culture time was reduced to 12 days (Duchez et al Cell Transplant 2012). That way, a mean expansion fold of 400, 80, and 150 was obtained for total cells, CD34+ cells and Colony Forming Cells – CFC, respectively. This amplification was associated with a slight enhancing effect on stem cells (Scid Repopulating Cells - SRC). These are the ultimate pre-clinical modifications of a clinical grade expansion protocol which is already employed in an ongoing clinical trial (adult allogeneic context) started in 2010). The preliminary results are encouraging – rapid and durable hematopoietic reconstitution after injection of one ex vivo expanded CB unit only.