Upon withdrawal of leukemia inhibitory factor (LIF) self-renewing pluripotent mouse embryonic cells (mESC) undergo reversible commitment, followed by irreversible commitment and heterogeneous cell differentiation or apoptosis, within three days. In this study we investigated bioenergetics profile of these different maturation stages. For this purpose mESC were cultivated in the presence or absence of LIF for 24, 48 and 72h and then analysed. Mitotracker green staining showed greater mitochondrial mass in the pluripotent than in the differentiated cells. To determine if this different mitochondrial content provides bioenergetics differences between this cells populations, we measured oxygen consumption rate (OCR) as indicator of principal mitochondrial respiration parameters and extracellular acidification rate as an indicator of glycolysis using XF24 analyzer. We found that basal OCR and ECAR declined in the order: pluripotent cells (+LIF) > reversible committed cells (-LIF 24h)> irreversible committed cells (-LIF 48h) > differentiated cells (-LIF72h). This was associated with mitochondrial ATP output, estimated as coupled rate (OCR linked to ATP synthesis), showing the same downtrend. Likewise, + LIF cells possessed the highest maximal respiratory capacity indicating their higher resistance to oxidative stress comparing to maturating stages. In addition, inhibition of mitochondrial respiration impaired proliferation of +LIF, but not –LIF 72h cells. Also, suppression of mitochondrial ATP synthesis, provoked compensatory increase of glycolysis in the + LIF cells which is impaired in the course of the maturation. Bioluminescent measurement of ATP content showed the highest level in the –LIF 72h cells, implying lessen ATP turnover in the more differentiated cells. Our results demonstrated that self-renewing pluripotent mESC have bioenergetics advantage reflecting in the high OXPHOS and glycolytic activity, which declines throughout their maturation.

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.

The role of Hypoxia and its key regulators, HIF-1 and HIF-2, remains to be defined for circulating Steady Sate Peripheral Blood (SSPB) Heamtopoietic Stem Cells HSC. In this work we tested the ability of Hypoxia and antioxidants-complemented medium to maintain the HSC activity inside SSPB CD34+ cell population. We first evidenced that Side Population (SP) activity of SSBP CD34+ cells is linked to very primitive cells according to proliferation, CFC content and engraftment capacity in NSG mice. By culturing SSBP CD34+ cells at atmospheric concentration (20% O2, control) and low O2 concentrations (3% and 1% O2), we showed that SP activity decreased by 10 fold at 20% and 3% O2 whereas the proportion and number of SP cells were increased by 2.6 fold at 1% O2. In this condition, the inhibition of hypoxia-induced stabilization of HIF-1α and HIF-2α using specific shRNA led to a 2.3 and 6.3 fold decrease of SP cells for shRNA/HIF-1α and shRNA/HIF-2α, respectively. Similarly, SSBP CD34+ transduced cells with either shRNA/HIF-1α or shRNA/HIF-2α failed to give a sustained hematopoietic reconstitution of NSG mice. By culturing SSBP CD34+ cells at 20% O2 with antioxidants-supplemented medium and cytokines inducing the stabilization of HIF-1α, we observed a 21 fold increase in SP cells number. The phenotypic characterization of these SP cells using CD34, CD38, CD90 and CD133 antibodies showed a 5.9 to 10 fold expansion depending on the phenotype. This result was corroborated by the xenotransplantation assays in NSG mice that showed an 11 fold increase in huCD45 chimerism after primary transplantation when cells were injected after 7 days of culture compared to uncultured cells. Besides, these cells displayed a secondary engraftment capacity. These data show that SSPB HSC fate is dependent on hypoxic regulatory pathways and environmental capacity to decrease the ROS content. This work strengthens the possibility to use SSBP as a source of HSC for research and cellular therapy.

Notre protocole d’expansion de cellules CD34+ de sang placentaire permet d’amplifier les progéniteurs engagés (PE) sans diminuer la quantité de cellules souches hématopoïétiques (CSH), assurant ainsi une greffe à court et à long terme. Ce protocole a fait l’objet d’un essai clinique avec des résultats prometteurs, dans un contexte de greffe allogénique adulte. La greffe de cellules de sang placentaire amplifiées ex vivo se confronte au problème de la cryoconservation du produit d’expansion afin d’assurer sa conservation lors du transport et pour une utilisation future. C’est pourquoi nous avons exploré la possibilité de maintenir les propriétés fonctionnelles des CSH et des PE après congélation et décongélation des cellules amplifiées ex vivo en comparant le protocole standard de cryoconservation (albumine humaine–(HSA)/DMSO) avec un protocole inédit basé sur une solution de congélation enrichie (milieu HP01 de grade clinique/DMSO). L’étude de l’indice de viabilité, du nombre de CD34+ et de cellules totales, et du rendement de progéniteurs engagés et de CSH indique que la cryoconservation et la décongélation des cellules amplifiées de sang placentaire par HSA/DMSO réduit de façon significative la quantité de progéniteurs engagés (40 %) et de CSH (diminution drastique de la capacité de greffe de souris NSG). En revanche, le protocole HP01/DMSO améliore la préservation des PE (> 60 %) sans altérer la capacité de greffe des CSH. Ainsi, le maintien fonctionnel des PE et des CSH après amplification ex vivo et cryoconservation est faisable en conditions compatibles avec les exigences de thérapie cellulaire humaine.