Zoran Ivanovic

The expansion of murine bone marrow cells preincubated in hypoxia as an in vitro indicator of their marrow-repopulating ability

In liquid cultures of murine bone marrow cells stimulated with interleukin-3 and granulocyte/macrophage colony-stimulating factor, hypoxia (1% oxygen) induced a reversible block of hematopoiesis, maintaining the progenitors’ expansion potential unreduced. Progenitors repopulating day-14 hypoxic cultures with cells or granulocyte/macrophage colony-forming units (CFU-GM) were found, on the basis of their maintenance in hypoxia (12% and 76%, respectively), to belong to different subsets, the latter being much more efficiently maintained. The maintenance in hypoxic cultures of progenitors detectable by marrow-repopulating ability (MRA) assay was 18% for MRAcell progenitors and 69% for MRACFU progenitors. Thus, the repopulation of hypoxic cultures with cells or CFU-GM closely reflected the presence of progenitors capable of repopulating, with cells or CFU-GM, the bone marrow of lethally irradiated syngeneic animals. Progenitors repopulating hypoxic cultures were, like MRA progenitors, significantly resistant to 5- fluorouracil, progenitors repopulating cultures with CFU-GM being two-fold more resistant than those repopulating cultures with cells. We concluded that the repopulation of day-14 hypoxic cultures occurring after their transfer to air is to be considered an indicator of the maintenance of MRA progenitors in hypoxia. The relevance of these results to stem cell biology and their potential practical applications are discussed.

Hypoxia Modifies Proliferation and Differentiation of CD34+ CML Cells

We previously showed that hypoxia (1% O2) favors the self‐renewal of murine and human normal hematopoietic stem cells. This study represents the first attempt to characterize the effects of hypoxia on the maintenance of chronic myeloid leukemia (CML) progenitors. CD34+ cells isolated from apheresis products of CML patients were incubated in hypoxia (1% O2) and normoxia (20% O2). After 8 days of culture, their proliferation, capacity for colony‐forming‐cell (CFC) generation in secondary cultures (pre‐CFC), and phenotype (CD34 and platelet‐activating factor receptor [PAF‐R]) were compared with those of normal cells, and tyrosine phosphorylation in CML cells was measured. Hypoxia inhibits the proliferation of CD34+ cells and preserves the pre‐CFC capacity and cell‐surface CD34 expression of CML cells better than normoxia. The PAF‐R expression, which was absent on freshly isolated cells, was detected at the cell surface in both populations after 8 days of culture, but with a lower percentage of positive cells in CML cell cultures. Incubation in hypoxia suppressed the PAF‐R expression of normal cells and increased it in CML cells, resulting in a similar expression in the two populations. These effects could be linked to inhibition by hypoxia of the tyrosine hyperphosphorylation of cellular proteins, a major hallmark of CML cells.

Combination of low O2 concentration and Mesenchymal Stromal Cells during culture of Cord Blood CD34 + Cells Improves the Maintenance and Proliferative Capacity of Hematopoietic Stem Cells

The physiological approach suggests that an environment associating the mesenchymal stromal cells (MSC) and low O2 concentration would be most favorable for the maintenance of hematopoietic stem cells (HSCs) in course of ex vivo expansion of hematopoietic grafts. To test this hypothesis, we performed a co‐culture of cord blood CD34+ cells with or without MSC in presence of cytokines for 10 days at 20%, 5%, and 1.5% O2 and assessed the impact on total cells, CD34+ cells, committed progenitors (colony‐forming cells—CFC) and stem cells activity (pre‐CFC and Scid repopulating cells—SRC). Not surprisingly, the expansion of total cells, CD34+ cells, and CFC was higher in co‐culture and at 20% O2 compared to simple culture and low O2 concentrations, respectively. However, co‐culture at low O2 concentrations provided CD34+ cell and CFC amplification similar to classical culture at 20% O2. Interestingly, low O2 concentrations ensured a better pre‐CFC and SRC preservation/expansion in co‐culture. Indeed, SRC activity in co‐culture at 1.5% O2 was higher than in freshly isolated CD34+ cells. Interleukin‐6 production by MSC at physiologically low O2 concentrations might be one of the factors mediating this effect. Our data demonstrate that association of co‐culture and low O2 concentration not only induces sufficient expansion of committed progenitors (with respect to the classical culture), but also ensures a better maintenance/expansion of hematopoietic stem cells (HSCs), pointing to the oxygenation as a physiological regulatory factor but also as a cell engineering tool. J. Cell. Physiol. 227: 2750–2758, 2012.

Angiotensin II That Reduces the Colony‐Forming Ability of Hematopoietic Progenitors in Serum Free Medium Has an Inverse Effect in Serum‐Supplemented Medium

Hematopoiesis is a complex process regulated by multi-ple factors: cellular interactions, cytokines, chemokines, andnumerous other molecules including vasoactive peptides(VAP). Angiotensin II (AII) and substance P (SP) wererecently described for their stimulatory effect onhematopoiesis [1-3]. Recently,

Interleukin-6 enhances the activity of in vivo long-term reconstituting hematopoietic stem cells in "hypoxic-like" expansion cultures ex vivo.

Since interleukin (IL)‐6 synergizes with the physiologically relevant O2 concentration in the maintenance of primitive hematopoietic stem cell (HSC) subpopulations, we hypothesized that its addition to our hypoxic response mimicking cultures (HRMCs), composed of an antioxidant‐supplied serum‐free xeno‐free medium supplemented with the cytokines stabilizing hypoxia‐inducible factor‐1α and balancing HSC self‐renewal and commitment, will result in a similar effect even if they are exposed to 20% O2.

The Inhibitory Effect of Human Macrophage Inflammatory Protein‐1α (LD78) on Acute Myeloid Leukemia Cells in Vitro

Macrophage inflammatory protein‐1α (MIP‐1α) has recently been shown to inhibit proliferation of immature hemopoietic progenitors. In addition, significant inhibition of early and mature leukemic progenitors in acute myeloid leukemia (AML) has been obtained with MIP‐1α. We performed a study of 25 AML patients at diagnosis to evaluate the effect of a human homolog of MIP‐1α (LD78) on bone marrow (BM) and peripheral blood (PB) leukemic progenitors (colony‐forming unit‐AML [CFU‐AML]) and AML cell proliferation. A methylcellulose culture system was used for CFU‐AML and incorporation of 3H‐TdR for AML cell proliferation. We found that LD78 inhibits CFU‐AML colony formation up to 100% for the BM in 14/16 samples studied with the average maximal inhibition of 62.7 ± 9.1% and up to 100% for the PB in 12/13 samples studied with the average maximal inhibition of 71.4 ± 9.9%. In addition to this, LD78 inhibited AML cell proliferation up to 60% for the BM in 10/18 samples studied with the average maximal inhibition of 17.8 ± 3.5%, and up to 87.1% for the PB cell proliferation in 10/16 samples studied with the average maximal inhibition of 27.5 ± 6.8%. Our results have shown that LD78 is more active on AML progenitors than on AML cell proliferation. Inhibition of the AML cells, although less than that of the progenitors, indicates that more limited activity of LD78 on more mature leukemic cells is present in AML.

Cord (placental) blood storage: extent and functional aspects

In 2010, two articles were published in TRANSFUSION dealing with the storage of umbilical (placental) cord blood cell units (UCB). Both articles studied the influence of temperature on maintenance of viable total nucleated cells, CD34+ cells, and committed progenitors (colony forming cells [CFCs]). They used, as a control group, the UCB stored at a temperature of 4°C, that is, the temperature employed in routine storage and transportation. With that respect, the first article showed that there were no significant differences during storage of 84 to 88 hours comparing to time zero values (n = 10) in the recoveries of total nucleated cells, CD34+ cells, and CFCs. However, in the second article, there was evidence of a difference for these three aspects of cells over time. However, this result was based on only three samples. Since this issue is of great importance for practice (the dilemma: could the storage of UCB be extended or not), we studied the storage of 12 UCB units for 72 hours to evaluate if there were any significant decrease for each of these cell populations. Cord blood was collected using Macopharma UCB collection and storage bags (Macopharma, Tourcoing, France) and maintained at 4°C. The viable cell count was obtained using an automatic counter (Cell-Dyn 3500R, Abbott, Ramsey, MN). The number of viable CD34+ cells was obtained using simultaneous staining with anti-CD34 and anti-CD45 as well as 7-aminoactinomycin-D (FACSCalibur flow cytometer, Becton Dickinson, San Jose, CA). CFC detection was made by a methylcellulose ready-touse kit (Stem Alpha ID, Stem Alpha, St Clément les Places, France; 150 ¥ 10 cells per dish; Nunc, Roskilde, Denmark) cultured for 14 days in water-saturated atmosphere at 37°C with 5% CO2. The number of total cells, CD34+ cells, and CFCs decreased for each individual sample after 72 hours of storage (Fig. 1). This series, limited in the number of samples, did not exhibit “Gaussian” distribution, and only two time points (0 and 72 hr) were compared. Thus we used a nonparametric Wilcoxon test for paired (dependent) samples. We found a significant decrease in the recovery for each of these three cell categories (Fig. 2). Concerning CFCs, which is the only functional variable in this study, it should be stressed that the mean recovery was below 80% at 72 hours, which represents an obvious detrimental effect of prolonged storage. In addition, in real use, these UCB cells would be volume reduced, frozen, and thawed. Each of these steps is associated with a cellular loss to some extent that could be increased by cell vulnerability that might be potentiated by length of storage (our experimental observations). Accordingly, to evaluate the possibility to extend the storage period to 72 hours, these variables (including CFCs) should be tested throughout the usual complete process through to the postthawing period. Furthermore, no data are available concerning the maintenance of stem cell populations

Energy Metabolism Rewiring Precedes UVB-Induced Primary Skin Tumor Formation

Although growing evidence indicates that bioenergetic metabolism plays an important role in the progression of tumorigenesis, little information is available on the contribution of reprogramming of energy metabolism in cancer initiation. By applying a quantitative proteomic approach and targeted metabolomics, we find that specific metabolic modifications precede primary skin tumor formation. Using a multistage model of ultraviolet B (UVB) radiation-induced skin cancer, we show that glycolysis, tricarboxylic acid (TCA) cycle, and fatty acid β-oxidation are decreased at a very early stage of photocarcinogenesis, while the distal part of the electron transport chain (ETC) is upregulated. Reductive glutamine metabolism and the activity of dihydroorotate dehydrogenase (DHODH) are both necessary for maintaining high ETC. Mice with decreased DHODH activity or impaired ETC failed to develop pre-malignant and malignant lesions. DHODH activity represents a major link between DNA repair efficiency and bioenergetic patterning during skin carcinogenesis.

Clinical-scale validation of a new efficient procedure for cryopreservation of ex vivo expanded cord blood hematopoietic stem and progenitor cells

Survival of ex vivo expanded hematopoietic stem cells (HSC) and progenitor cells is low with the standard cryopreservation procedure. We recently showed that the efficiency of cryopreservation of these cells may be greatly enhanced by adding a serum-free xeno-free culture medium (HP01 Macopharma), which improves the antioxidant and biochemical properties of the cryopreservation solution. Here we present the clinical-scale validation of this cryopreservation procedure. The hematopoietic cells expanded in clinical-scale cultures were cryopreserved applying the new HP01-based procedure. The viability, apoptosis rate and number of functional committed progenitors (methyl-cellulose colony forming cell test), short-term repopulating HSCs (primary recipient NSG mice) and long-term HSCs (secondary recipient NSG mice) were tested before and after thawing. The efficiency of clinical-scale procedure reproduced the efficiency of cryopreservation obtained earlier in miniature sample experiments. Furthermore, the full preservation of short- and long-term HSCs was obtained in clinical scale conditions. Because the results obtained in clinical-scale volume are comparable to our earlier results in miniature-scale cultures, the clinical-scale procedure should be considered validated. It allows cryopreservation of the whole ex vivo expanded culture content, conserving full short- and long-term HSC activity.

Ex vivo amplification kinetics of cord blood hematopoietic progenitor cells in one- and two-step hypoxic response-mimicking cultures (HRMC)

In order to facilitate the transfer of our technique of expansion of cord blood hematopoietic cells to the industrial scale, we investigated if our two-step ex vivo expansion cultures can be transformed in one-step cultures. For this purpose, the cultures are initiated with the definitive medium volume and with the cell concentrations proportionally decreased. It turned out that the one-step cultures have different kinetics of cell expansion comparing to two-step cultures. However, the one-step cultures can produce more committed progenitors if maintained for 12 days than two-step ones, which can be a non-negligible advantage. To use these cultures in clinical trials it is necessary to check the activity of the hematopoietic stem cells after expansion using in vivo repopulation models.