Vincent Praloran

Very Low O2 Concentration (0.1%) Favors G0 Return of Dividing CD34+ Cells

Physiological bone marrow oxygen concentrations are everywhere lower than 4% and almost null in some areas. We compared the effects of 20%, 3%, and 0.1% O2 concentrations on cord blood CD34+ cell survival, cycle, and functionality in serum‐free cultures for 72 hours with or without interleukin‐3 (IL‐3). As from 24 hours, IL‐3 improved cell survival and proliferation in all conditions. After 72 hours, cells were 1.5 and 2.5 times more in quiescence (G0) at 3% and 0.1% O2, respectively, than at 20%; transforming growth factor‐β signaling seemed not to be involved. To explore cell cycle further, fresh CD34+ cells were stained with PKH26 and cultured for 72 hours, and then undivided and divided cells were sorted. At 0.1% O2, 46.5% ± 19.1% of divided cells returned to G0 compared with 7.9% ± 0.3% at 20%. Colony formation and nonobese diabetic/severe combined immunodeficient mice engraftment efficiency were similar after 3 days at 20% and 0.1% O2 concentrations but lower than at T0. In conclusion, a low O2 concentration, close to those found in bone marrow stem cell niches, induces the G0 return of CD34+ cells without impairing their functional capacity.

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.

Interleukin-6 (IL-6) and low O2 concentration (1%) synergize to improve the maintenance of hematopoietic stem cells (pre-CFC)

Low O2 concentration (1%) favors the self‐renewal of hematopoietic stem cells and inhibits committed progenitors (CFC). Since IL‐6 influences both stem cells and committed progenitors at 20% O2, we studied its effects in cultures at 1% O2. The pre‐CFC activity in Lin− population of mouse bone marrow was analyzed following 10 days of serum‐free culture in medium (LC1) supplemented with IL‐3 with and without IL‐6, at 20 and 1% O2 and phenotypic differentiation and proliferative history monitored. The IL‐6 receptor expression and initiation of VEGF‐A synthesis were also investigated. At 20% O2, the effects of IL‐6 on pre‐CFC were negligible but effects on CFC were apparent; conversely, at 1% O2, the IL‐6 enhances activity of pre‐CFC but not of CFC. Unlike at 20% O2, at 1% O2 a subpopulation of cells remained Lin− in spite of extensive proliferation. However, the absolute number of Lin− cells, did not correlate with pre‐CFC activity. A relative increase in VEGF transcripts at 1% O2 in presence of IL‐3 alone was enhanced by the addition of IL‐6. IL‐6 enhanced pre‐CFC activity at 1% O2 and this was correlated to the induction of VEGF. These data reinforce the concept that physiologically low oxygenation of bone marrow is a regulator of stem cell maintenance. Since the 20% O2 does not exist in tissues in vivo, further studies in vitro at lower O2 concentrations should revise our knowledge relating to cytokine effects on stem and progenitor cells. J. Cell. Physiol. 212: 68–75, 2007.

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.

γδ T Cells Confer Protection against Murine Cytomegalovirus (MCMV)

Cytomegalovirus (CMV) is a leading infectious cause of morbidity in immune-compromised patients. γδ T cells have been involved in the response to CMV but their role in protection has not been firmly established and their dependency on other lymphocytes has not been addressed. Using C57BL/6 αβ and/or γδ T cell-deficient mice, we here show that γδ T cells are as competent as αβ T cells to protect mice from CMV-induced death. γδ T cell-mediated protection involved control of viral load and prevented organ damage. γδ T cell recovery by bone marrow transplant or adoptive transfer experiments rescued CD3ε−/− mice from CMV-induced death confirming the protective antiviral role of γδ T cells. As observed in humans, different γδ T cell subsets were induced upon CMV challenge, which differentiated into effector memory cells. This response was observed in the liver and lungs and implicated both CD27+ and CD27− γδ T cells. NK cells were the largely preponderant producers of IFNγ and cytotoxic granules throughout the infection, suggesting that the protective role of γδ T cells did not principally rely on either of these two functions. Finally, γδ T cells were strikingly sufficient to fully protect Rag−/−γc−/− mice from death, demonstrating that they can act in the absence of B and NK cells. Altogether our results uncover an autonomous protective antiviral function of γδ T cells, and open new perspectives for the characterization of a non classical mode of action which should foster the design of new γδ T cell based therapies, especially useful in αβ T cell compromised patients.

Plasminogen- and colony-stimulating factor-1-associated markers in bladder carcinoma: diagnostic value of urokinase plasminogen activator receptor and plasminogen activator inhibitor type-2 using immunocytochemical analysis

Abstract. The expression of plasminogen- and colony-stimulating factor-1-associated markers was first investigated in seven bladder carcinoma cell lines and in 15 primary bladder tumors using RT-PCR (mRNAs), zymography (protein activity), ELISA and immunocytochemistry analysis (ICC) (protein levels). The mRNAs expression, the activity and the levels of the secreted proteins were not informative. Only urokinase plasminogen activator receptor (uPA-R/CD87) and possibly plasminogen activator inhibitor type-2 (PAI2) antigen expression at the cellular levels seem to be useful markers. uPA-R antigen expression correlated with the secretion of hepatocyte growth factor (HGF) (P=0.016) and the motility of the bladder tumor cells (P=0.014), two markers associated with a poor prognosis in bladder carcinoma. To validate our technique and confirm these preliminary results, uPA-R and PAI2 antigen expression was determined in the imprints from 129 resected bladder carcinoma fragments. uPA-R correlated with the grade (P=0.002), tumor invasion (P=0.003) and the ploidy (P=0.05) of the bladder carcinomas and with the low overall survival (P=0.045) of the patients. PAI2 correlated only with the stage (P=0.02) and low overall survival (P=0.038). We conclude that in bladder carcinomas, studying the transcripts of PAs, PAIs, CSF-1 and its receptor, as well as measuring their concentration or activity in culture supernatants was of no clinical interest in terms of diagnostic or prognostic value. Only the ICC of uPA-R, which correlated with the major histopathological parameters of tumors and the low overall survival, proved to be a diagnostic and prognostic marker.

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,

Expression of Platelet‐Activating Factor Receptor Transcript‐1 but Not Transcript‐2 by Human Bone Marrow Cells

The presence of platelet‐activating factor receptor (PAF‐R) transcripts 1 and 2 was investigated in human bone marrow cells by a reverse transcriptase polymerase chain reaction (RT‐PCR) procedure which detected their simultaneous presence. RT‐PCR experiments reveal PAF‐R transcript 1 (but not 2) in freshly isolated mononuclear marrow cells, CD34+ hematopoietic stem/progenitor cells and cultured marrow stromal cells. For these experiments, the 5637 human bladder carcinoma cell line is used as a positive control for the presence of PAF‐R transcripts 1 and 2. Flow cytometry experiments confirm the presence of PAF‐R on marrow stromal cells and CD34+ stem/progenitor cells. In conclusion, the expression of PAF‐R transcript 1, which mainly exists in circulating leukocytes, is also found in CD34+ stem/progenitor cells and cells of the marrow microenvironment, strengthening the potential role of PAF during marrow hematopoiesis.