Fluctuation of the SP/non-SP phenotype in the C6 glioma cell line

AbstractUsing the C6 glioma cell as a paradigm, we found that (i) the clonogenicity of C6 cells is several orders of magnitude higher than the percentage of SP cells; (ii) non-SP cells are able to generate SP cells, and conversely SP cells generate non-SP cells; (iii) non-SP sorted cells behave as tumorigenic cells. Hence, in C6 cells cultured in serum-containing medium, SP cells can be generated from non-SP cells. This dynamic equilibrium explains in C6 cells the maintenance of the SP phenotype with cell passaging and demonstrates the existence of tumorigenic non-SP cells

Persistent mRNA and miRNA expression changes in irradiated baboons

Abstract We examined the transcriptome/post-transcriptome for persistent gene expression changes after radiation exposure in a baboon model. Eighteen baboons were irradiated with a whole body equivalent dose of 2.5 or 5 Gy. Blood samples were taken before, 7, 28 and 75–106 days after radiation exposure. Stage I was a whole genome screening for mRNA combined with a qRT-PCR platform for detection of 667 miRNAs. Candidate mRNAs and miRNAs differentially up- or down-regulated in stage I were chosen for validation in stage II using the remaining samples. Only 12 of 32 candidate genes provided analyzable results with two mRNAs showing significant 3–5-fold differences in gene expression over the reference (p < 0.0001). From 667 candidate miRNAs, 290 miRNA were eligible for analysis with 21 miRNAs independently validated using qRT-PCR. These miRNAs showed persistent expression changes on each day and over days 7–106 days after exposure (n = 7). In particular miR-212 involved in radiosensitivity and immune modulation appeared persistently and 48–77-fold up-regulated over the entire time period. We are finally trying to put our results into a context of clinical implications and provide possible hints on underlying molecular mechanisms to be examined in future studies

Soluble Vascular Endothelial Cadherin as a New Biomarker of Irradiation in Highly Irradiated Baboons with Bone Marrow Protection

International audienceVascular endothelial cadherin is the main component of adherens junctions enabling cohesion of the endothelial monolayer in vessels. The extracellular part of vascular endothelial cadherin (VE-cadherin) can be cleaved, releasing soluble fragments in blood (sVE-cadherin). In some diseases with endothelial dysfunction, a correlation between increased blood sVE-cadherin levels and disease state has been proposed. Irradiation is known to induce endothelial damage, but new serum biomarkers are needed to evaluate endothelial damage after irradiation. Here, the authors investigated whether sVE-cadherin may be an interesting biomarker of irradiation in highly irradiated baboons with bone marrow protection. sVE-cadherin was detected in the plasma of young as well as old baboons. Plasma sVE-cadherin levels significantly decrease a few days after irradiation but recover in the late time after irradiation. Kinetic analysis of plasma sVE-cadherin levels suggests a correlation with white blood cell counts in both the acute phase of irradiation and during hematopoietic recovery, suggesting that plasma sVE-cadherin levels may be partly linked to the disappearance and recovery of white blood cells. Interestingly, after hematopoietic recovery was completed, sVE-cadherin levels were found to exceed control values, suggesting that plasma sVE-cadherin may represent a new biomarker of endothelial damage or neovascularization in the late time after irradiation

Pre-Exposure Gene Expression in Baboons with and without Pancytopenia after Radiation Exposure

Radiosensitivity differs in humans and likely among primates. The reasons are not well known. We examined pre-exposure gene expression in baboons (n = 17) who developed haematologic acute radiation syndrome (HARS) without pancytopenia or a more aggravated HARS with pancytopenia after irradiation. We evaluated gene expression in a two stage study design where stage I comprised a whole genome screen for messenger RNAs (mRNA) (microarray) and detection of 667 microRNAs (miRNA) (real-time quantitative polymerase chain reaction (qRT-PCR) platform). Twenty candidate mRNAs and nine miRNAs were selected for validation in stage II (qRT-PCR). None of the mRNA species could be confirmed during the validation step, but six of the nine selected candidate miRNA remained significantly different during validation. In particular, miR-425-5p (receiver operating characteristic = 0.98; p = 0.0003) showed nearly complete discrimination between HARS groups with and without pancytopenia. Target gene searches of miR-425-5p identified new potential mRNAs and associated biological processes linked with radiosensitivity. We found that one miRNA species examined in pre-exposure blood samples was associated with HARS characterized by pancytopenia and identified new target mRNAs that might reflect differences in radiosensitivity of irradiated normal tissue

Lack of evidence of sustained hematopoietic reconstitution after transplantation of unmanipulated adult liver stem cells in monkeys.

International audienceThe aim of this study was to search for hematopoietic potential in the liver of non-human primates. Lethally irradiated (2 x 5 Gy gamma) macaque monkeys were given autologous hepatic mononuclear cells (HMNC) isolated from a liver lobe by perfusion and digestion with 0.1% collagenase. Two monkeys were given intramedullary injections of HMNC (18.6 x 10(6)/kg, 20.4 x 10(6)/kg) and two others were co-transplanted with HMNC (14.35 x 10(6)/kg, 96.5 x 10(6)/kg) and bone marrow mesenchymal stem cells (0.42 x 10(6)/kg, 1.16 x 10(6)/kg). All monkeys exhibited a transient neutrophil recovery from day 22 for 10 days, but failed to produce platelets and remained transfusion-dependent. In conclusion, adult liver stem cells from a monkey model show a low level of in vivo hematopoietic potential, suggesting ex vivo manipulation will be required before clinical use of such cells

Mesenchymal stem cells rescue CD34+ cells from radiation-induced apoptosis and sustain hematopoietic reconstitution after coculture and cografting in lethally irradiated baboons: is autologous stem cell therapy in nuclear accident settings hype or reality?

International audienceAutologous stem cell therapy (ACT) has been proposed to prevent irradiated victims from bone marrow (BM) aplasia by grafting hematopoietic stem and progenitor cells (HSPCs) collected early after damage, provided that a functional graft of sufficient size could be produced ex vivo. To address this issue, we set up a baboon model of cell therapy in which autologous peripheral blood HSPCs collected before lethal total body irradiation were irradiated in vitro (2.5 Gy, D0 1 Gy) to mimic the cell damage, cultured in small numbers for a week in a serum-free medium in the presence of antiapoptotic cytokines and mesenchymal stem cells (MSCs) and then cografted. Our study shows that baboons cografted with expanded cells issued from 0.75 and 1 x 10(6)/kg irradiated CD34+ cells and MSCs (n=2) exhibited a stable long-term multilineage engraftment. Hematopoietic recovery became uncertain when reducing the CD34+ cell input (0.4 x 10(6)/kg CD34+ cells; n=3). However, platelet recovery was accelerated in all surviving cografted animals, when compared with baboons transplanted with unirradiated, unmanipulated CD34+ cells (0.5-1 x 10(6)/kg, n=4). Baboons grafted with MSCs alone (n=3) did not recover. In all cases, the nonhematopoietic toxicity remained huge. This baboon study suggests that ACT feasibility is limited

Revisiting Biomarkers of Total-Body and Partial-Body Exposure in a Baboon Model of Irradiation.

In case of a mass casualty radiation event, there is a need to distinguish total-body irradiation (TBI) and partial-body irradiation (PBI) to concentrate overwhelmed medical resources to the individuals that would develop an acute radiation syndrome (ARS) and need hematologic support (i.e., mostly TBI victims). To improve the identification and medical care of TBI versus PBI individuals, reliable biomarkers of exposure could be very useful. To investigate this issue, pairs of baboons (n = 18) were exposed to different situations of TBI and PBI corresponding to an equivalent of either 5 Gy 60Co gamma irradiation (5 Gy TBI; 7.5 Gy left hemibody/2.5 right hemibody TBI; 5.55 Gy 90% PBI; 6.25 Gy 80% PBI; 10 Gy 50% PBI, 15 Gy 30% PBI) or 2.5 Gy (2.5 Gy TBI; 5 Gy 50% PBI). More than fifty parameters were evaluated before and after irradiation at several time points up to 200 days. A partial least square discriminant analysis showed a good distinction of TBI from PBI situations that were equivalent to 5 Gy. Furthermore, all the animals were pooled in two groups, TBI (n = 6) and PBI (n = 12), for comparison using a logistic regression and a non parametric statistical test. Nine plasmatic biochemical markers and most of hematological parameters turned out to discriminate between TBI and PBI animals during the prodromal phase and the manifest illness phase. The most significant biomarkers were aspartate aminotransferase, creatine kinase, lactico dehydrogenase, urea, Flt3-ligand, iron, C-reactive protein, absolute neutrophil count and neutrophil-to-lymphocyte ratio for the early period, and Flt3-ligand, iron, platelet count, hemoglobin, monocyte count, absolute neutrophil count and neutrophil-to-lymphocyte ratio for the ARS phase. These results suggest that heterogeneity could be distinguished within a range of 2.5 to 5 Gy TBI