Stem cell therapy From bench to bedside

International audienceSeveral countries have increased efforts to develop medical countermeasures to protect against radiation toxicity due to acts of bioterrorism as well as cancer treatment. Both acute radiation injuries and delayed effects such as cutaneous effects and impaired wound repair depend, to some extent, on angiogenesis deficiency. Vascular damage influences levels of nutrients, oxygen available to skin tissue and epithelial cell viability. Consequently, the evolution of radiation lesions often becomes uncontrolled and surgery is the final option-amputation leading to a disability. Therefore, the development of strategies designed to promote healing of radiation injuries is a major therapeutic challenge. Adult mesenchymal stem cell therapy has been combined with surgery in some cases and not in others and successfully applied in patients with accidental radiation injuries. Although research in the field of radiation skin injury management has made substantial progress in the past 10 y, several strategies are still needed in order to enhance the beneficial effect of stem cell therapy and to counteract the deleterious effect of an irradiated tissue environment. This review summarises the current and evolving advances concerning basic and translational research based on stem cell therapy for the management of radiological burns. © World Health Organization 2012. All rights reserved

New biological indicators to evaluate and monitor radiation-induced damage: an accident case report.

On March 11th, 2006, a radiation accident occurred in an industrial radiation facility, in the city of Fleurus (Belgium). An operator entered an irradiation room without noticing that the cobalt-60 source (activity of 3x104 TBq) was out of the security position. Few hours after the exposure, the victim had nausea and vomiting, but these clinical signs were confounded with a benign gastroenteritis. Eighteen days later, the patient consulted for persistent nausea and headache, transitory and refractory diarrhoea and hair loss. The occurrence of a radiation accident was then proposed as an explanation for these symptoms, and the patient was taken in charge by the medical staff of Percy hospital (Clamart, France). Cytogenetic dosimetry was used to define the radiation dose and heterogeneity received by the victim. This cytogenetic analysis indicated a mean radiation dose of 4.5 Gy, based upon dicentric chromosome frequency, reciprocal translocations and total translocations. However, the presence of 18.8% of cells without dicentric chromosomes suggested of an heterogeneous irradiation. This was confirmed by the physical dosimetry, which suggested an anterior-posterior gradient of dose. The patient was then followed by the mean of several new biological indicators of radiation-induced damages to specific organs and physiological systems. The blood Flt3 ligand concentration was used to evaluate and follow the hematopoietic syndrome. Initial measurement gave <2500 pg FL/ml of plasma, indicating a severe hematopoietic syndrome, with some evidence of a residual hematopoiesis. As soon as the patient received a cytokine treatment, a rapid and sustained hematopoietic recovery was observed. Citrulline concentration was used to evaluate damage to the mucosal epithelium of the small bowel. However, citrulline concentration was in the range of normal values, indicating the absence of significant damage to the mucosal epithelium. Several oxysterols were used to evaluate damages to the cardiovascular system and to the liver lipid metabolism. The decrease over time observed in 7α-hydroxycholesterol (7α-OH-Chol) concentration was indicative of a liver damage, as confirmed by decrease in AST and ALT concentrations and in Apolipoproteins A1 and B. The 27α-OH-Chol was strongly decreased over time, in parallel to an increase in CK-MB. This suggested radiation-induced damages to the cardiovascular system. Overall, our results show that these new biological indicators are useful for the initial evaluation of radiation-induced damages to specific organs and physiological systems, but also for the monitoring of pathophysiological evolution of the patient, in combination with existing scoring for radiation accident victims

New biological indicators to evaluate and monitor radiation-induced damage: An accident case report

The aim of this work was to use several new biological indicators to evaluate damage to the main physiological systems in a victim exposed accidentally to ionizing radiation. Blood samples were used for biological dosimetry and for measurement of the plasma concentrations of several molecules: Flt3 ligand to assess the hematopoietic system, citrulline as an indicator of the digestive tract, and several oxysterols as lipid metabolism and vascular markers. The cytogenetic evaluation estimated the dose to the victim to be between 4.2 and 4.8 Gy, depending on the methodology used. Monitoring the Flt3 ligand demonstrated the severity of bone marrow aplasia. In contrast, the citrulline concentration showed the absence of gastrointestinal damage. Variations in oxysterol concentrations suggested radiation-induced damage to the liver and the cardiovascular system. These results were correlated with those from classic biochemical markers, which demonstrated severe damage to the hematopoietic system and suggested the appearance of subclinical damage to the liver and cardiovascular system. These results demonstrate for the first time the importance of a multiparameter biological approach in the evaluation of radiation damage after accidental irradiation. © 2008 by Radiation Research Society

Therapeutic potential of gingival fibroblasts for cutaneous radiation syndrome Comparison to bone marrow-mesenchymal stem cell grafts

International audienceMesenchymal stem cell (MSC) therapy has recently been investigated as a potential treatment for cutaneous radiation burns. We tested the hypothesis that injection of local gingival fibroblasts (GFs) would promote healing of radiation burn lesions and compared results with those for MSC transplantation. Human clinical-grade GFs or bone marrow-derived MSCs were intradermally injected into mice 21 days after local leg irradiation. Immunostaining and real-time PCR analysis were used to assess the effects of each treatment on extracellular matrix remodeling and inflammation in skin on days 28 and 50 postirradiation. GFs induced the early development of thick, fully regenerated epidermis, skin appendages, and hair follicles, earlier than MSCs did. The acceleration of wound healing by GFs involved rearrangement of the deposited collagen, modification of the Col/MMP/TIMP balance, and modulation of the expression and localization of tenascin-C and of the expression of growth factors (VEGF, EGF, and FGF7). As MSC treatment did, GF injection decreased the irradiation-induced inflammatory response and switched the differentiation of macrophages toward an M2-like phenotype, characterized by CD163+ macrophage infiltration and strong expression of arginase-1. These findings indicate that GFs are an attractive target for regenerative medicine, for easier to collect, can grow in culture, and promote cutaneous wound healing in irradiation burn lesions. © 2015, Mary Ann Liebert, Inc

New approach to radiation burn treatment by dosimetry-guided surgery combined with autologous mesenchymal stem cell therapy

The therapeutic management of severe radiation burns remains a challenging issue. Conventional surgical treatment (excision and skin autograft or rotation flap) often fails to prevent unpredictable and uncontrolled extension of the radiation necrotic process. We report here an innovative therapeutic strategy applied to the victim of a radiation accident (December 15, 2005) with an iridium gammagraphy radioactive source (192Ir, 3.3 TBq). The approach combined numerical dosimetry-guided surgery with cellular therapy using mesenchymal stem cells. A very severe buttock radiation burn (2000 Gy at the center of the skin surface lesion) of a 27-year-old Chilean victim was widely excised (10 cm in diameter) using a physical and anatomical dose reconstruction in order to better define the limit of the surgical excision in apparently healthy tissues. A secondary extension of the radiation necrosis led to a new excision of fibronecrotic tissues associated with a local cellular therapy using autologous expanded mesenchymal stem cells as a source of trophic factors to promote tissue regeneration. Bone marrow-derived mesenchymal stem cells were expanded according to a clinical-grade technique using closed culture devices and serum-free medium enriched in human platelet lysate. The clinical evolution (radiation pain and healing progression) was favorable and no recurrence of radiation inflammatory waves was observed during the 11 month patient's follow-up. This novel multidisciplinary therapeutic approach combining physical techniques, surgical procedures and cellular therapy with adult stem cells may be of clinical relevance for improving the medical management of severe localized irradiations. It may open new prospects in the field of radiotherapy complications. © 2007 Future Medicine Ltd

Development of proteomic tools to study protein adsorption on a biomaterial, titanium grafted with poly(sodium styrene sulfonate)

1 - ArticleIt is known that protein adsorption is the initial interaction between implanted biomaterials and biological environment. Generally, a complex protein layer will be formed on material surfaces within a few minutes and the composition of this layer at the interface determines the biological response to the implanted material, and therefore the long-term compatibility of the biomaterial. Despite different techniques exist to observe protein adsorption on biomaterials, none of them led to the identification of adsorbed proteins. In this paper, we report a chromatographic technique coupled to proteomics to analyse and identify proteins from complex biological samples adsorbed on biomaterial surfaces. This approach is based on (1) elaboration of the chromatographic support containing the biomaterial (2) a chromatography step involving adsorption of proteins on the biomaterial (3) the high-resolution separation of eluted proteins by 2-DE gel and (4) the identification of proteins by mass spectrometry. Experiments were performed with proteins from platelets rich plasma (PRP) adsorbed on a biomaterial which consist in titanium bioactivated with PolyNaSS. Our results show that chromatographic approach combined to 2-DE gels and mass spectrometry provides a powerful tool for the analysis and identification of proteins adsorbed on various surfaces

Effect of Lentivirus-induced shRNA Silencing CXCR4 Gene on Proliferation and Apoptosis in Human Esophageal Carcinoma Cell Line Eca109

OBJECTIVE To discuss the application of the slow virus- induced short-hairpin RNA (vshRNA) to silence the expression of CXCR4 in EsCa cell lines Eca109, and observe the effect of silencing CXCR4 on the proliferation and apoptosis of Eca109 cells in vitro. METHODS The expression plasmid of vshRNA targeting CXCR4 was constructed, with a concurrent construction of negative vshRNA expression plasmid, and without targeting any known mRNA. Real-time quantitative PCR and Western blot assay were used to determine the change of CXCR4 expression in the post-transfected EsCa cell Eca109, and MTT assay was conducted to detect the change of proliferation in EsCa Eca109 cell after silencing the CXCR4. The flow cytometry was used to detect the change of the cell cycle and apoptosis in the post-silenced EsCa Eca109 cell in different groups. RESULTS The transfection rate was respectively (87.3 ± 1.2)% and (90.1 ± 1.4)% in the CXCR4- RNAi-LV (silent group) and NC-GFP-RNAi-LV (negative control group) cellular plasmids. The vshRNA interference resulted in a down-regulation of the CXCR4 gene mRNA and protein expressions in Eca109 cells. CXCL12 promoted the proliferation of EsCa cell lines Eca109. The speed of EsCa cell proliferation became slower in the silencing group than in the normal control (also the control) and the negative control groups (P < 0.05). However, there was no significant difference in comparison of the proliferation speeds between the negative control and the normal control groups (P > 0.05). In the silencing group, the proportion of the cells in phase G0/G1, phase S and phase G2/M was respectively (69.9 ± 5.0)%, (17.1 ± 2.5)% and (13.0 ± 7.4)%, and the apoptotic rate achieved (7.27 ± 0.50)%. In the normal control group, the proportion of the cells in phase G0/G1, S and G2/M was respectively (55.9 ± 4.6)%, (30.2 ± 3.9)% and (13.8 ± 1.4)%, and the apoptotic rate was (3.30 ± 0.70)%. In the negative control group, the proportion of cells in phase G0/G1, S and G2/M was respectively (52.7 ± 7.8)%, (25.3 ± 2.3)% and (21.9 ± 7.4)%, with an apoptotic rate of (4.03 ± 1.37)%. Compared with the normal control and negative control groups, there was an apparent growth of cells in the phase G0/G1 (P < 0.05), and a greatly increased number of cells in phase S (P < 0.05) in the silencing group. There was no significant difference in comparison of those between the normal control and negative control groups (P > 0.05). The apoptotic rate was obviously higher in the cells of the silencing group than in the normal control and the negative control groups (P < 0.05). There was no significant difference in comparison of the apoptotic rate between the normal control and the negative control groups (P >0.05). CONCLUSION CXCR4-vshRNA can specifically and effectively inhibit CXCR4 expression of Eca109 cells. CXCR4-vshRNA can inhibit the proliferation and enhance the apoptosis rate of Eca109 cells through intervening the expression of CXCR4, suggesting that CXCL12/CXCR4 might have an important role in the progression of Escc. This slow virus-induced shRNA can effectively silence the expression of CXCR4 gene in the EsCa cells; block up the biological effect of CXCL12/CXCR4 axle; and effectively inhibit the potency of proliferation in the EsCa cell line Eca109, thus advancing apoptosis. It suggests that the CXCL12/CXCR4 plays an important role in the progression of EsCa