Dual SP/ALDH functionalities refine the human hematopoietic Lin-CD34+CD38- stem/progenitor cell compartment.

Identification of prevalent specific markers is crucial to stem/progenitor cell purification. Determinants such as the surface antigens CD34 and CD38 are traditionally used to analyze and purify hematopoietic stem/progenitor cells (HSCs/HPCs). However, the variable expression of these membrane antigens poses some limitations to their use in HSC/HPC purification. Techniques based on drug/stain efflux through the ATP-binding cassette (ABC)G2 pump (side population [SP] phenotype) or on detection of aldehyde dehydrogenase (ALDH) activity have been independently developed and distinguish the SP and ALDH(Bright) (ALDH(Br)) cell subsets for their phenotype and proliferative capability. In this study, we developed a multiparametric flow cytometric method associating both SP and ALDH activities on human lineage negative (Lin(-)) bone marrow cells and sorted different cell fractions according to their SP/ALDH activity level. We find that Lin(-)CD34(+)CD38(Low/-) cells are found throughout the spectrum of ALDH expression and are enriched especially in ALDH(Br) cells when associated with SP functionality (SP/ALDH(Br) fraction). Furthermore, the SP marker identified G(0) cells in all ALDH fractions, allowing us to sort quiescent cells regardless of ALDH activity. Moreover, we show that, within the Lin(-)CD34(+)CD38(-)ALDH(Br) population, the SP marker identifies cells with higher primitive characteristics, in terms of stemness-related gene expression and in vitro and in vivo proliferative potential, than the Lin(-)CD34(+) CD38(-)ALDH(Br) main population cells. In conclusion, our study shows that the coexpression of SP and ALDH markers refines the Lin(-)CD34(+)CD38(-) hematopoietic compartment and identifies an SP/ALDH(Br) cell subset enriched in quiescent primitive HSCs/HPCs.

Emerging therapy for improving wound repair of severe radiation burns using local bone marrow-derived stem cell administrations.

The therapeutic management of severe radiation burns remains a challenging issue today. Conventional surgical treatment including excision, skin autograft, or flap often fails to prevent unpredictable and uncontrolled extension of the radiation-induced necrotic process. In a recent very severe accidental radiation burn, we demonstrated the efficiency of a new therapeutic approach combining surgery and local cellular therapy using autologous mesenchymal stem cells (MSC), and we confirmed the crucial place of the dose assessment in this medical management. The patient presented a very significant radiation lesion located on the arm, which was first treated by several surgical procedures: iterative excisions, skin graft, latissimus muscle dorsi flap, and forearm radial flap. This conventional surgical therapy was unfortunately inefficient, leading to the use of an innovative cell therapy strategy. Autologous MSC were obtained from three bone marrow collections and were expanded according to a clinical-grade protocol using platelet-derived growth factors. A total of five local MSC administrations were performed in combination with skin autograft. After iterative local MSC administrations, the clinical evolution was favorable and no recurrence of radiation inflammatory waves occurred during the patient's 8-month follow-up. The benefit of this local cell therapy could be linked to the "drug cell" activity of MSC by modulating the radiation inflammatory processes, as suggested by the decrease in the C-reactive protein level observed after each MSC administration. The success of this combined treatment leads to new prospects in the medical management of severe radiation burns and more widely in the improvement of wound repair.

[Skin engineering for burns treatment]. / L'ingéniérie cutanée pour le traitement des brûlures graves.

Severely burned patients need effective and permanent wound coverage. The outcome of massive burn injuries has improved with the use of cultured epithelial autografts (CEA), despite their fragility, frequent failure to take, high cost and long-term tendency to contract. Combining CEA with dermal substitutes provides earlier skin closure and satisfactory functional results. Another promising line of research is skin regeneration with epidermal stem cells, which have the capacity to differentiate into keratinocytes, to promote wound repair, and to regenerate skin appendages. Human mesenchymal stem cells have been evaluated in radiation-induced skin damage.

Bone marrow stromal cells spontaneously produce Flt3-ligand: influence of ionizing radiations and cytokine stimulation.

PURPOSE: To define the ability of human bone marrow (BM) stromal cells to produce fms-like tyrosine kinase 3 (Flt3)-ligand (FL), and the effect of irradiation, tumour necrosis factor-alpha (TNFalpha) or tumour growth factor beta (TGFbeta) on FL production. MATERIAL AND METHODS: Primary BM stromal cell cultures were irradiated at 2-10 Gy or were stimulated with TNFalpha or TGFbeta1. The presence of FL was tested in culture supernatants and in cell lysate. The presence of a membrane-bound form of FL and the level of gene expression were also tested. RESULTS: Primary BM stromal cells spontaneously released FL. This production was increased by TNFalpha but not by TGFbeta1 or by irradiation. Chemical induction of osteoblastic differentiation from BM stromal cells also induced an increase in FL release. CONCLUSIONS: Our results suggest that the observed increase in FL concentration after in vivo irradiation is an indirect effect. The possible implication of BM stromal cells in these mechanisms is discussed.

Striatal and Extrastriatal Dopamine Transporter Availability in Schizophrenia and Its Clinical Correlates: A Voxel-Based and High-Resolution PET Study.

Neuroimaging studies investigating dopamine (DA) function widely support the hypothesis of presynaptic striatal DA hyperactivity in schizophrenia. However, published data on the striatal DA transporter (DAT) appear less consistent with this hypothesis, probably partly due to methodological limitations. Moreover, DAT in extrastriatal regions has been very poorly investigated in the context of schizophrenia. In order to address these issues, we used a high resolution positron emission tomograph and the selective DAT radioligand [11C]PE2I, coupled with a whole brain voxel-based analysis method to investigate DAT availability in striatal but also extra-striatal regions in 21 male chronic schizophrenia patients compared to 30 healthy male controls matched by age. We found higher DAT availability in schizophrenia patients in midbrain, striatal, and limbic regions. DAT availability in amygdala/hippocampus and putamen/pallidum was positively correlated with hallucinations and suspiciousness/persecution, respectively. These results are consistent with an increase of presynaptic DA function in patients with schizophrenia, and support the involvement of both striatal and extrastriatal DA dysfunction in positive psychotic symptoms. The study also highlights the whole brain voxel-based analysis method to explore DA dysfunction in schizophrenia.

Use of flt3 ligand to evaluate residual hematopoiesis after heterogeneous irradiation in mice.

We evaluated the possibility of using plasma Flt3 ligand (FL) concentration as a biological indicator of bone marrow function after heterogeneous irradiation. Mice were irradiated with 4, 7.5 or 11 Gy with 25, 50, 75 or 100% of the bone marrow in the field of irradiation. This model of irradiation resulted in graded and controlled damage to the bone marrow. Mice exhibited a pancytopenia correlated with both the radiation dose and the percentage of bone marrow irradiated. The FL concentration in the blood increased with the severity of bone marrow aplasia. Nonlinear regression analysis showed that the FL concentration was strongly correlated with the total number of residual colony-forming cells 3 days after irradiation, allowing a precise estimate of residual hematopoiesis. Moreover, the FL concentration on day 3 postirradiation was correlated with the duration and severity of subsequent pancytopenia, suggesting that variations in FL concentrations might be used as a predictive indicator of bone marrow aplasia, especially by the use of linear regression equations describing these correlations. Our results provide a rationale for the use of FL concentration as a biological indicator of residual hematopoiesis after heterogeneous irradiation.

Kinetics of plasma FLT3 ligand concentration in hematopoietic stem cell transplanted patients.

The present study aimed to follow-up variations in plasma Flt3 ligand (FL) concentration after hematopoietic stem cell transplantation and to compare the influence of conditioning regimens on variations in FL concentration. Ten patients undergoing a conditioning regimen, including BEAM, cyclophosphamide (Cy) + total body irradiation or Cy + anti-thymocyte globulins (ATG), which was then followed by hematopoietic stem cell transplantation, were studied. Plasma FL concentrations, white blood cell (WBC) expression of both FL mRNA and the membrane-bound form of FL were carried out at different times post-treatment. The results indicated that plasma FL concentration increased rapidly after the conditioning regimen in all patients, in correlation with the decrease in number of WBCs. The area under the curve of FL according to time was directly correlated with the duration of pancytopenia, except when ATG was included in the conditioning regimen. Although the number of patients was limited in this study, the comparison of ATG-treated patients and other patients suggests that plasma FL concentration is regulated by a complex mechanism partly involving circulating blood cells.

Comparison of autologous cell therapy and granulocyte-colony stimulating factor (G-CSF) injection vs. G-CSF injection alone for the treatment of acute radiation syndrome in a non-human primate model.

PURPOSE: To compare the efficacy of autologous cell therapy after irradiation combined with granulocyte-colony stimulating factor (G-CSF) injections with G-CSF treatment alone in a heterogeneous model of irradiation representative of an accidental situation. MATERIAL AND METHODS: Non-human primates were irradiated at 8.7 Gy whole-body dose with the right arm shielded to receive 4.8 Gy. The first group of animals received G-CSF (lenograstim) injections starting 6 h after irradiation, and a second group received a combination of G-CSF (lenograstim) injections and autologous expanded hematopoietic cells. Animals were followed up for blood cell counts, circulating progenitors, and bone marrow cellularity. RESULTS: No significant differences were seen between the two treatment groups, whatever the parameter observed: time to leukocyte or platelet recovery and duration and severity of aplasia. CONCLUSION: Our results indicated that identical recovery kinetic was observed when irradiated animals are treated with G-CSF independently of the reinjection of ex vivo expanded autologous hematopoietic cells. Thus G-CSF injections might be chosen as a first-line therapeutic strategy in the treatment of accidental acute radiation victims.

Radiation-induced increase in plasma Flt3 ligand concentration in mice: evidence for the implication of several cell types.

Circulating T lymphocytes were proposed as the main producer of Flt3 ligand. However, during aplasia, there is a drastic reduction in the number of T lymphocytes, while plasma Flt3 ligand concentration is increased. This contradiction prompted us to compare variations in plasma Flt3 ligand during radiation-induced aplasia in BALB/c mice and in T-lymphocyte-deficient NOD-SCID mice to delineate the role of T lymphocytes in the increase in Flt3 ligand concentration. The results showed that plasma Flt3 ligand concentration was increased similarly in the two strains of mice, and that Flt3 ligand concentration was negatively correlated to the number of residual hematopoietic progenitors. Moreover, the Flt3 ligand mRNA expression and Flt3 ligand protein concentration were similar in the two strains of mice in all organs tested, i.e. thymus, spleen, bone marrow, liver, brain and blood cells. These results confirm that Flt3 ligand concentration in the blood is a reflection of bone marrow function and that T lymphocytes are not the main regulator of Flt3 ligand variations during aplasia.

Application of autologous hematopoietic cell therapy to a nonhuman primate model of heterogeneous high-dose irradiation.

We developed a model of heterogeneous irradiation in a nonhuman primate to test the feasibility of autologous hematopoietic cell therapy for the treatment of radiation accident victims. Animals were irradiated either with 8 Gy to the body with the right arm shielded to obtain 3.4 Gy irradiation or with 10 Gy total body and 4.4 Gy to the arm. Bone marrow mononuclear cells were harvested either before irradiation or after irradiation from an underexposed area of the arm and were expanded in previously defined culture conditions. We showed that hematopoietic cells harvested after irradiation were able to expand and to engraft when reinjected 7 days after irradiation. Recovery was observed in all 8-Gy-irradiated animals, and evidence for a partial recovery was observed in 10-Gy-irradiated animals. However, in 10-Gy-irradiated animals, digestive disease was observed from day 16 and resulted in the death of two animals. Immunohistological examinations showed damage to the intestine, lungs, liver and kidneys and suggested radiation damage to endothelial cells. Overall, our results provide evidence that such an in vivo model of heterogeneous irradiation may be representative of accidental radiation exposures and may help to define the efficacy of therapeutic interventions such as autologous cell therapy in radiation accident victims.

Plasma Flt-3 ligand concentration correlated with radiation-induced bone marrow damage during local fractionated radiotherapy.

PURPOSE: To determine whether variations in the plasma Flt-3 ligand (FL) concentration after radiotherapy (RT) may serve as a biomarker for radiation-induced bone marrow damage. METHODS AND MATERIALS: Twenty-seven patients were followed during RT. The irradiated bone marrow volume was determined. The blood cell counts and plasma FL concentrations were evaluated before and after RT. The expression of membrane-bound FL and mRNA expression were also defined in circulating blood cells. RESULTS: We found a negative correlation between the plasma FL concentration and the number of circulating white blood cells and platelets during RT. Moreover, the overall amount of FL in the blood of patients during RT correlated directly with both the cumulated radiation dose and the proportion of irradiated bone marrow. CONCLUSIONS: We demonstrated that the variations in plasma FL concentration directly reflect the radiation-induced bone marrow damage during fractionated local RT. We suggest a possible use for FL monitoring as a means to predict the occurrence of Grade 3-4 leukopenia or thrombocytopenia during the course of RT.

In vivo quantitation of tumour vascularisation assessed by Doppler sonography in rat mammary tumours.

This study was designed to evaluate high-frequency power Doppler (PDS) and to quantify treatment-induced changes in an experimental autochthonous mammary tumour model in rats. A total of 13 rats with N-methyl, N-nitroso urea-induced mammary tumours were split into three courses; 6 rats were treated with epirubicin, 3 received a placebo injection and 4 had irradiation of their tumour with a direct electron beam using a single dose of 18 Gy. In all groups, treatment began when the tumour area reached at least 1 cm(2) and was preceded by the first power Doppler sonography study of the tumour (Echo #1). Echo #2 was carried out in the middle of the placebo or epirubicin treatment (after 3 weeks) or 7 days after irradiation in the irradiated group. Echo #3 was carried out at the end of placebo or epirubicin treatment or 28 days after irradiation. Then colour pixel density (CPD) and vascularity index (VI) were quantitated. Intraobserver and interobserver variability of the CPD and VI quantitation was low (r = 0.99 and 0.97, respectively, for intraobserver and interobserver variability of the CPD values). The monitoring of CPD and VI showed an increase with time during the observation period. No increase in CPD or VI was observed in treated mammary tumours (p < 0.01). Power Doppler sonography quantitation of angiogenesis is reproducible, noninvasive and feasible in this in vivo breast cancer model. The monitoring of angiogenesis according to different treatments is feasible in real-time. Further studies are needed to investigate the predictive value of CPD and VI on sensitivity of mammary tumours to anticancer treatment.

A new platelet cryoprecipitate glue promoting bone formation after ectopic mesenchymal stromal cell-loaded biomaterial implantation in nude mice.

INTRODUCTION: This study investigated the promising effect of a new Platelet Glue obtained from Cryoprecipitation of Apheresis Platelet products (PGCAP) used in combination with Mesenchymal Stromal Cells (MSC) loaded on ceramic biomaterials to provide novel strategies enhancing bone repair. METHODS: PGCAP growth factor content was analyzed by ELISA and compared to other platelet and plasma-derived products. MSC loaded on biomaterials (65% hydroxyapatite/35% beta-TCP or 100% beta-TCP) were embedded in PGCAP and grown in presence or not of osteogenic induction medium for 21 days. Biomaterials were then implanted subcutaneously in immunodeficient mice for 28 days. Effect of PGCAP on MSC was evaluated in vitro by proliferation and osteoblastic gene expression analysis and in vivo by histology and immunohistochemistry. RESULTS: We showed that PGCAP, compared to other platelet-derived products, allowed concentrating large amount of growth factors and cytokines which promoted MSC and osteoprogenitor proliferation. Next, we found that PGCAP improves the proliferation of MSC and osteogenic-induced MSC. Furthermore, we demonstrated that PGCAP up-regulates the mRNA expression of osteogenic markers (Collagen type I, Osteonectin, Osteopontin and Runx2). In vivo, type I collagen expressed in ectopic bone-like tissue was highly enhanced in biomaterials embedded in PGCAP in the absence of osteogenic pre-induction. Better results were obtained with 65% hydroxyapatite/35% beta-TCP biomaterials as compared to 100% beta-TCP. CONCLUSIONS: We have demonstrated that PGCAP is able to enhance in vitro MSC proliferation, osteoblastic differentiation and in vivo bone formation in the absence of osteogenic pre-induction. This clinically adaptable platelet glue could be of interest for improving bone repair.

Repeated autologous bone marrow-derived mesenchymal stem cell injections improve radiation-induced proctitis in pigs.

The management of proctitis in patients who have undergone very-high-dose conformal radiotherapy is extremely challenging. The fibrosis-necrosis, fistulae, and hemorrhage induced by pelvic overirradiation have an impact on morbidity. Augmenting tissue repair by the use of mesenchymal stem cells (MSCs) may be an important advance in treating radiation-induced toxicity. Using a preclinical pig model, we investigated the effect of autologous bone marrow-derived MSCs on high-dose radiation-induced proctitis. Irradiated pigs received repeated intravenous administrations of autologous bone marrow-derived MSCs. Immunostaining and real-time polymerase chain reaction analysis were used to assess the MSCs' effect on inflammation, extracellular matrix remodeling, and angiogenesis, in radiation-induced anorectal and colon damages. In humans, as in pigs, rectal overexposure induces mucosal damage (crypt depletion, macrophage infiltration, and fibrosis). In a pig model, repeated administrations of MSCs controlled systemic inflammation, reduced in situ both expression of inflammatory cytokines and macrophage recruitment, and augmented interleukin-10 expression in rectal mucosa. MSC injections limited radiation-induced fibrosis by reducing collagen deposition and expression of col1a2/col3a1 and transforming growth factor-ß/connective tissue growth factor, and by modifying the matrix metalloproteinase/TIMP balance. In a pig model of proctitis, repeated injections of MSCs effectively reduced inflammation and fibrosis. This treatment represents a promising therapy for radiation-induced severe rectal damage.

Phenotypic study of human gingival fibroblasts in a medium enriched with platelet lysate.

BACKGROUND: The modulation abilities of gingival fibroblasts open new therapeutic strategies for the treatment of vascular diseases (e.g., aneurism) and irradiation burns. Culture media are classically supplemented with animal sera to provide nutriments. Unfortunately, because of their potential for interspecies transmission of microorganisms, these media are not used for cells destined for human transplantation. This preliminary phenotypic study aims to test a serum-free (SF) culture medium for human gingival fibroblasts (hGF) supplemented with human platelet lysates (PLs) for rapid cell expansion. METHODS: An SF medium was first elaborated to compete with hGF proliferation in a reference medium containing 10% fetal bovine serum (BSmedium). Adhesion, proliferation, and doubling kinetics were run in the presence of PLs (SF+PL). Cytoskeletal proteins were analyzed and chromosomal abnormalities were evaluated by karyotype analyses. The SF+PL influence on secretion of molecules implied in tissue remodeling (i.e., matrix metalloproteinases [MMPs], their tissue inhibitors [TIMPs], and several growth factors) was studied. RESULTS: SF+PL increased the proliferation rate 1.5-fold in a week compared to BSmedium. Cytoskeleton protein expression was similar in BSmedium and in SF+PL. Chromosomal abnormalities were rare in SF+PL. MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, TIMP-1, and the growth factors interleukin-1ß and -4 and transforming growth factor-ß1 secretions were stable during the experiment. TIMP-2 and interleukin-6 were slightly decreased in SF+PL compared to BSmedium. CONCLUSION: While waiting confirmation from a proteomic approach, this SF culture medium could allow a secured faster hGF proliferation adapted for human cell transplant therapy.

New emerging concepts in the medical management of local radiation injury.

Treatment of severe radiation burns remains a difficult medical challenge. The response of the skin to ionizing radiation results in a range of clinical manifestations. The most severe manifestations are highly invalidating. Although several therapeutic strategies (excision, skin grafting, skin or muscle flaps) have been used with some success, none have proven entirely satisfying. The concept that stem cell injections could be used for reducing normal tissue injury has been discussed for a number of years. Mesenchymal stem cells therapy may be a promising therapeutic approach for improving radiation-induced skin and muscle damages. Pre-clinical and clinical benefit of mesenchymal stem cell injection for ulcerated skin and muscle restoration after high dose radiation exposure has been successfully demonstrated. Three first patients suffering from severe radiological syndrome were successfully treated in France based on autologous human grade mesenchymal stem cell injection combined to plastic surgery or skin graft. Stem cell therapy has to be improved to the point that hospitals can put safe, efficient, and reliable clinical protocols into practice.

Correlation between plasma Flt3-ligand concentration and hematopoiesis during G-CSF-induced CD34+ cell mobilization.

This study aimed to correlate blood Flt3-ligand (FL) concentration with CD34(+) cell number in blood and bone marrow (BM) during granulocyte colony-stimulating factor (G-CSF) mobilization. Nonhuman primates were injected with 10 microg/kg of G-CSF (Lenograstim) daily over a period of 5 days. Daily blood sampling and repeated BM sampling showed that FL concentration before mobilization was negatively correlated to the absolute number of BM CD34(+) cells, but also to the number of G-CSF-mobilized CD34(+) cells on days 3-5 of treatment. This showed that FL concentration in the blood reflected BM status before mobilization, and suggested that this parameter could be used as a predictive indicator of G-CSF-induced CD34(+) cell mobilization.

Enhanced radiosensitivity of rat autochthonous mammary tumors by dietary docosahexaenoic acid.

Dietary docosahexaenoic acid (DHA), which integrates into tumor cell membranes, has been reported to enhance the efficacy against tumors of cytotoxic drugs that induce reactive oxygen species (ROS). Because ionizing radiation also generate ROS, we initiated a study to determine whether dietary DHA might sensitize mammary tumors to irradiation. Mammary tumors were induced by N-methylnitrosourea (NMU) in Sprague-Dawley rats. The optimal dose of radiation to examine the effect of DHA on tumor response to irradiation was determined to be 18 grays (Gy) using a 4-6 MeV electron beam (according to the depth of the target volume) delivered in a single fraction from a linear accelerator. Two groups of rats were fed a basal diet containing 7% of a mixture of peanut and rapeseed oils enriched with 8% of an oil containing either a low (palm oil) or high (DHASCO oil containing 40% DHA) DHA content. DHA group was equally subdivided into 2 groups without or with addition of vitamin E (100 IU/kg diet). Irradiation was carried out when the first tumor in each rat reached 1.5 cm2 and subsequent change in tumor size was documented over time. DHA level in adipose tissue, taken as a biomarker, was higher in the DHA supplemented group compared to the control group. Vitamin E level in liver, the best storage for this compound, was higher in the vitamin E supplemented DHA group compared to the DHA group. Tumor size decreased by 60% at 12 days after irradiation in the DHA group vs. 31% in the control group (p = 0.03) and 36% in the DHA plus vitamin E group. Therefore, dietary DHA sensitized mammary tumors to radiation. The addition of vitamin E inhibited the beneficial effect of DHA, suggesting that this effect might be mediated by oxidative damage to the peroxidizable lipids.