A human postnatal lymphoid progenitor capable of circulating and seeding the thymus

Identification of a thymus-seeding progenitor originating from human bone marrow (BM) constitutes a key milestone in understanding the mechanisms of T cell development and provides new potential for correcting T cell deficiencies. We report the characterization of a novel lymphoid-restricted subset, which is part of the lineage-negative CD34+CD10+ progenitor population and which is distinct from B cell–committed precursors (in view of the absence of CD24 expression). We demonstrate that these Lin−CD34+CD10+CD24− progenitors have a very low myeloid potential but can generate B, T, and natural killer lymphocytes and coexpress recombination activating gene 1, terminal deoxynucleotide transferase, PAX5, interleukin 7 receptor α, and CD3ε. These progenitors are present in the cord blood and in the BM but can also be found in the blood throughout life. Moreover, they belong to the most immature thymocyte population. Collectively, these findings unravel the existence of a postnatal lymphoid-polarized population that is capable of migrating from the BM to the thymus

A method to sequence and quantify DNA integration for monitoring outcome in gene therapy

Human genetic diseases have been successfully corrected by integration of functional copies of the defective genes into human cells, but in some cases integration of therapeutic vectors has activated proto-oncogenes and contributed to leukemia. For this reason, extensive efforts have focused on analyzing integration site populations from patient samples, but the most commonly used methods for recovering newly integrated DNA suffer from severe recovery biases. Here, we show that a new method based on phage Mu transposition in vitro allows convenient and consistent recovery of integration site sequences in a form that can be analyzed directly using DNA barcoding and pyrosequencing. The method also allows simple estimation of the relative abundance of gene-modified cells from human gene therapy subjects, which has previously been lacking but is crucial for detecting expansion of cell clones that may be a prelude to adverse events

DNA bar coding and pyrosequencing to analyze adverse events in therapeutic gene transfer

Gene transfer has been used to correct inherited immunodeficiencies, but in several patients integration of therapeutic retroviral vectors activated proto-oncogenes and caused leukemia. Here, we describe improved methods for characterizing integration site populations from gene transfer studies using DNA bar coding and pyrosequencing. We characterized 160 232 integration site sequences in 28 tissue samples from eight mice, where Rag1 or Artemis deficiencies were corrected by introducing the missing gene with gamma-retroviral or lentiviral vectors. The integration sites were characterized for their genomic distributions, including proximity to proto-oncogenes. Several mice harbored abnormal lymphoproliferations following therapy—in these cases, comparison of the location and frequency of isolation of integration sites across multiple tissues helped clarify the contribution of specific proviruses to the adverse events. We also took advantage of the large number of pyrosequencing reads to show that recovery of integration sites can be highly biased by the use of restriction enzyme cleavage of genomic DNA, which is a limitation in all widely used methods, but describe improved approaches that take advantage of the power of pyrosequencing to overcome this problem. The methods described here should allow integration site populations from human gene therapy to be deeply characterized with spatial and temporal resolution

Risk Factor Analysis of Outcomes after Unrelated Cord Blood Transplantation in Patients with Hurler Syndrome

AbstractAllogeneic stem cell transplantation (SCT) is considered effective in preventing disease progression in patients with Hurler syndrome (HS). Unrelated umbilical cord blood (UCB) grafts are suggested as an alternative to bone marrow (BM) or peripheral blood stem cells (PBSC). We studied 93 HS patients receiving an UCB graft to analyze risk factors for outcomes. The median time from diagnosis to transplant was 4.6 months, median follow-up was 29 months, and median number of nucleated CB cells infused was 7.6 × 107/kg. Most of the patients received 1 or 2 HLA disparate grafts, and the most frequently used conditioning regimen was cyclophosphamide + busulfan (Bu/Cy). All patients received anti-T cell antibody. At post transplant day +60, the cumulative incidence of neutrophil engraftment was 85%. A younger age at transplant and a higher CD34+ dose at infusion were favorably associated with engraftment. With the exception of 2 patients, all engrafted patients achieved full and sustained donor chimerism. The 3-year event-free survival (EFS) and 3-year overall survival (OS) rates were 70% and 77%, respectively. In a multivariate analyses, use of Bu/Cy and a shorter interval from diagnosis to transplant were predictors for improved EFS rate (82% for patients transplanted within 4.6 months after diagnosis compared to 57% for the rest). Improved outcomes from early transplantation and immediate availability of CB unit lead us to conclude that CB transplantation is a beneficial option, which should be considered expediently for children with HS

Real-Time Definition of Non-Randomness in the Distribution of Genomic Events

Features such as mutations or structural characteristics can be non-randomly or non-uniformly distributed within a genome. So far, computer simulations were required for statistical inferences on the distribution of sequence motifs. Here, we show that these analyses are possible using an analytical, mathematical approach. For the assessment of non-randomness, our calculations only require information including genome size, number of (sampled) sequence motifs and distance parameters. We have developed computer programs evaluating our analytical formulas for the real-time determination of expected values and p-values. This approach permits a flexible cluster definition that can be applied to most effectively identify non-random or non-uniform sequence motif distribution. As an example, we show the effectivity and reliability of our mathematical approach in clinical retroviral vector integration site distribution

Approche de thérapie cellulaire pour maladies du sytème hématopoietique en utilisant des cellules souches foetales isolées à partir de liquide amniotique

In the present study we investigated the possibility of differentiating AFS cells towards the hematopoietic pathway. We achieved a reproducible erythroid differentiation by culturing hAFSCs as embryoid bodies (EBs) under serum free conditions with haematopoietic cytokines. Furthermore, human erythrocytes (human CD235a) were isolated from bone marrow and spleen of sublethally irradiated NOD/SCID mice at 3 months after the injection of hAFSCs. We compared the hematopoietic potential of mAFKL and mAmKL to Fetal Liver KL, the main source of fetal HSC. When cultivated immediatly after their sorting, freshly isolated murine AFKL and AmKL cells gave rise to all the different hematopoietic lineages both in vitro and in vivo. Experiments with freshly isolated hAFKL gave good results in the in vitro assays being able to give rise to erythroid, myeloid and lymphoid lineages, but failed to reconstitute the hematopoietic system in irradiated NOD/SCID mice, probably due to the poor amount of cells injected. This is the first report demonstrating that AFKL and AmKL do have an haematopoietic potential, supporting the idea that AF and Am may be an excellent source for therapeutic application.Dans la présente étude, nous avons étudié la possibilité de différencier les cellules souches du liquide amniotique humain (hAFSC) et murin (mAFSC) vers la voie hématopoïétique à la fois in vitro et in vivo. Nous avons de manière reproductible réalisé une différenciation érythroïde par culture des hAFSCs en corps embryoïdes (EB). Plus de 70% des cellules constituant les EB coexprimaient des marqueurs erythroïdes. De plus, 3 mois après l'injection de hAFSC à des souris NOD/SCID irradiées, nous avons pu détecter dans la rate et la moelle osseuse des receveurs des érythrocytes humains. Nous avons ainsi comparé le potentiel hématopoïétique des mAFKL, des mAmKL aux KL issues du foie foetal (mFLKL), qui constituent la source principale de cellules souches hématopoïétiques à ce stade de développement. In vivo, nous avons retrouvé dans le sang de souris déficientes pour RAG1, des cellules appartenant aux trois lignées hématopoïétiques (lymphoïde, erythroïdes et myéloïdes) 4 semaines seulement après la greffe de mAFKL et mAmKL. Analysées quatre mois plus tard, les souris greffées présentent des lymphocytes, des érythrocytes et des cellules myéloïdes provenant des mAFKL et mAmKL dans tous les organes hématopoïétiques. Le succès de transplantations secondaires a confirmé que les mAFKL et mAmKL comprennent des progéniteurs hématopoïétiques capables d'auto-renouvellement, ce qui correspond à la définition d'une cellule souche hématopoïétique.PARIS5-BU Méd.Cochin (751142101) / SudocSudocFranceF

Integration of retroviruses: a fine balance between efficiency and danger.

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