T Lymphocyte Potential Marks the Emergence of Definitive Hematopoietic Progenitors in Human Pluripotent Stem Cell Differentiation Cultures

SummaryThe efficient generation of hematopoietic stem cells from human pluripotent stem cells is dependent on the appropriate specification of the definitive hematopoietic program during differentiation. In this study, we used T lymphocyte potential to track the onset of definitive hematopoiesis from human embryonic and induced pluripotent stem cells differentiated with specific morphogens in serum- and stromal-free cultures. We show that this program develops from a progenitor population with characteristics of hemogenic endothelium, including the expression of CD34, VE-cadherin, GATA2, LMO2, and RUNX1. Along with T cells, these progenitors display the capacity to generate myeloid and erythroid cells. Manipulation of Activin/Nodal signaling during early stages of differentiation revealed that development of the definitive hematopoietic progenitor population is not dependent on this pathway, distinguishing it from primitive hematopoiesis. Collectively, these findings demonstrate that it is possible to generate T lymphoid progenitors from pluripotent stem cells and that this lineage develops from a population whose emergence marks the onset of human definitive hematopoiesis

Primitive Erythropoiesis Is Regulated by miR-126 via Nonhematopoietic Vcam-1+ Cells

SummaryPrimitive erythropoiesis defines the onset of hematopoiesis in the yolk sac of the early embryo and is initiated by the emergence of progenitors assayed as colony-forming cells (EryP-CFCs). EryP-CFCs are detected for only a narrow window during embryonic development, suggesting that both their initiation and termination are tightly controlled. Using the embryonic stem differentiation system to model primitive erythropoiesis, we found that miR-126 regulates the termination of EryP-CFC development. Analyses of miR-126 null embryos revealed that this miR also regulates EryP-CFCs in vivo. We identified vascular cell adhesion molecule-1 (Vcam-1) expressed by a mesenchymal cell population as a relevant target of miR-126. Interaction of EryP-CFCs with Vcam-1 accelerated their maturation to ßh1-globin+ and Ter119+ cells through a Src family kinase. These findings uncover a cell nonautonomous regulatory pathway for primitive erythropoiesis that may provide insight into the mechanism(s) controlling the developmental switch from primitive to definitive hematopoiesis

Clonal Characterization of Rat Muscle Satellite Cells: Proliferation, Metabolism and Differentiation Define an Intrinsic Heterogeneity

Satellite cells (SCs) represent a distinct lineage of myogenic progenitors responsible for the postnatal growth, repair and maintenance of skeletal muscle. Distinguished on the basis of their unique position in mature skeletal muscle, SCs were considered unipotent stem cells with the ability of generating a unique specialized phenotype. Subsequently, it was demonstrated in mice that opposite differentiation towards osteogenic and adipogenic pathways was also possible. Even though the pool of SCs is accepted as the major, and possibly the only, source of myonuclei in postnatal muscle, it is likely that SCs are not all multipotent stem cells and evidences for diversities within the myogenic compartment have been described both in vitro and in vivo. Here, by isolating single fibers from rat flexor digitorum brevis (FDB) muscle we were able to identify and clonally characterize two main subpopulations of SCs: the low proliferative clones (LPC) present in major proportion (∼75%) and the high proliferative clones (HPC), present instead in minor amount (∼25%). LPC spontaneously generate myotubes whilst HPC differentiate into adipocytes even though they may skip the adipogenic program if co-cultured with LPC. LPC and HPC differ also for mitochondrial membrane potential (ΔΨm), ATP balance and Reactive Oxygen Species (ROS) generation underlying diversities in metabolism that precede differentiation. Notably, SCs heterogeneity is retained in vivo. SCs may therefore be comprised of two distinct, though not irreversibly committed, populations of cells distinguishable for prominent differences in basal biological features such as proliferation, metabolism and differentiation. By these means, novel insights on SCs heterogeneity are provided and evidences for biological readouts potentially relevant for diagnostic purposes described

Approccio di terapia cellulare mediante l'utilizzo di cellule fetali isolate dal liquido amniotico per malattie del sistema ematopoieico

Cell therapy is an attractive perspective for the treatment of life threatening disorders. In this context, foetal tissues are gaining interest as sources of cells for auto- and allo-transplantation, because of their pluripotency, proliferative capability and their low, if any, immunogenicity. Recently a pluripotent stem cell population has been isolated from Amniotic Fluid (AF). It is able to proliferate for more than 18 months, maintaining their differentiative ability as well as a normal karyotype. In term of differentiation potential, we succeeded in obtaining in vitro mesenchymal-, ectodermal- and endodermal-derived tissues from human Amniotic Fluid Stem (AFS) cells. Furthermore, murine AFS cells injected in blastocytes took part to the formation not only of several different foetal organs, but also of the placenta and the umbilical cord. In the present study we investigated the possibility of differentiating AFS cells towards the hematopoietic pathway. AFS cells isolated from human amniotic fluid, collected during routine diagnostic procedures and obtained under informed consent, were firstly expanded in vitro and selected on the basis of their ckit expression. We achieved a reproducible erythroid differentiation by culturing hAFSCs as embryoid bodies (EBs) under serum free conditions with haematopoietic cytokines. Erythroid cells expressing CD235a constituted 70% of the total hAFSCs forming EBs showing also a co-expression of CD36 and CD71. 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. To determine if the expansion procedure had led to a restriction of the hematopoietic potential towards the erythroid pathway, we compared expanded AFSCs and freshly isolated cKit+ Lin- (AFKL) cells. We also harvested cKit+ Lin- KL cells from the membrane surrounding the AF, the Amnion, in search for a possible origin. 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. Actually, when cocultivated with OP9(d)1 cells, AFKL and AmKL undergo complete T cell differentiation within 2 weeks. They also generate myeloid and erythroid colonies when cultivated in methylcellulose for clonogenic assay. The erythroid restricted potential of human AFS cells was thus probably linked to the in vitro expansion procedure. Moreover, cells belonging to all the three hematopoietic lineages (lymphoid, myeloid and erythroid) and arising from freshly isolated mAFKL and mAmKL are found in the peripheral blood of sublethally irradiated RAG1 deficient mice only 4 weeks after transplantation. Four month later, transplanted mice showed mAFKL-derived lymphoid, myeloid and erythroid cells, in all the hematopoietic organs. Successful econdary transplantation strongly suggest that mAFKL and mAmKL comprise HSC, with self-renewal ability. Those results were very similar to those obtained with mFLKL, confirming the strong hematopoietic potential of mAFKL and mAmKL. 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

Let Me Speak! A Reviewers’ Guide to Writing a Successful Meeting Abstract

Being able to summarize properly your work is not an easy task. But learning the skill of writing a good abstract is very important, as it can open many doors, including the possibility to be selected as a speaker at conferences. As meeting abstract reviewers, here we are writing to give you insights into the abstract review process and insiders' tips to help increase your chances of landing on that podium. : Learning how to write a great abstract is essential to one’s career and key to being selected as an abstract-selected speaker. As meeting abstract reviewers, we are writing to give you key insights into the abstract review process and tips to help increase your chances of landing on that podium

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

Systemic delivery of therapeutic nucleic acids into skeletal muscle by means of lipid-based vectors

Introduction : Lipid-based vectors are considered a promising tool when a systemic delivery is desirable. Although not as efficient as viral vectors, lipoplexes present some major advantages, as they are non immunogenic, non mutagenic, as well as easy and cheap to prepare. We had previously shown that skeletal muscles are poorly accessible to lipoplexes via intra-venous administration, whereas local intra-arterial delivery yielded good preliminary results. Objectives : Optimization of a lipid-based, systemic gene delivery system capable of introducing therapeutic nucleic acids into skeletal muscle. Methods : We use lipid-based vehicles containing the cationic lipid DODAC, formulated either as conventional or encapsulated PEG-ylated lipoplexes. Results : We injected lipoplexes in the femoral artery of adult rats in which muscle regeneration was chemically induced in the TA muscles. Using an encapsulated formulation (SPLP, developed by Protiva Bioteherapeutics) we obtained up to 10% of GFP+ve fibers after a single injection. High transfection levels were maintained for at least three weeks. At the same time, we showed that SPLPs only induced a slight delay in the process of muscle regeneration and did not lead to any germ-line transmission of the transgene. More recently we began to apply our delivery protocol to the development of an exon-skipping approach for the correction of mutations in the dystrophin gene. In particular, we are using DODACbased lipoplexes to deliver an exon 23-specific morpholino AO into the muscles of mdx mice. Our initial data showed the production of a significant amount (i.e., as abundant as the wild type or higher) of the desired skipped mRNA in the leg muscle groups of the injected limbs. The effect of the treatment on dystrophin production is presently being assessed. Conclusions : Our data indicate that both encapsulated and conventional lipoplexes have the potential to be used for the development of therapeutic protocols for muscle diseases

Regulatory mutations in transforming growth factor-beta3 gene cause arrhythmogenic right ventricular cardiomyopathy type 1.

OBJECTIVE: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a genetically heterogeneous disorder characterized by fibro-fatty replacement of the right ventricular myocardium, associated with high risk of sudden death. The objective of this study is to identify the gene involved in ARVD1, which has been elusive ever since its locus was mapped to chromosome 14q24.3. METHODS AND RESULTS: Mutation screening of the promoter and untranslated regions (UTRs) of the transforming growth factor-beta3 (TGFbeta3) gene was performed by direct sequencing of genomic DNA of one index case belonging to an ARVD1 family including 38 members in four generations. We detected a nucleotide substitution (c.-36G>A) in 5' UTR of TGFbeta3 gene, invariably associated with the typical ARVC clinical phenotype in the affected family members, according to the established diagnostic criteria. Investigation extended to 30 unrelated ARVC patients, performed by denaturing high-performance liquid chromatography (DHPLC), led to the identification of an additional mutation (c.1723C>T) in the 3' UTR of one proband. Neither nucleotide change was found in 300 control subjects. In vitro expression assays with constructs containing the mutations showed that mutated UTRs were twofold more active than wild-types. CONCLUSION: We identified TGFbeta3 as the disease gene involved in ARVD1. The identification of a novel ARVC gene will increase the power of the genetic screening for early diagnosis of asymptomatic carriers among relatives of ARVC patients