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In pathologies of the esophagus such as esophageal atresia, cancers, and caustic injuries, methods for full thickness esophageal replacement require the sacrifice of healthy intra-abdominal organs such as the stomach and the colon and are associated with high morbidity, mortality, and poor functional results. To overcome these problems, tissue engineering methods are developed to create a substitute with scaffolds and cells. The aim of this study was to develop a simple and safe decellularization process in order to obtain a clinical grade esophageal extracellular matrix. Following the decontamination step, porcine esophagi were decellularized in a bioreactor with sodium dodecyl sulfate and ethylenediaminetetraacetic acid for 3 days and were rinsed with deionized water. DNA was eliminated by a 3-hr DNase treatment. To remove any residual detergent, the matrix was then incubated with an absorbing resin. The resulting porcine esophageal matrix was characterized by the assessment of the efficiency of the decellularization process (DNA quantification), evaluation of sterility and absence of cytotoxicity, and its composition and biomechanical properties, as well as the possibility to be reseeded with mesenchymal stem cells. Complete decellularization with the preservation of the general structure, composition, and biomechanical properties of the native esophageal matrix was obtained. Sterility was maintained throughout the process, and the matrix showed no cytotoxicity. The resulting matrix met clinical grade criteria and was successfully reseeded with mesenchymal stem cells.., (© 2019 John Wiley & Sons, Ltd.)

Natural regulatory T cells (nTregs) ensure the control of self-tolerance and are currently used in clinical trials to alleviate autoimmune diseases and graft-versus-host disease after hematopoietic stem cell transfer. Based on CD39/CD26 markers, blood nTreg analysis revealed the presence of five different cell subsets, each representing a distinct stage of maturation. Ex vivo added microenvironmental factors, including IL-2, TGFβ, and PGE2, direct the conversion from naive precursor to immature memory and finally from immature to mature memory cells, the latest being a no-return stage. Phenotypic and genetic characteristics of the subsets illustrate the structural parental maturation between subsets, which further correlates with the expression of regulatory factors. Regarding nTreg functional plasticity, both maturation stage and microenvironmental cytokines condition nTreg activities, which include blockade of autoreactive immune cells by cell-cell contact, Th17 and IL-10 Tr1-like activities, or activation of TCR-stimulating dendritic cell tolerization. Importantly, blood nTreg CD39/CD26 profile remained constant over a 2-y period in healthy persons but varied from person to person. Preliminary data on patients with autoimmune diseases or acute myelogenous leukemia illustrate the potential use of the nTreg CD39/CD26 profile as a blood biomarker to monitor chronic inflammatory diseases. Finally, we confirmed that naive conventional CD4 T cells, TCR-stimulated under a tolerogenic conditioned medium, could be ex vivo reprogrammed to FOXP3 lineage Tregs, and further found that these cells were exclusively committed to suppressive function under all microenvironmental contexts., Competing Interests: Conflict of interest statement: D.Z. is a cofounder of Neovacs, and R.C. is an employee of eBioscience., (Copyright © 2019 the Author(s). Published by PNAS.)

Thromboembolic events are the main cause of mortality in BCR-ABL1-negative myeloproliferative neoplasms (MPNs) but their underlying mechanisms are largely unrecognized. The Janus kinase 2 (JAK2) V617F mutation is the most frequent genetic alteration leading to MPN. Usually found in haematopoietic progenitors and stem cells, this mutation has also been described in endothelial cells (ECs) of MPN patients. In this study, we have questioned the impact of the JAK2 V617F mutation on EC phenotype and functions. We developed an induced pluripotent stem cells strategy to compare JAK2 mutant and wild-type ECs. Transcriptomic assays showed that several genes and pathways involved in inflammation, cell adhesion and thrombotic events were over-represented in JAK2 V617F ECs and expression levels of von Willebrand factor and P-selectin (CD62P) proteins were increased. Finally, we found that leucocytes from MPN patients adhere more tightly to JAK2 V617F ECs. Our results show that JAK2 V617F ECs have a pro-inflammatory and pro-thrombotic phenotype and were functionally pro-adherent., Competing Interests: None., (Georg Thieme Verlag KG Stuttgart · New York.)

The beneficial effect of autologous peripheral blood stem cell transplantation (APBSCT) may be compromised by acute vascular complications related to hypercoagulability. We studied the impact of graft product on thrombin generation of normal plasma and the expression of tissue factor (TF) and procoagulant platelet-derived procoagulant microparticles (Pd-MPs) in samples of graft products. Graft products from 10 patients eligible for APBSCT were mixed with platelet-poor plasma (PPP) or platelet-rich plasma (PRP) from healthy volunteers and assessed for in vitro thrombin generation. In control experiments, thrombin generation was assessed in (1) PPP and PRP without any exogenous TF and/or procoagulant phospholipids, (2) PPP with the addition of TF (5 pM) and procoagulant phospholipids (4 μM), (3) in PRP with the addition of TF (5 pM). Graft products were assessed with Western blot assay for TF expression, with a specific clotting assay for TF activity and with flow cytometry assay for Pd-MPs. The graft product enhanced thrombin generation and its procoagulant activity was related to the presence of Pd-MPs and TF. The concentration of Pd-MPs in the graft product was characterized by a significant interindividual variability. The present study reveals the need for a thorough quality control of the graft products regarding their procoagulant potential.

Distal lung diseases, such as pulmonary fibrosis or acute lung injury, are commonly associated with local alveolar hypoxia that may be deleterious through the stimulation of alveolar epithelial cell (AEC) apoptosis. In various murine models of alveolar injury, administration of allogenic human mesenchymal stem cells (hMSCs) exerts an overall protective paracrine effect, limiting lung inflammation and fibrosis. However, the precise mechanisms on lung cells themselves remain poorly understood. Here, we investigated whether hMSC-conditioned medium (hMSC-CM) would protect AECs from hypoxia-induced apoptosis and explored the mechanisms involved in this cytoprotective effect. Exposure of rat primary AECs to hypoxia (1.5% O 2 for 24 h) resulted in hypoxia-inducible factor (HIF)-1α protein stabilization, partly dependent on reactive oxygen species (ROS) accumulation, and in a twofold increase in AEC apoptosis that was prevented by the HIF inhibitor 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl-indazole and the antioxidant drug N-acetyl cysteine. Incubation of AECs with hMSC-CM significantly reduced hypoxia-induced apoptosis. hMSC-CM decreased HIF-1α protein expression, as well as ROS accumulation through an increase in antioxidant enzyme activities. Expression of Bnip3 and CHOP, two proapoptotic targets of HIF-1α and ROS pathways, respectively, was suppressed by hMSC-CM, while Bcl-2 expression was restored. The paracrine protective effect of hMSC was partly dependent on keratinocyte growth factor and hepatocyte growth factor secretion, preventing ROS and HIF-1α accumulation.

After 30 years of hematopoietic stem cell use for various indications, umbilical cord blood is considered as an established source of cells with marrow and postmobilization peripheral blood. The limited number of cells still remains a problematic element restricting their use, especially in adults who require to be grafted with a higher cell number. Improving the quality of harvested cord blood, at least in terms of volume and amount of cells, is essential to decrease the number of discarded units. In this review, we examine several variables related to parturient, pregnancy, labor, delivery, collection, the newborn, umbilical cord, and placenta. We aim to understand the biologic mechanisms that can impact cord blood quality. This knowledge will ultimately allow targeting donors, which could provide a rich graft and improve the efficiency of the collection., (© 2017 AABB.)

Background: In addition to scalability, human embryonic stem cells (hESCs) have the unique advantage of allowing their directed differentiation toward lineage-specific cells., Objectives: This study tested the feasibility of leveraging the properties of hESCs to generate clinical-grade cardiovascular progenitor cells and assessed their safety in patients with severe ischemic left ventricular dysfunction., Methods: Six patients (median age 66.5 years [interquartile range (IQR): 60.5 to 74.7 years]; median left ventricular ejection fraction 26% [IQR: 22% to 32%]) received a median dose of 8.2 million (IQR: 5 to 10 million) hESC-derived cardiovascular progenitors embedded in a fibrin patch that was epicardially delivered during a coronary artery bypass procedure. The primary endpoint was safety at 1 year and focused on: 1) cardiac or off-target tumor, assessed by imaging (computed tomography and fluorine-18 fluorodeoxyglucose positron emission tomography scans); 2) arrhythmias, detected by serial interrogations of the cardioverter-defibrillators implanted in all patients; and 3) alloimmunization, assessed by the presence of donor-specific antibodies. Patients were followed up for a median of 18 months., Results: The protocol generated a highly purified (median 97.5% [IQR: 95.5% to 98.7%]) population of cardiovascular progenitors. One patient died early post-operatively from treatment-unrelated comorbidities. All others had uneventful recoveries. No tumor was detected during follow-up, and none of the patients presented with arrhythmias. Three patients developed clinically silent alloimmunization. All patients were symptomatically improved with an increased systolic motion of the cell-treated segments. One patient died of heart failure after 22 months., Conclusions: This trial demonstrates the technical feasibility of producing clinical-grade hESC-derived cardiovascular progenitors and supports their short- and medium-term safety, thereby setting the grounds for adequately powered efficacy studies. (Transplantation of Human Embryonic Stem Cell-derived Progenitors in Severe Heart Failure [ESCORT]; NCT02057900)., (Copyright © 2018 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Tissue engineering appears promising as an alternative technique for esophageal replacement. Mesenchymal stem cells (MSCs) could be of interest for esophageal regeneration. Evaluation of the ability of an acellular matrix seeded with autologous MSCs to promote tissue remodeling toward an esophageal phenotype after circumferential replacement of the esophagus in a mini pig model. A 3 cm long circumferential replacement of the abdominal esophagus was performed with an MSC-seeded matrix (MSC group, n = 10) versus a matrix alone (control group, n = 10), which has previously been matured into the great omentum. The graft area was covered with an esophageal removable stent. A comparative histological analysis of the graft area after animals were euthanized sequentially is the primary outcome of the study. Histological findings after maturation, overall animal survival, and postoperative morbidity were also compared between groups. At postoperative day 45 (POD 45), a mature squamous epithelium covering the entire surface of the graft area was observed in all the MSC group specimens but in none of the control group before POD 95. Starting at POD 45, desmin positive cells were seen in the graft area in the MSC group but never in the control group. There were no differences between groups in the incidence of surgical complications and postoperative death. In this model, MSCs accelerate the mature re-epitheliazation and early initiation of muscle cell colonization. Further studies will focus on the use of cell tracking tools in order to analyze the becoming of these cells and the mechanisms involved in this tissue regeneration.

Technical progress in materials science and bioprinting has for the past few decades fostered considerable advances in medicine. More recently, the understanding of the processes of self-organization of cells into three-dimensional multicellular structures and the study of organoids have opened new perspectives for tissue engineering. Here, we review microengineering approaches for building functional tissues, and discuss recent progress in the understanding of morphogenetic processes and in the ability to steer them in vitro. On the basis of biological and technical considerations, we emphasize the achievements and remaining challenges of bringing together microengineering and morphogenesis. Our viewpoint underlines the importance of cellular self-organization for the success of tissue engineering in therapeutic applications. We reason that directed self-organization, at the convergence of microengineering and cellular self-organization, is a promising direction for the manufacturing of complex functional tissues.

The classical model of hematopoiesis established in the mouse postulates that lymphoid cells originate from a founder population of common lymphoid progenitors. Here, using a modeling approach in humanized mice, we showed that human lymphoid development stemmed from distinct populations of CD127 - and CD127 + early lymphoid progenitors (ELPs). Combining molecular analyses with in vitro and in vivo functional assays, we demonstrated that CD127 - and CD127 + ELPs emerged independently from lympho-mono-dendritic progenitors, responded differently to Notch1 signals, underwent divergent modes of lineage restriction, and displayed both common and specific differentiation potentials. Whereas CD127 - ELPs comprised precursors of T cells, marginal zone B cells, and natural killer (NK) and innate lymphoid cells (ILCs), CD127 + ELPs supported production of all NK cell, ILC, and B cell populations but lacked T potential. On the basis of these results, we propose a "two-family" model of human lymphoid development that differs from the prevailing model of hematopoiesis., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Peripheral arterial disease results from the chronic obstruction of arteries leading to critical hindlimb ischemia. The aim was to develop a new therapeutic strategy of revascularization by using biodegradable and biocompatible polysaccharides-based microparticles (MP) to treat the mouse hindlimb ischemia. For this purpose, we deliver the pro-angiogenic chemokine Regulated upon Activation, Normal T-cell Expressed and Secreted (RANTES)/CCL5 in the mouse ischemic hindlimb, in solution or incorporated into polysaccharide-based microparticles. We demonstrate that RANTES-loaded microparticles improve the clinical score, induce the revascularization and the muscle regeneration in injured mice limb. To decipher the mechanisms underlying RANTES effects in vivo, we demonstrate that RANTES increases the spreading, the migration of human endothelial progenitor cells (EPC) and the formation of vascular network. The main receptors of RANTES i.e. CCR5, syndecan-4 and CD44 expressed at endothelial progenitor cell surface are involved in RANTES-induced in vitro biological effects on EPC. By using two RANTES mutants, [E66A]-RANTES with impaired ability to oligomerize, and [ 44 AANA 47 ]-RANTES mutated in the main RANTES-glycosaminoglycan binding site, we demonstrate that both chemokine oligomerization and binding site to glycosaminoglycans are essential for RANTES-induced angiogenesis in vitro. Herein we improved the muscle regeneration and revascularization after RANTES-loaded MP local injection in mice hindlimb ischemia.

Efforts to implement family cord blood banking have been developed in the past decades for siblings requiring stem cell transplantation for conditions such as sickle cell disease. However, public banks are faced with challenging decisions about the units to be stored, discarded, or used for other endeavors. We report here 20 years of experience in family cord blood banking for sickle cell disease in two dedicated public banks. Participants were pregnant women who had a previous child diagnosed with homozygous sickle cell disease. Participation was voluntary and free of charge. All mothers underwent mandatory serological screening. Cord blood units were collected in different hospitals, but processed and stored in two public banks. A total of 338 units were stored for 302 families. Median recipient age was six years (11 months-15 years). Median collected volume and total nucleated cell count were 91 mL (range 23-230) and 8.6×10 8 (range 0.7-75×10 8 ), respectively. Microbial contamination was observed in 3.5% (n=12), positive hepatitis B serology in 25% (n=84), and homozygous sickle cell disease in 11% (n=37) of the collections. Forty-four units were HLA-identical to the intended recipient, and 28 units were released for transplantation either alone (n=23) or in combination with the bone marrow from the same donor (n=5), reflecting a utilization rate of 8%. Engraftment rate was 96% with 100% survival. Family cord blood banking yields good quality units for sibling transplantation. More comprehensive banking based on close collaboration among banks, clinical and transplant teams is recommended to optimize the use of these units., (Copyright© Ferrata Storti Foundation.)

The capacity of hematopoietic stem cells (HSC) to generate B lymphocytes declines with age, contributing to impaired immune function in the elderly. Here we show that the histone methyltransferase SUV39H1 plays an important role in human B lymphoid differentiation and that expression of SUV39H1 decreases with age in both human and mouse HSC, leading to a global reduction in H3K9 trimethylation and perturbed heterochromatin function. Further, we demonstrate that SUV39H1 is a target of microRNA miR-125b, a known regulator of HSC function, and that expression of miR-125b increases with age in human HSC. Overexpression of miR-125b and inhibition of SUV39H1 in young HSC induced loss of B cell potential. Conversely, both inhibition of miR-125 and enforced expression of SUV39H1 improved the capacity of HSC from elderly individuals to generate B cells. Our findings highlight the importance of heterochromatin regulation in HSC aging and B lymphopoiesis., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Cell-based therapies are being explored as a therapeutic option for patients with chronic heart failure following myocardial infarction. Extracellular vesicles (EV), including exosomes and microparticles, secreted by transplanted cells may orchestrate their paracrine therapeutic effects. We assessed whether post-infarction administration of EV released by human embryonic stem cell-derived cardiovascular progenitors (hESC-Pg) can provide equivalent benefits to administered hESC-Pg and whether hESC-Pg and EV treatments activate similar endogenous pathways., Methods: Mice underwent surgical occlusion of their left coronary arteries. After 2-3 weeks, 95 mice included in the study were treated with hESC-Pg, EV, or Minimal Essential Medium Alpha Medium (alpha-MEM; vehicle control) delivered by percutaneous injections under echocardiographic guidance into the peri-infarct myocardium. functional and histologic end-points were blindly assessed 6 weeks later, and hearts were processed for gene profiling. Genes differentially expressed between control hearts and hESC-Pg-treated and EV-treated hearts were clustered into functionally relevant pathways., Results: At 6 weeks after hESC-Pg administration, treated mice had significantly reduced left ventricular end-systolic (-4.20 ± 0.96 µl or -7.5%, p = 0.0007) and end-diastolic (-4.48 ± 1.47 µl or -4.4%, p = 0.009) volumes compared with baseline values despite the absence of any transplanted hESC-Pg or human embryonic stem cell-derived cardiomyocytes in the treated mouse hearts. Equal benefits were seen with the injection of hESC-Pg-derived EV, whereas animals injected with alpha-MEM (vehicle control) did not improve significantly. Histologic examination suggested a slight reduction in infarct size in hESC-Pg-treated animals and EV-treated animals compared with alpha-MEM-treated control animals. In the hESC-Pg-treated and EV-treated groups, heart gene profiling identified 927 genes that were similarly upregulated compared with the control group. Among the 49 enriched pathways associated with these up-regulated genes that could be related to cardiac function or regeneration, 78% were predicted to improve cardiac function through increased cell survival and/or proliferation or DNA repair as well as pathways related to decreased fibrosis and heart failure., Conclusions: In this post-infarct heart failure model, either hESC-Pg or their secreted EV enhance recovery of cardiac function and similarly affect cardiac gene expression patterns that could be related to this recovery. Although the mechanisms by which EV improve cardiac function remain to be determined, these results support the idea that a paracrine mechanism is sufficient to effect functional recovery in cell-based therapies for post-infarction-related chronic heart failure., (Copyright © 2016 International Society for Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.)

The cornea provides two thirds of the refractive power of the eye and protection against insults such as infection and injury. The outermost tissue of the cornea is renewed by stem cells located in the limbus. Depletion or destruction of these stem cells may lead to blinding limbal stem cell deficiency (LSCD) that concerns millions of patients around the world. Innovative strategies based on adult stem cell therapies have been developed in the recent years but they are still facing numerous unresolved issues, and the long term results can be deceiving. Today there is a clear need to improve these therapies, and/or to develop new approaches for the treatment of LSCD. Here, we review the current cell-based therapies used for the treatment of ocular diseases, and discuss the potential of pluripotent stem cells (embryonic and induced pluripotent stem cells) in corneal repair. As the secretion of paracrine factors is known to have a crucial role in maintaining stem cell homeostasis and in wound repair, we also consider the therapeutic potential of a promising novel pathway, the exosomes. Exosomes are nano-sized vesicles that have the ability to transfer RNAs and proteins to recipient cells, and several studies demonstrated their role in cell protection and wound healing. Exosomes could circumvent the hurdles of stem-cell based approaches, and they could become a strong candidate as an alternative therapy for ocular surface diseases.

Thrombopoietin (TPO) and its receptor Mpl (CD110) play a crucial role in the regulation of hematopoietic stem cells (HSCs). Functional study of Mpl-expressing HSCs has, however, been hampered by the lack of efficient monoclonal antibodies, explaining the very few data available on Mpl(+) HSCs during human embryonic development and after birth. Investigating the main monoclonal antibodies used so far to sort CD110(+) cells from cord blood (CB) and adult bone marrow (BM), we found that only the recent monoclonal antibody 1.6.1 engineered by Immunex Corporation was specific. Using in vitro functional assays, we found that this antibody can be used to sort a CD34(+)CD38(-)CD110(+) population enriched in hematopoietic progenitor stem cells, both in CB and in adult BM. In vivo injection into NSG mice further indicated that the CB CD34(+)CD38(-)CD110(+) population is highly enriched in HSCs compared with both CD34(+)CD38(-)CD110(-) and CD34(+)CD38(-) populations. Together our results validate MAb1.6.1 as an important tool, which has so far been lacking, in the HSC field., (Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.)

Administration of bone marrow-derived human mesenchymal stem cells (hMSC) reduces lung inflammation, fibrosis, and mortality in animal models of lung injury, by a mechanism not completely understood. We investigated whether hMSC would prevent epithelial-mesenchymal transition (EMT) induced by hypoxia in primary rat alveolar epithelial cell (AEC). In AEC cultured on semipermeable filters, prolonged hypoxic exposure (1.5% O2 for up to 12 days) induced phenotypic changes consistent with EMT, i.e., a change in cell morphology, a decrease in transepithelial resistance (Rte) and in the expression of epithelial markers [zonula occludens-1 (ZO-1), E-cadherin, AQP-5, TTF-1], together with an increase in mesenchymal markers [vimentin, α-smooth muscle actin (α-SMA)]. Expression of transcription factors driving EMT such as SNAIL1, ZEB1, and TWIST1 increased after 2, 24, and 48 h of hypoxia, respectively. Hypoxia also induced TGF-β1 mRNA expression and the secretion of active TGF-β1 in apical medium, and the expression of connective tissue growth factor (CTGF), two inducers of EMT. Coculture of AEC with hMSC partially prevented the decrease in Rte and in ZO-1, E-cadherin, and TTF-1 expression, and the increase in vimentin expression induced by hypoxia. It also abolished the increase in TGF-β1 expression and in TGF-β1-induced genes ZEB1, TWIST1, and CTGF. Finally, incubation with human recombinant KGF at a concentration similar to what was measured in hMSC-conditioned media restored the expression of TTF-1 and prevented the increase in TWIST1, TGF-β1, and CTGF in hypoxic AEC. Our results indicate that hMSC prevent hypoxia-induced alveolar EMT through the paracrine modulation of EMT signaling pathways and suggest that this effect is partly mediated by KGF., (Copyright © 2016 the American Physiological Society.)

Limited data are available on the effects of stem cells in non-ischemic dilated cardiomyopathy (DCM). Since the diffuse nature of the disease calls for a broad distribution of cells, this study investigated the scaffold-based delivery of human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CM) in a mouse model of DCM. Nanofibrous scaffolds were produced using a clinical grade atelocollagen which was electrospun and cross-linked under different conditions. As assessed by scanning electron microscopy and shearwave elastography, the optimum crosslinking conditions for hiPS-CM colonization proved to be a 10% concentration of citric acid crosslinking agent and 150 min of post-electrospinning baking. Acellular collagen scaffolds were first implanted in both healthy mice and those with induced DCM by a cardiac-specific invalidation of serum response factor (SRF). Seven and fourteen days after implantation, the safety of the scaffold was demonstrated by echocardiography and histological assessments. The subsequent step of implantation of the scaffolds seeded with hiPS-CM in DCM induced mice, using cell-free scaffolds as controls, revealed that after fourteen days heart function decreased in controls while it remained stable in the treated mice. This pattern was associated with an increased number of endothelial cells, in line with the greater vascularity of the scaffold. Moreover, a lesser degree of fibrosis consistent with the upregulation of several genes involved in extracellular matrix remodeling was observed. These results support the interest of the proposed hiPS-CM seeded electrospun scaffold for the stabilization of the DCM outcome with potential for its clinical use in the future., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: In spite of the recent discovery of genetic mutations in most myelodysplasic (MDS) patients, the pathophysiology of these disorders still remains poorly understood, and only few in vivo models are available to help unravel the disease., Methods: We performed global specific gene expression profiling and functional pathway analysis in purified Sca1+ cells of two MDS transgenic mouse models that mimic human high-risk MDS (HR-MDS) and acute myeloid leukemia (AML) post MDS, with NRASD12 and BCL2 transgenes under the control of different promoters MRP8NRASD12/tethBCL-2 or MRP8[NRASD12/hBCL-2], respectively., Results: Analysis of dysregulated genes that were unique to the diseased HR-MDS and AML post MDS mice and not their founder mice pointed first to pathways that had previously been reported in MDS patients, including DNA replication/damage/repair, cell cycle, apoptosis, immune responses, and canonical Wnt pathways, further validating these models at the gene expression level. Interestingly, pathways not previously reported in MDS were discovered. These included dysregulated genes of noncanonical Wnt pathways and energy and lipid metabolisms. These dysregulated genes were not only confirmed in a different independent set of BM and spleen Sca1+ cells from the MDS mice but also in MDS CD34+ BM patient samples., Conclusions: These two MDS models may thus provide useful preclinical models to target pathways previously identified in MDS patients and to unravel novel pathways highlighted by this study.

Background: Expansion protocols aim at both increasing the number of umbilical cord blood (UCB) hematopoietic stem cells and progenitor cells (HSPCs) and reducing the period of neutropenia in UCB HSPC graft. Because glycosaminoglycans (GAGs) are known to be important components of the hematopoietic niche and to modulate growth factor effects, we explored the use of GAG mimetic OTR4131 to potentiate HSPC's in vitro expansion and in vivo engraftment., Methods: UCB CD34+ cells were expanded with serum-free medium, SCF, TPO, FLT3-lig and G-CSF during 12 days in the absence or the presence of increasing OTR4131 concentrations (0-100 μg/mL). Proliferation ratio, cell viability and phenotype, functional assays, migration capacity and NOD-scid/γc(-/-) mice engraftment were assessed after expansion., Results: At Day 12, ratios of cell expansion were not significantly increased by OTR4131 treatment. Better total nucleated cell viability was observed with the use of 1 μg/mL GAG mimetic compared to control (89.6 % ± 3.7 % and 79.9 % ± 3.3 %, respectively). Phenotype analysis showed a decrease of monocyte lineage in the presence of OTR4131 and HSPC migration capacity was diminished when GAG mimetic was used at 10 μg/mL (10.9 % ± 4.1 % vs. 52.9 % ± 17.9 % for control). HSPC clonogenic capacities were similar whatever the culture conditions. Finally, in vivo experiments revealed that mice successfully engrafted in all conditions, even if some differences were observed during the first month. Three months after graft, bone marrow chimerism and blood subpopulations were similar in both groups., Conclusions: UCB HSPCs ex-vivo expansion in the presence of OTR4131 is a safe approach that did not modify cell function and engraftment capacities. In our experimental conditions, the use of a GAG mimetic did not, however, allow increasing cell expansion or optimizing their in vivo engraftment.

Many of the components that regulate the circadian clock have been identified in organisms and humans. The influence of circadian rhythm (CR) on the regulation of stem cells biology began to be evaluated. However, little is known on the role of CR on human mesenchymal stem cell (hMSCs) properties. The objective of this study was to investigate the influence of CR on the differentiation capacities of bone marrow hMSCs, as well as the regulation of cell cycle and migration capabilities. To that, we used both a chemical approach with a GSK-3β specific inhibitor (2'E,3'Z-6-bromoindirubin-3'-oxime, BIO) and a knockdown of CLOCK and PER2, two of the main genes involved in CR regulation. In these experimental conditions, a dramatic inhibition of adipocyte differentiation was observed, while osteoblastic differentiation capacities were not modified. In addition, cell migration was decreased in PER2-/- cells. Lastly, downregulation of circadian clock genes induced a modification of the hMSCs cell cycle phase distribution, which was shown to be related to a change of the cyclin expression profile. Taken together, these data showed that CR plays a role in the regulation of hMSCs differentiation and division, and likely represent key factor in maintaining hMSCs properties.

Background: Cardiac-committed cells and biomimetic scaffolds independently improve the therapeutic efficacy of stem cells. In this study we tested the long-term effects of their combination., Methods: Eighty immune-deficient rats underwent permanent coronary artery ligation. Five to 7 weeks later, those with an echocardiographically measured ejection fraction (EF) ≤55% were re-operated on and randomly allocated to receive a cell-free fibrin patch (n = 25), a fibrin patch loaded with 700,000 human embryonic stem cells (ESC) pre-treated to promote early cardiac differentiation (SSEA-1(+) progenitors [n = 30]), or to serve as sham-operated animals (n = 25). Left ventricular function was assessed by echocardiography at baseline and every month thereafter until 4 months. Hearts were then processed for assessment of fibrosis and angiogenesis and a 5-component heart failure score was constructed by integrating the absolute change in left ventricular end-systolic volume (LVESV) between 4 months and baseline, and the quantitative polymerase chain reaction (qPCR)-based expression of natriuretic peptides A and B, myosin heavy chain 7 and periostin. All data were recorded and analyzed in a blinded manner., Results: The cell-treated group consistently yielded better functional outcomes than the sham-operated group (p = 0.002 for EF; p = 0.01 for LVESV). Angiogenesis in the border zone was also significantly greater in the cell-fibrin group (p = 0.006), which yielded the lowest heart failure score (p = 0.04 vs sham). Engrafted progenitors were only detected shortly after transplantation; no grafted cells were identified after 4 months. There was no teratoma identified., Conclusions: A fibrin scaffold loaded with ESC-derived cardiac progenitors resulted in sustained improvement in contractility and attenuation of remodeling without sustained donor cell engraftment. A paracrine effect, possibly on innate reparative responses, is a possible mechanism for this enduring effect., (Copyright © 2015 International Society for Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.)

Aims: Comparative studies suggest that stem cells committed to a cardiac lineage are more effective for improving heart function than those featuring an extra-cardiac phenotype. We have therefore developed a population of human embryonic stem cell (ESC)-derived cardiac progenitor cells., Methods and Results: Undifferentiated human ESCs (I6 line) were amplified and cardiac-committed by exposure to bone morphogenetic protein-2 and a fibroblast growth factor receptor inhibitor. Cells responding to these cardio-instructive cues express the cardiac transcription factor Isl-1 and the stage-specific embryonic antigen SSEA-1 which was then used to purify them by immunomagnetic sorting. The Isl-1(+) SSEA-1(+) cells were then embedded into a fibrin scaffold which was surgically delivered onto the infarct area in a 68-year-old patient suffering from severe heart failure [New York Heart Association [NYHA] functional Class III; left ventricular ejection fraction (LVEF): 26%]. A coronary artery bypass was performed concomitantly in a non-infarcted area. The implanted cells featured a high degree of purity (99% were SSEA-1(+)), had lost the expression of Sox-2 and Nanog, taken as markers for pluripotency, and strongly expressed Isl-1. The intraoperative delivery of the patch was expeditious. The post-operative course was uncomplicated either. After 3 months, the patient is symptomatically improved (NYHA functional Class I; LVEF: 36%) and a new-onset contractility is echocardiographically evident in the previously akinetic cell/patch-treated, non-revascularized area. There have been no complications such as arrhythmias, tumour formation, or immunosuppression-related adverse events., Conclusion: This observation demonstrates the feasibility of generating a clinical-grade population of human ESC-derived cardiac progenitors and combining it within a tissue-engineered construct. While any conclusion pertaining to efficacy would be meaningless, the patient's functional outcome yet provides an encouraging hint. Beyond this case, the platform that has been set could be useful for generating different ESC-derived lineage-specific progenies., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2015. For permissions please email: journals.permissions@oup.com.)

Background: Esophageal replacement by the colon or the stomach for malignant and nonmalignant esophageal diseases exposes to significant morbidity and mortality. In this setting, tissue engineering seems to be a seductive alternative., Methods: In a porcine model, we performed a 5-cm-long circumferential replacement of the cervical esophagus by a tubulized acellular matrix (small intestinal submucosa) cellularized with autologous skeletal myoblasts and covered by a human amniotic membrane seeded with autologous oral epithelial cells. The substitute was grown for 2 weeks in the great omentum before esophageal replacement. Eighteen minipigs (divided into 3 groups: group A [substitute with esophageal endoprothesis; n = 6], group B [substitute alone; n = 6], and group C [endoprothesis alone; n = 6]) were included. The esophageal endoprothesis was removed at 6 months. Animals were killed sequentially over a 12 month-period. Clinical, endoscopic, radiologic and histologic outcomes were analyzed., Results: All animals except 1 of in groups B and C died during the first 2 months owing to refractory esophageal stenosis or endoprothesis extrusion. Nutritional autonomy without endoprothesis was observed in all animals of group A with a follow-up of >6 months (n = 3). A phenotype similar to that of native esophagus, consisting of a mature epithelium, submucosal glands, and a circular muscular layer, was observed after 9 months., Conclusion: In this model, the circumferential replacement of the cervical esophagus by a tube-shaped tissue-engineered substitute under the temporary cover of an esophageal endoprothesis allowed nutritional autonomy and tissue remodeling toward an esophageal phenotype., (Copyright © 2015 Elsevier Inc. All rights reserved.)

As part of a program targeted at developing a resorbable valved tube for replacement of the right ventricular outflow tract, we compared three biopolymers (polyurethane [PU], polyhydroxyalkanoate (the poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-4-hydroxyvalerate) [PHBVV]), and polydioxanone [PDO]) and two biofunctionalization techniques (using adipose-derived stem cells [ADSCs] or the arginine-glycine-aspartate [RGD] peptide) in a rat model of partial inferior vena cava (IVC) replacement. Fifty-three Wistar rats first underwent partial replacement of the IVC with an acellular electrospun PDO, PU, or PHBVV patch, and 31 nude rats subsequently underwent the same procedure using a PDO patch biofunctionalized either by ADSC or RGD. Results were assessed both in vitro (proliferation and survival of ADSC seeded onto the different materials) and in vivo by magnetic resonance imaging (MRI), histology, immunohistochemistry [against markers of vascular cells (von Willebrand factor [vWF], smooth muscle actin [SMA]), and macrophages ([ED1 and ED2] immunostaining)], and enzyme-linked immunosorbent assay (ELISA; for the expression of various cytokines and inducible NO synthase). PDO showed the best in vitro properties. Six weeks after implantation, MRI did not detect significant luminal changes in any group. All biopolymers were evenly lined by vWF-positive cells, but only PDO and PHBVV showed a continuous layer of SMA-positive cells at 3 months. PU patches resulted in a marked granulomatous inflammatory reaction. The ADSC and RGD biofunctionalization yielded similar outcomes. These data confirm the good biocompatibility of PDO and support the concept that appropriately peptide-functionalized polymers may be successfully substituted for cell-loaded materials.

Aim: There is now compelling evidence that cells committed to a cardiac lineage are most effective for improving the function of infarcted hearts. This has been confirmed by our pre-clinical studies entailing transplantation of human embryonic stem cell (hESC)-derived cardiac progenitors in rat and non-human primate models of myocardial infarction. These data have paved the way for a translational programme aimed at a phase I clinical trial., Methods and Results: The main steps of this programme have included (i) the expansion of a clone of pluripotent hESC to generate a master cell bank under good manufacturing practice conditions (GMP); (ii) a growth factor-induced cardiac specification; (iii) the purification of committed cells by immunomagnetic sorting to yield a stage-specific embryonic antigen (SSEA)-1-positive cell population strongly expressing the early cardiac transcription factor Isl-1; (iv) the incorporation of these cells into a fibrin scaffold; (v) a safety assessment focused on the loss of teratoma-forming cells by in vitro (transcriptomics) and in vivo (cell injections in immunodeficient mice) measurements; (vi) an extensive cytogenetic and viral testing; and (vii) the characterization of the final cell product and its release criteria. The data collected throughout this process have led to approval by the French regulatory authorities for a first-in-man clinical trial of transplantation of these SSEA-1(+) progenitors in patients with severely impaired cardiac function., Conclusion: Although several facets of this manufacturing process still need to be improved, these data may yet provide a useful platform for the production of hESC-derived cardiac progenitor cells under safe and cost-effective GMP conditions., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2014. For permissions please email: journals.permissions@oup.com.)

The pathophysiology of acute graft-versus-host disease (GVHD) remains poorly understood in humans. Although T cell subsets have been identified to play a major role in disease initiation in rodents, clinical data on the effect of these different subsets are scarce and conflicting. To address this question, immunophenotyping analyses were performed on the graft in 210 patients. The onset of acute GVHD was retrospectively correlated with these subpopulations. In an adjusted analysis, only the absolute count of CD45lo/CD62Llo CD8(+) T cells (effector memory T cells) was significantly associated with the onset of grade 2 to 4 acute GVHD. Thus, in contrast to experimental data, we found that the number of effector memory but not of naïve T cells was associated with the onset of GVHD. These results should be kept in mind while clinical trials, which aim to deplete naïve T cells, are underway in several institutions., (Copyright © 2015 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

Hematopoietic stem cells (HSCs) are the most commonly used cell type in cell-based therapy. However, the investigation of their behavior in vitro has been limited by the difficulty of monitoring these non-adherent cells under classical culture conditions. Indeed, fluid flow moves cells away from the video-recording position and prevents single cell tracking over long periods of time. Here we describe a large array of 2D no-flow chambers allowing the monitoring of single HSCs for several days. The chamber design has been optimized to facilitate manufacturing and routine use. The chip contains a single inlet and 800 chambers. The chamber medium can be renewed by diffusion within a few minutes. This allowed us to stain live human HSCs with fluorescent primary antibodies in order to reveal their stage in the hematopoiesis differentiation pathway. Thus we were able to correlate human HSCs' growth rate, polarization and migration to their differentiation stage.

Mesenchymal stromal cells (MSCs) or their media (MSC-M) were reported to reverse acute lung injury (ALI)-induced decrease of alveolar fluid clearance. To determine the mechanisms by which MSC-M exert their beneficial effects, an in vitro model of alveolar epithelial injury was created by exposing primary rat alveolar epithelial cells (AECs) to hypoxia (3% O2) plus cytomix, a combination of IL-1β, TNF-α, and IFN-γ. MSC-M were collected from human MSCs exposed for 12 h to either normoxia (MSC-M) or to hypoxia plus cytomix (HCYT-MSC-M). This latter condition was used to model the effect of alveolar inflammation and hypoxia on paracrine secretion of MSCs in the injured lung. Comparison of paracrine soluble factors in MSC media showed that the IL-1 receptor antagonist and prostaglandin E2 were markedly increased while keratinocyte growth factor (KGF) was twofold lower in HCYT-MSC-M compared with MSC-M. In AECs, hypoxia plus cytomix increased protein permeability, reduced amiloride-sensitive short-circuit current (AS-Isc), and also decreased the number of α-epithelial sodium channel (α-ENaC) subunits in the apical membrane. To test the effects of MSC media, MSC-M and HCYT-MSC-M were added for an additional 12 h to AECs exposed to hypoxia plus cytomix. MSC-M and HCYT-MSC-M completely restored epithelial permeability to normal. MSC-M, but not HCYT-MSC-M, significantly prevented the hypoxia plus cytomix-induced decrease of ENaC activity and restored apical α-ENaC channels. Interestingly, KGF-deprived MSC-M were unable to restore amiloride-sensitive sodium transport, indicating a possible role for KGF in the beneficial effect of MSC-M. These results indicate that MSC-M may be a preferable therapeutic option for ALI., (Copyright © 2014 the American Physiological Society.)

Oculopharyngeal muscular dystrophy (OPMD) is a late-onset autosomal dominant genetic disease mainly characterized by ptosis and dysphagia. We conducted a phase I/IIa clinical study (ClinicalTrials.gov NCT00773227) using autologous myoblast transplantation following myotomy in adult OPMD patients. This study included 12 patients with clinical diagnosis of OPMD, indication for cricopharyngeal myotomy, and confirmed genetic diagnosis. The feasibility and safety end points of both autologous myoblast transplantation and the surgical procedure were assessed by videoendoscopy in addition to physical examinations. Potential therapeutic benefit was also assessed through videoendoscopy and videofluoroscopy of swallowing, quality of life score, dysphagia grade, and a drink test. Patients were injected with a median of 178 million myoblasts following myotomy. Short and long-term (2 years) safety and tolerability were observed in all the patients, with no adverse effects. There was an improvement in the quality of life score for all 12 patients, and no functional degradation in swallowing was observed for 10 patients. A cell dose-dependant improvement in swallowing was even observed in this study. This trial supports the hypothesis that a local injection of autologous myoblasts in the pharyngeal muscles is a safe and efficient procedure for OPMD patients.

Biased DNA segregation is a mitotic event in which the chromatids carrying the original template DNA strands and those carrying the template copies are not segregated randomly into the two daughter cells. Biased segregation has been observed in several cell types, but not in human mesenchymal stem cells (hMSCs), and the factors affecting this bias have yet to be identified. Here, we have investigated cell adhesion geometries as a potential parameter by plating hMSCs from healthy donors on fibronectin-coated micropatterns. On symmetric micropatterns, the segregation of sister chromatids to the daughter cells appeared random. In contrast, on asymmetric micropatterns, the segregation was biased. This sensitivity to asymmetric extracellular cues was reproducible in cells from all donors but was not observed in human skin-derived fibroblasts or in a fibroblastic cell line used as controls. We conclude that the asymmetry of cell adhesion is a major factor in the regulation of biased DNA segregation in hMSCs., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: Tissue engineering represents a promising approach for esophageal replacement, considering the complexity and drawbacks of conventional techniques., Aim: To create the components necessary to reconstruct in vitro or in vivo an esophageal wall, we analyzed the feasibility and the optimal conditions of human and pig skeletal myoblast (HSM and PSM) and porcine oral epithelial cell (OEC) culture on biologic scaffolds., Materials and Methods: PSM and HSM were isolated from striated muscle and porcine OECs were extracted from oral mucosa biopsies. Myoblasts were seeded on an acellular scaffold issue from porcine small intestinal submucosa (SIS) and OEC on decellularized human amniotic membrane (HAM). Seeding conditions (cell concentrations [0.5×10(6) versus 10(6) cells/cm(2)] and culture periods [7, 14 and 21 days]), were analyzed using the methyl thiazoltetrazolium assay, quantitative PCR, flow cytometry, and immunohistochemistry., Results: Phenotypic stability was observed after cellular expansion for PSM and HSM (85% and 97% CD56-positive cells, respectively), and OECs (90% AE1/AE3- positive cells). After PSM and HSM seeding, quantities of viable cells were similar whatever the initial cell concentration used and remained stable at all time points. During cell culture on SIS, a decrease of CD56-positive cells was observed (76% and 76% by D7, 56% and 70% by D14, 28% and 60% by D21, for PSM and HSM, respectively). Multilayered surface of α-actin smooth muscle and Desmine-positive cells organized in bundles was seen as soon as D7, with no evidence of cell within the SIS. Myoblasts fusion was observed at D21. Pax3 and Pax7 expression was downregulated and MyoD expression upregulated, at D14.OEC proliferation was observed on HAM with both cell concentrations from D7 to D21. The cell metabolism activity was more important on matrix seeded by 10(6) cells/cm(2). With 0.5×10(6) OEC/cm(2), a single layer of pancytokeratin-positive cells was seen at D7, which became pluristratified by D14, while when 10(6) OEC/cm(2) were used, a pluristratified epithelial structure was seen as soon as D7. Proliferative cells (Proliferating Cell Nuclear Antigen staining) were mainly located at the basal layer., Conclusion: In this model, the optimal conditions of cell seeding in terms of cell concentration and culture duration were 0.5×10(6) myoblasts/cm(2) and 10(6) OEC/cm(2), and 7 days.

Circulating endothelial progenitor cells (cEPC) are capable of homing to neovascularisation sites, in which they proliferate and differentiate into endothelial cells. Transplantation of cEPC-derived cells, in particular those isolated from umbilical cord blood (UCB), has emerged as a promising approach in the treatment of cardio-vascular diseases. After in vivo transplantation, these cells may be exposed to local or systemic inflammation or pathogens, of which they are a common target. Because Toll-like receptors (TLR) are critical in detecting pathogens and in initiating inflammatory responses, we hypothesized that TLR may govern UCB cEPC-derived cells function. While these cells expressed almost all TLR, we found that only TLR3 dramatically impaired cell properties. TLR3 activation inhibited cell proliferation, modified cell cycle entry, impaired the in vitro angiogenic properties and induced pro-inflammatory cytokines production. The anti-angiogenic effect of TLR3 activation was confirmed in vivo in a hind-limb ischemic mice model. Moreover, TLR3 activation consistently leads to an upregulation of miR-29b, -146a and -155 and to a deregulation of cytoskeleton and cell cycle regulator. Hence, TLR3 activation is likely to be a key regulator of cEPC-derived cells properties.

Gaucher disease (GD) is an autosomal recessive disorder characterized by lysosomal glucocerebrosidase (GBA) deficiency leading to hematological and skeletal manifestations. Mechanisms underlying these symptoms have not yet been elucidated. In vivo, bone marrow (BM) mesenchymal stem cells (MSCs) have important role in the regulation of bone mass and in the support of hematopoiesis, thus representing potential candidate that could contribute to the disease. GBA deficiency may also directly impair hematopoietic stem/progenitors cells (HSPCs) intrinsic function and induce hematological defect. In order to evaluate the role of BM stem cells in GD pathophysiology, we prospectively analyzed BM-MSCs and HSPCs properties in a series of 10 patients with type 1 GD. GBA activity was decreased in all tested cell subtypes. GD-MSCs had an impaired growth potential, morphological and cell cycle abnormalities, decreased capacities to differentiate into osteoblasts. Moreover, GD-MSCs secreted soluble factors that stimulated osteoclasts resorbing activities. In vitro and in vivo primitive and mature hematopoiesis were similar between patients and controls. However, GD-MSCs had a lower hematopoietic supportive capacity than those from healthy donors. These data suggest that BM microenvironment is altered in GD and that MSCs are key components of the manifestations observed in GD.

Objectives: The present work aimed to evaluate the expression of transforming growth factor-β (TGF-β) receptors on bone marrow-derived multipotent mesenchymal stromal cells (MSCs) in patients with systemic sclerosis (SSc) and the consequences of TGF-β activation in these cells, since MSC have potential therapeutic interest for SSc patients and knowing that TGF-β plays a critical role during the development of fibrosis in SSc., Design: This is a prospective research study using MSC samples obtained from SSc patients and compared with MSC from healthy donors., Setting: One medical hospital involving collaboration between an internal medicine department for initial patient recruitment, a clinical biotherapeutic unit for MSC preparation and an academic laboratory for research., Participants: 9 patients with diffuse SSc for which bone marrow (BM) aspiration was prescribed by sternum aspiration before haematopoietic stem cell transplantation, versus nine healthy donors for normal BM., Primary and Secondary Outcome Measures: TGF-β, TGF-β receptor types I (TBRI) and II (TBRII) mRNA and protein expression were assessed by quantitative PCR and flow cytometry, respectively, in MSC from both SSc patients and healthy donors. MSC were exposed to TGF-β and assessed for collagen 1α2 synthesis and Smad expression. As positive controls, primary cultures of dermal fibroblasts were also analysed., Results: Compared with nine controls, MSC from nine SSc patients showed significant increase in mRNA levels (p<0.002) and in membrane expression (p<0.0001) of TBRII. In response to TGF-β activation, a significant increase in collagen 1α synthesis (p<0.05) and Smad-3 phosphorylation was upregulated in SSc MSC. Similar results were obtained on eight SSc-derived dermal fibroblasts compared to six healthy controls., Conclusions: TBRII gene and protein expression defect in MSC derived from SSc patients may have pathological significance. These findings should be taken into account when considering the use of MSC-based therapies in an autologous setting.