Human erythroid differentiation requires VDAC1-mediated mitochondrial clearance

Erythroblast maturation in mammals is dependent on organelle clearance throughout terminal erythropoiesis. We studied the role of the outer mitochondrial membrane protein voltage-dependent anion channel-1 (VDAC1) in human terminal erythropoiesis. We show that short hairpin (shRNA)-mediated downregulation of VDAC1 accelerates erythroblast maturation. Thereafter, erythroblasts are blocked at the orthochromatic stage, exhibiting a significant decreased level of enucleation, concomitant with an increased cell death. We demonstrate that mitochondria clearance starts at the transition from basophilic to polychromatic erythroblast, and that VDAC1 downregulation induces the mitochondrial retention. In damaged mitochondria from non-erythroid cells, VDAC1 was identified as a target for Parkin-mediated ubiquitination to recruit the phagophore. Here, we showed that VDAC1 is involved in phagophore’s membrane recruitment regulating selective mitophagy of still functional mitochondria from human erythroblasts. These findings demonstrate for the first time a crucial role for VDAC1 in human erythroblast terminal differentiation, regulating mitochondria clearance.Fil: Moras, Martina. Universite de Paris; Francia. Institut National de Transfusion Sanguine; Francia. Laboratoire d’Excellence GR-Ex; FranciaFil: Hattab, Claude. Universite de Paris; Francia. Institut National de Transfusion Sanguine; Francia. Laboratoire d’Excellence GR-Ex; FranciaFil: Gonzalez Menendez, Pedro. Laboratoire d’Excellence GR-Ex; Francia. Université Montpellier II; Francia. Centre National de la Recherche Scientifique; FranciaFil: Fader Kaiser, Claudio Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina. Universidad Nacional de Cuyo. Facultad de Odontologia; ArgentinaFil: Dussiot, Michael. Universite de Paris; Francia. Laboratoire d’Excellence GR-Ex; FranciaFil: Larghero, Jerome. Hôpital Saint-Louis. Unité de Thérapie cellulaire; FranciaFil: Le Van Kim, Caroline. Universite de Paris; Francia. Institut National de Transfusion Sanguine; Francia. Laboratoire d’Excellence GR-Ex; FranciaFil: Kinet, Sandrina. Laboratoire d’Excellence GR-Ex; Francia. Université Montpellier II; Francia. Centre National de la Recherche Scientifique; FranciaFil: Taylor, Naomi. Laboratoire d’Excellence GR-Ex; Francia. Centre National de la Recherche Scientifique; Francia. Université Montpellier II; Francia. Center for Cancer Research; Estados UnidosFil: Lefevre, Sophie D.. Universite de Paris; Francia. Institut National de Transfusion Sanguine; Francia. Laboratoire d’Excellence GR-Ex; FranciaFil: Ostuni, Mariano. Universite de Paris; Francia. Institut National de Transfusion Sanguine; Francia. Laboratoire d’Excellence GR-Ex; Franci

Age-adjusted recipient pretransplantation telomere length and treatment-related mortality after hematopoietic stem cell transplantation

Telomere attrition induces cell senescence and apoptosis. We hypothesized that age-adjusted pretransplantation telomere length might predict treatment-related mortality (TRM) after hematopoietic stem cell transplantation (HSCT). Between 2000 and 2005, 178 consecutive patients underwent HSCT from HLA-identical sibling donors after myeloablative conditioning regimens, mainly for hematologic malignancies (n = 153). Blood lymphocytes' telomere length was measured by real-time quantitative PCR before HSCT. Age-adjusted pretransplantation telomere lengths were analyzed for correlation with clinical outcomes. After age adjustment, patients' telomere-length distribution was similar among all 4 quartiles except for disease stage. There was no correlation between telomere length and engraftment, GVHD, or relapse. The overall survival was 62% at 5 years (95% confidence interval [CI], 54-70). After a median follow-up of 51 months (range, 1-121 months), 43 patients died because of TRM. The TRM rate inversely correlated with telomere length. TRM in patients in the first (lowest telomere length) quartile was significantly higher than in patients with longer telomeres (P = .017). In multivariate analysis, recipients' age (hazard ratio, 1.1; 95% CI, .0-1.1; P = .0001) and age-adjusted telomere length (hazard ratio, 0.4; 95% CI; 0.2-0.8; P = .01) were independently associated with TRM. In conclusion, age-adjusted recipients' telomere length is an independent biologic marker of TRM after HSCT. (Blood. 2012;120(16):3353-3359)National Institutes of Health Intramural Research Program, National Heart, Lung, and Blood InstituteAA and Myelodysplastic Syndrome International FoundationFrance HPNFAPES

Effect of HLA-matching recipients to donor non-inherited maternal antigens on outcomes after mismatched umbilical cord blood transplantation for hematologic malignancy

peer reviewe

The HIV proteins Tat and Nef promote human bone marrow mesenchymal stem cell senescence and alter osteoblastic differentiation

International audienceTo maintain bone mass turnover and bone mineral density (BMD), bone marrow (BM) mesenchymal stem cells (MSCs) are constantly recruited and subsequently differentiated into osteo-blasts. HIV-infected patients present lower BMD than non-HIV infected individuals and a higher prevalence of osteopenia/ osteoporosis. In antiretroviral treatment (ART)-naive patients, encoded HIV proteins represent pathogenic candidates. They are released by infected cells within BM and can impact on neighbouring cells. In this study, we tested whether HIV proteins Tat and/or Nef could induce senescence of human BM-MSCs and reduce their capacity to differentiate into osteoblasts. When compared to nontreated cells, MSCs chronically treated with Tat and/or Nef up to 30 days reduced their proliferative activity and underwent early senescence, associated with increased oxidative stress and mitochondrial dysfunction. The antioxidant molecule N-acetyl-cysteine had no or minimal effects on Tat-or Nef-induced senescence. Tat but not Nef induced an early increase in NF-jB activity and cytokine/chemokine secretion. Tat-induced effects were prevented by the NF-jB inhibitor parthenolide, indicating that Tat triggered senescence via NF-jB activation leading to oxidative stress. Otherwise, Nef-but not Tat-treated cells displayed early inhibition of autophagy. Rapamycin, an autophagy inducer, reversed Nef-induced senescence and oxida-tive stress. Moreover, Tat+Nef had cumulative effects. Finally, Tat and/or Nef decreased the MSC potential of osteoblastic differentiation. In conclusion, our in vitro data show that Tat and Nef could reduce the number of available precursors by inducing MSC senescence, through either enhanced inflammation or reduced autophagy. These results offer new insights into the pathophys-iological mechanisms of decreased BMD in HIV-infected patients

Convergence of microengineering and cellular self-organization towards functional tissue manufacturing

International audienceTechnical 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

Feasibility of an Acoustophoresis-based System for a High-Throughput Cell Washing: Application to Bioproduction

Background These last decades have seen the emergence and development of cell-based therapies, notably those based on mesenchymal stromal cells (MSCs). The advancement of these promising treatments requires increasing the throughput of processed cell for industrialization in order to reduce production costs. Among the various bioproduction challenges, downstream processing (DSP), including medium exchange, cell washing, cell harvesting and volume reduction, remains a critical step on which improvements are needed. Typically, these processes are performed by centrifugation. However, this approach limits the automation, especially in small batch productions where it is performed manually in open system. Methods An acoustophoresis-based system was developed for cell washing. The cells were transferred from one stream to another via the acoustic forces and were collected in a different medium. The optimal flow rates of the different streams were assessed using Red Blood Cells (RBCs) suspended in an albumin solution. Finally, the impact of acoustic washing on adipose tissue-derived MSCs (AD-MSCs) transcriptome was investigated by RNA-sequencing. Results With a single passage through the acoustic device at input flow rate of 45 mL/h, the albumin removal was up to 90% while recovering 99% of RBCs. To further increase the protein removal, a loop washing in two steps was performed and has allowed an albumin removal ≥99% and a RBCs/AD-MSCs recovery of 99%. After loop washing of AD-MSCs, only 2 genes, HES4 and MIR-3648-1, were differently expressed compared to the input. Conclusion In this study, we developed a continuous cell washing system based on acoustophoresis. The process allows a high cell throughput while inducing little gene expression changes. These results highly suggest that cell washing based on acoustophoresis is a relevant and promising solution for numerous applications in cell manufacturing

Gene expression in knockdown hMSCs.

<p>(A) <i>CLOCK</i>, <i>PER2</i> and <i>GAPDH</i> mRNA levels in hMSCs, normalized to shGFP. Data are expressed as the mean of 2^(–ΔΔCt) ± SEM. Endogenous gene: <i>HMBS</i>. Bars represent means of 3 independent experiments. *: p<0.05. (B) Expression profiles of clock-associated genes -<i>CLOCK</i> (a), <i>BMAL1</i> (b), <i>PER1</i> (c), <i>PER2</i> (d), <i>GSK-3β</i> (e)- and <i>GAPDH</i> (f), <i>PPARγ</i> (g) and <i>OSTEOCALCIN</i> (h) in WT, shClock and shPer2 hMSCs (T0 = serum shock). Data are expressed as the mean of ΔCt ± SEM calculated from the Ct value of each gene T0. Bars represent means of 2 independent experiments.</p

Microtubules control nuclear shape and gene expression during early stages of hematopoietic differentiation

International audienceHematopoietic stem and progenitor cells (HSPC) can differentiate into all hematopoietic lineages to support hematopoiesis. Cells from the myeloid and lymphoid lineages fulfill distinct functions with specific shapes and intra-cellular architectures. The role of cytokines in the regulation of HSPC differentiation has been intensively studied but our understanding of the potential contribution of inner cell architecture is relatively poor. Here, we show that large invaginations are generated by microtubule constraints on the swelling nucleus of human HSPC during early commitment toward the myeloid lineage. These invaginations are associated with a local reduction of lamin B density, local loss of heterochromatin H3K9me3 and H3K27me3 marks, and changes in expression of specific hematopoietic genes. This establishes the role of microtubules in defining the unique lobulated nuclear shape observed in myeloid progenitor cells and suggests that this shape is important to establish the gene expression profile specific to this hematopoietic lineage. It opens new perspectives on the implications of microtubule-generated forces, in the early commitment to the myeloid lineage

Human Bone Marrow Mesenchymal Stem Cells Regulate Biased DNA Segregation in Response to Cell Adhesion Asymmetry

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

Construction of functional biliary epithelial branched networks with predefined geometry using digital light stereolithography

International audienc