Pbx1 Regulates Self-Renewal of Long-Term Hematopoietic Stem Cells by Maintaining Their Quiescence

SummarySelf-renewal is a defining characteristic of stem cells; however, the molecular pathways underlying its regulation are poorly understood. Here, we demonstrate that conditional inactivation of the Pbx1 proto-oncogene in the hematopoietic compartment results in a progressive loss of long-term hematopoietic stem cells (LT-HSCs) that is associated with concomitant reduction in their quiescence, leading to a defect in the maintenance of self-renewal as assessed by serial transplantation. Transcriptional profiling revealed that multiple stem cell maintenance factors are perturbed in Pbx1-deficient LT-HSCs, which prematurely express a large subset of genes, including cell-cycle regulators, normally expressed in non-self-renewing multipotent progenitors. A significant proportion of Pbx1-dependent genes is associated with the TGF-β pathway, which serves a major role in maintaining HSC quiescence. Prospectively isolated, Pbx1-deficient LT-HSCs display altered transcriptional responses to TGF-β stimulation in vitro, suggesting a possible mechanism through which Pbx1 maintenance of stem cell quiescence may in part be achieved

Editorial: Ecotechnologies for Wastewater Treatment - Impacting the environment with innovation in wastewater treatment

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IL-3 or IL-7 Increases ex Vivo Gene Transfer Efficiency in ADA-SCID BM CD34 + Cells while Maintaining in Vivo Lymphoid Potential

To improve maintenance and gene transfer of human lymphoid progenitors for clinical use in gene therapy of adenosine deaminase (ADA)-deficient SCID we investigated several gene transfer protocols using various stem cell-enriched sources. The lymphoid differentiation potential was measured by an in vitro clonal assay for B/NK cells and in the in vivo SCID-hu mouse model. Ex vivo culture with the cytokines TPO, FLT3-ligand, and SCF (T/F/S) plus IL-3 or IL-7 substantially increased the yield of transduced bone marrow (BM) CD34+ cells purified from ADA-SCID patients or healthy donors, compared to T/F/S alone. Moreover, the use of IL-3 or IL-7 significantly improved the maintenance of in vitro B cell progenitors from ADA-SCID BM cells and allowed the efficient transduction of B and NK cell progenitors. Under these optimized conditions transduced CD34+ cells were efficiently engrafted into SCID-hu mice and gave rise to B and T cell progeny, demonstrating the maintenance of in vivo lymphoid reconstitution capacity. The protocol based on the T/F/S + IL-3 combination was included in a gene therapy clinical trial for ADA-SCID, resulting in long-term engraftment of stem/progenitor cells. Remarkably, gene-corrected BM CD34+ cells obtained from one patient 4 and 11 months after gene therapy were capable of repopulating the lymphoid compartment of SCID-hu hosts

MicroRNA-127-3p controls murine hematopoietic stem cell maintenance by limiting differentiation

The balance between self-renewal and differentiation is crucial to ensure the homeostasis of the hematopoietic system, and is a hallmark of hematopoietic stem cells. However, the underlying molecular pathways, including the role of micro-RNA, are not completely understood. To assess the contribution of micro-RNA, we performed micro-RNA profiling of hematopoietic stem cells and their immediate downstream progeny multi-potent progenitors from wild-type control and Pbx1-conditional knockout mice, whose stem cells display a profound self-renewal defect. Unsupervised hierarchical cluster analysis separated stem cells from multi-potent progenitors, suggesting that micro-RNA might regulate the first transition step in the adult hematopoietic development. Notably, Pbx1-deficient and wild-type cells clustered separately, linking micro-RNAs to self-renewal impairment. Differential expression analysis of micro-RNA in the physiological stem cell-to-multi-potent progenitor transition and in Pbx1-deficient stem cells compared to control stem cells revealed miR-127-3p as the most differentially expressed. Furthermore, miR-127-3p was strongly stem cell-specific, being quickly down-regulated upon differentiation and not re-expressed further downstream in the bone marrow hematopoietic hierarchy. Inhibition of miR-127-3p function in Lineage-negative cells, achieved through a lentiviral-sponge vector, led to severe stem cell depletion, as assessed with serial transplantation assays. miR-127-3p-sponged stem cells displayed accelerated differentiation, which was uncoupled from proliferation, accounting for the observed stem cell reduction. miR-127-3p overexpression in Lineage-negative cells did not alter stem cell pool size, but gave rise to lymphopenia, likely due to lack of miR-127-3p physiological downregulation beyond the stem cell stage. Thus, tight regulation of miR-127-3p is crucial to preserve the self-renewing stem cell pool and homeostasis of the hematopoietic system

Molecular purging of multiple myeloma cells by ex-vivo culture and retroviral transduction of mobilized-blood CD34+ cells

<p>Abstract</p> <p>Background</p> <p>Tumor cell contamination of the apheresis in multiple myeloma is likely to affect disease-free and overall survival after autografting.</p> <p>Objective</p> <p>To purge myeloma aphereses from tumor contaminants with a novel culture-based purging method.</p> <p>Methods</p> <p>We cultured myeloma-positive CD34⁺PB samples in conditions that retained multipotency of hematopoietic stem cells, but were unfavourable to survival of plasma cells. Moreover, we exploited the resistance of myeloma plasma cells to retroviral transduction by targeting the hematopoietic CD34⁺cell population with a retroviral vector carrying a selectable marker (the truncated form of the human receptor for nerve growth factor, ΔNGFR). We performed therefore a further myeloma purging step by selecting the transduced cells at the end of the culture.</p> <p>Results</p> <p>Overall recovery of CD34⁺cells after culture was 128.5%; ΔNGFR transduction rate was 28.8% for CD34⁺cells and 0% for CD138-selected primary myeloma cells, respectively. Recovery of CD34⁺cells after ΔNGFR selection was 22.3%. By patient-specific Ig-gene rearrangements, we assessed a decrease of 0.7–1.4 logs in tumor load after the CD34⁺cell selection, and up to 2.3 logs after culture and ΔNGFR selection.</p> <p>Conclusion</p> <p>We conclude that <it>ex-vivo </it>culture and retroviral-mediated transduction of myeloma leukaphereses provide an efficient tumor cell purging.</p

Expanded circulating hematopoietic stem/progenitor cells as novel cell source for the treatment of TCIRG1 osteopetrosis

Allogeneic hematopoietic stem cell transplantation is the treatment of choice for autosomal recessive osteopetrosis caused by defects in the TCIRG1 gene. Despite recent progress in conditioning, a relevant number of patients are not eligible for allogeneic stem cell transplantation because of the severity of the disease and significant transplant-related morbidity. We exploited peripheral CD34+ cells, known to circulate at high frequency in the peripheral blood of TCIRG1-deficient patients, as a novel cell source for autologous transplantation of gene corrected cells. Detailed phenotypical analysis showed that circulating CD34+ cells have a cellular composition that resembles bone marrow, supporting their use in gene therapy protocols. Transcriptomic profile revealed enrichment in genes expressed by hematopoietic stem and progenitor cells (HSPCs). To overcome the limit of bone marrow harvest/ HSPC mobilization and serial blood drawings in TCIRG1 patients, we applied UM171-based ex-vivo expansion of HSPCs coupled with lentiviral gene transfer. Circulating CD34+ cells from TCIRG1-defective patients were transduced with a clinically-optimized lentiviral vector (LV) expressing TCIRG1 under the control of phosphoglycerate promoter and expanded ex vivo. Expanded cells maintained long-term engraftment capacity and multi-lineage repopulating potential when transplanted in vivo both in primary and secondary NSG recipients. Moreover, when CD34+ cells were differentiated in vitro, genetically corrected osteoclasts resorbed the bone efficiently. Overall, we provide evidence that expansion of circulating HSPCs coupled to gene therapy can overcome the limit of stem cell harvest in osteopetrotic patients, thus opening the way to future gene-based treatment of skeletal diseases caused by bone marrow fibrosis

Maize mono-digestion efficiency: results from laboratory tests

A laboratory experimental campaign was carried out in order to assess the optimal configuration for the anaerobic digestion of a mixture of sweet corn and ensiled maize. Batch hydrolysis tests were conducted at 35 and 55 WC and at four different particle sizes (2, 5, 20 and 50 mm) obtained by manual chopping and sieving. Chemical pre-treatment by 24 h incubation at various acid and alkaline pH was also considered for its potential to increase the maize methane yield. Results suggest that the hydrolytic phase proceeds significantly faster under thermophilic conditions. Significant differences in the solubilization rate were also observed when comparing coarse (20–50 mm) with fine (2–5 mm) particles, while 2 and 5 mm particles were solubilized at similar rates. No advantages from the chemical pre-treatment, in terms of solubilization efficiency and biomethanization potential were observed. According to these preliminary results, a two-stage semi-continuous laboratory plant consisting in a thermophilic hydrolytic reactor followed by a mesophilic methanogenic reactor was operated for 110 days. Steady state loading parameters were: influent concentration (maize mixture diluted in tap waster) of 46 g VS/L, hydraulic retention time of 31 d, organic loading rate of 1.5 g VS/L/d. Alkalinity was dosed to the methanogenic reactor to avoid pH drops. Collected data allowed to estimate the average biodegradation efficiency which resulted to be around 60–65%

Evaluating the effect of post-treated solid recirculation via BMP tests and simplified AD model

The biogas production through anaerobic digestion (AD) of agricultural residues, animal manure, energy crops and combination thereof (codigestion) has become an important aspect of the agricultural sector of the European Community. Within this context, technologies/strategies improving the energetic efficiency of AD plants are expected to have a great impact on the productivity/profitability, and the environmental footprint of this industrial sector. The recirculation of the solid separated fraction of the digestate (DigSS) would result in the reduction of undigested solid residues and in the increase of the energy recovery of the AD process. To enhance the DigSS biodegradability, a post-pretreatment step could be applied prior to digestate recirculation. The overall impact on the biogas plant would be a higher specific methane production and higher operational costs for solid/liquid separation, mixing and pumping. The optimal amount of DigSS to be recirculated is plant-specific and would depend on the effectiveness of the post-treatment in terms of anaerobic degradability (ultimate biochemical methane potential, BMPu) and degradation kinetics. A simplified AD model was then implemented to be used for scenarios analyses. In this work both the DigSS used in the post-treatment experiments and the data used for calibration and validation of the simplified AD model came from a full-scale mesophilic AD fed with a mixture of animal manure (cow and turkey), whey and corn silage. A methodology for calibration is presented based on simple experimental measurements, widening the applicability of the model. In view of maximizing economical sustainability, the types of post-treatment considered in this study were limited to those seen as most easily and economically applicable at farm scale, such as mild thermal treatment (40-80ºC), dilute-alkali treatment and combination thereof

Optimizing resource recovery from wastewater with algae-bacteria membrane reactors

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