Maintenance of Leukemia-Initiating Cells Is Regulated by the CDK Inhibitor Inca1

Functional differences between healthy progenitor and cancer initiating cells may provide unique opportunities for targeted therapy approaches. Hematopoietic stem cells are tightly controlled by a network of CDK inhibitors that govern proliferation and prevent stem cell exhaustion. Loss of Inca1 led to an increased number of short-term hematopoietic stem cells in older mice, but Inca1 seems largely dispensable for normal hematopoiesis. On the other hand, Inca1-deficiency enhanced cell cycling upon cytotoxic stress and accelerated bone marrow exhaustion. Moreover, AML1-ETO9a-induced proliferation was not sustained in Inca1-deficient cells in vivo. As a consequence, leukemia induction and leukemia maintenance were severely impaired in Inca1−/− bone marrow cells. The re-initiation of leukemia was also significantly inhibited in absence of Inca1−/− in MLL—AF9- and c-myc/BCL2-positive leukemia mouse models. These findings indicate distinct functional properties of Inca1 in normal hematopoietic cells compared to leukemia initiating cells. Such functional differences might be used to design specific therapy approaches in leukemia

Epiblast Stem Cell Subpopulations Represent Mouse Embryos of Distinct Pregastrulation Stages

SummaryEmbryonic stem cells (ESCs) comprise at least two populations of cells with divergent states of pluripotency. Here, we show that epiblast stem cells (EpiSCs) also comprise two distinct cell populations that can be distinguished by the expression of a specific Oct4-GFP marker. These two subpopulations, Oct4-GFP positive and negative EpiSCs, are capable of converting into each other in vitro. Oct4-GFP positive and negative EpiSCs are distinct from ESCs with respect to global gene expression pattern, epigenetic profile, and Oct4 enhancer utilization. Oct4-GFP negative cells share features with cells of the late mouse epiblast and cannot form chimeras. However, Oct4-GFP positive EpiSCs, which only represent a minor EpiSC fraction, resemble cells of the early epiblast and can readily contribute to chimeras. Our findings suggest that the rare ability of EpiSCs to contribute to chimeras is due to the presence of the minor EpiSC fraction representing the early epiblast

Lima1 mediates the pluripotency control of membrane dynamics and cellular metabolism.

Lima1 is an extensively studied prognostic marker of malignancy and is also considered to be a tumour suppressor, but its role in a developmental context of non-transformed cells is poorly understood. Here, we characterise the expression pattern and examined the function of Lima1 in mouse embryos and pluripotent stem cell lines. We identify that Lima1 expression is controlled by the naïve pluripotency circuit and is required for the suppression of membrane blebbing, as well as for proper mitochondrial energetics in embryonic stem cells. Moreover, forcing Lima1 expression enables primed mouse and human pluripotent stem cells to be incorporated into murine pre-implantation embryos. Thus, Lima1 is a key effector molecule that mediates the pluripotency control of membrane dynamics and cellular metabolism

A New Mint1 Isoform, but Not the Conventional Mint1, Interacts with the Small GTPase Rab6

Small GTPases of the Rab family are important regulators of a large variety of different cellular functions such as membrane organization and vesicle trafficking. They have been shown to play a role in several human diseases. One prominent member, Rab6, is thought to be involved in the development of Alzheimer’s Disease, the most prevalent mental disorder worldwide. Previous studies have shown that Rab6 impairs the processing of the amyloid precursor protein (APP), which is cleaved to β-amyloid in brains of patients suffering from Alzheimer’s Disease. Additionally, all three members of the Mint adaptor family are implied to participate in the amyloidogenic pathway. Here, we report the identification of a new Mint1 isoform in a yeast two-hybrid screening, Mint1 826, which lacks an eleven amino acid (aa) sequence in the conserved C-terminal region. Mint1 826, but not the conventional Mint1, interacts with Rab6 via the PTB domain. This interaction is nucleotide-dependent, Rab6-specific and influences the subcellular localization of Mint1 826. We were able to detect and sequence a corresponding proteolytic peptide derived from cellular Mint1 826 by mass spectrometry proving the absence of aa 495–505 and could show that the deletion does not influence the ability of this adaptor protein to interact with APP. Taking into account that APP interacts and co-localizes with Mint1 826 and is transported in Rab6 positive vesicles, our data suggest that Mint1 826 bridges APP to the small GTPase at distinct cellular sorting points, establishing Mint1 826 as an important player in regulation of APP trafficking and processing

Pressure Drives Rapid Burst-Like Coordinated Cellular Motion from 3D Cancer Aggregates

A key behavior observed during morphogenesis, wound healing, and cancer invasion is that of collective and coordinated cellular motion. Hence, understanding the different aspects of such coordinated migration is fundamental for describing and treating cancer and other pathological defects. In general, individual cells exert forces on their environment in order to move, and collective motion is coordinated by cell-cell adhesion-based forces. However, this notion ignores other mechanisms that encourage cellular movement, such as pressure differences. Here, using model tumors, it is found that increased pressure drove coordinated cellular motion independent of cell-cell adhesion by triggering cell swelling in a soft extracellular matrix (ECM). In the resulting phenotype, a rapid burst-like stream of cervical cancer cells emerged from 3D aggregates embedded in soft collagen matrices (0.5 mg mL(-1)). This fluid-like pushing mechanism, recorded within 8 h after embedding, shows high cell velocities and super-diffusive motion. Because the swelling in this model system critically depends on integrin-mediated cell-ECM adhesions and cellular contractility, the swelling is likely triggered by unsustained mechanotransduction, providing new evidence that pressure-driven effects must be considered to more completely understand the mechanical forces involved in cell and tissue movement as well as invasion

On the origin of Fe/S cluster biosynthesis in eukaryotes

Iron and sulfur are indispensable elements of every living cell, but on their own these elements are toxic and require dedicated machineries for the formation of Fe/S clusters. In eukaryotes, proteins requiring Fe/S clusters (Fe/S proteins) are found in or associated with various organelles including the mitochondrion, endoplasmic reticulum, cytosol and the nucleus. These proteins are involved in several pathways indispensable for the viability of each living cell including DNA maintenance, protein translation and metabolic pathways. Thus, the formation of Fe/S clusters and their delivery to these proteins has a fundamental role in the functions and the evolution of the eukaryotic cell. Currently, most eukaryotes harbor two (located in cytosol and mitochondrion) or three (located in plastid) machineries for the assembly of Fe/S clusters, but certain anaerobic microbial eukaryotes contain Sulfur Mobilization (SUF) machineries that were previously thought to be present only in archaeal linages. These machineries could not only stipulate which pathway was present in the last eukaryotic common ancestor (LECA), but they could also provide clues regarding presence of an Fe/S cluster machinery in the proto-eukaryote and evolution of Fe/S cluster assembly machineries in all eukaryotes

Electrical breakdown in tissue electroporation

Electroporation, the permeabilization of the cell membrane by brief, high electric fields, has become an important technology in medicine for diverse application ranging from gene transfection to tissue ablation. There is ample anecdotal evidence that the clinical application of electroporation is often associated with loud sounds and extremely high currents that exceed the devices design limit after which the devices cease to function. The goal of this paper is to elucidate and quantify the biophysical and biochemical basis for this phenomenon. Using an experimental design that includes clinical data, a tissue phantom, sound, optical, ultrasound and MRI measurements, we show that the phenomenon is caused by electrical breakdown across ionized electrolysis produced gases near the electrodes. The breakdown occurs primarily near the cathode. Electrical breakdown during electroporation is a biophysical phenomenon of substantial importance to the outcome of clinical applications. It was ignored, until now

HDAC7 Is a Repressor of Myeloid Genes Whose Downregulation Is Required for Transdifferentiation of Pre-B Cells into Macrophages

B lymphopoiesis is the result of several cell-commitment, lineage-choice, and differentiation processes. Every differentiation step is characterized by the activation of a new, lineage-specific, genetic program and the extinction of the previous one. To date, the central role of specific transcription factors in positively regulating these distinct differentiation processes to acquire a B cell-specific genetic program is well established. However, the existence of specific transcriptional repressors responsible for the silencing of lineage inappropriate genes remains elusive. Here we addressed the molecular mechanism behind repression of non-lymphoid genes in B cells. We report that the histone deacetylase HDAC7 was highly expressed in pre-B cells but dramatically down-regulated during cellular lineage conversion to macrophages. Microarray analysis demonstrated that HDAC7 re-expression interfered with the acquisition of the gene transcriptional program characteristic of macrophages during cell transdifferentiation; the presence of HDAC7 blocked the induction of key genes for macrophage function, such as immune, inflammatory, and defense response, cellular response to infections, positive regulation of cytokines production, and phagocytosis. Moreover, re-introduction of HDAC7 suppressed crucial functions of macrophages, such as the ability to phagocytose bacteria and to respond to endotoxin by expressing major pro-inflammatory cytokines. To gain insight into the molecular mechanisms mediating HDAC7 repression in pre-B cells, we undertook co-immunoprecipitation and chromatin immunoprecipitation experimental approaches. We found that HDAC7 specifically interacted with the transcription factor MEF2C in pre-B cells and was recruited to MEF2 binding sites located at the promoters of genes critical for macrophage function. Thus, in B cells HDAC7 is a transcriptional repressor of undesirable genes. Our findings uncover a novel role for HDAC7 in maintaining the identity of a particular cell type by silencing lineage-inappropriate genes

Randomized controlled phase 2 trial of hydroxychloroquine in childhood interstitial lung disease

Background No results of controlled trials are available for any of the few treatments offered to children with interstitial lung diseases (chILD). We evaluated hydroxychloroquine (HCQ) in a phase 2, prospective, multicentre, 1:1-randomized, double-blind, placebo-controlled, parallel-group/crossover trial. HCQ (START arm) or placebo were given for 4 weeks. Then all subjects received HCQ for another 4 weeks. In the STOP arm subjects already taking HCQ were randomized to 12 weeks of HCQ or placebo (= withdrawal of HCQ). Then all subjects stopped treatment and were observed for another 12 weeks. Results 26 subjects were included in the START arm, 9 in the STOP arm, of these four subjects participated in both arms. The primary endpoint, presence or absence of a response to treatment, assessed as oxygenation (calculated from a change in transcutaneous O 2 -saturation of ≥ 5%, respiratory rate ≥ 20% or level of respiratory support), did not differ between placebo and HCQ groups. Secondary endpoints including change of O 2 -saturation ≥ 3%, health related quality of life, pulmonary function and 6-min-walk-test distance, were not different between groups. Finally combining all placebo and all HCQ treatment periods did not identify significant treatment effects. Overall effect sizes were small. HCQ was well tolerated, adverse events were not different between placebo and HCQ. Conclusions Acknowledging important shortcomings of the study, including a small study population, the treatment duration, lack of outcomes like lung function testing below age of 6 years, the small effect size of HCQ treatment observed requires careful reassessments of prescriptions in everyday practice (EudraCT-Nr.: 2013-003714-40, www.clinicaltrialsregister.eu , registered 02.07.2013)

Human skeletal muscle organoids model fetal myogenesis and sustain uncommitted PAX7 myogenic progenitors

peer reviewedIn vitro culture systems that structurally model human myogenesis and promote PAX7+ myogenic progenitor maturation have not been established. Here we report that human skeletal muscle organoids can be differentiated from induced pluripotent stem cell lines to contain paraxial mesoderm and neuromesodermal progenitors and develop into organized structures reassembling neural plate border and dermomyotome. Culture conditions instigate neural lineage arrest and promote fetal hypaxial myogenesis toward limb axial anatomical identity, with generation of sustainable uncommitted PAX7 myogenic progenitors and fibroadipogenic (PDGFRa+) progenitor populations equivalent to those from the second trimester of human gestation. Single-cell comparison to human fetal and adult myogenic progenitor /satellite cells reveals distinct molecular signatures for non-dividing myogenic progenitors in activated (CD44High/CD98+/MYOD1+) and dormant (PAX7High/FBN1High/SPRY1High) states. Our approach provides a robust 3D in vitro developmental system for investigating muscle tissue morphogenesis and homeostasis