Slowing the onset of hypoxia increases colony forming efficiency of connective tissue progenitor cells \u3ci\u3ein vitro\u3c/i\u3e

Background: Survival and colony formation by transplanted tissue derived connective tissue progenitor cells (CTPs) are thought to be important factors in the success of clinical tissue engineering strategies for bone regeneration. Transplantation of cells into defects larger than a few millimeters expose cells to a profoundly hypoxic environment. This study tested the hypothesis that delaying the onset of hypoxia will improve the survival and performance of CTPs in vitro. Methods: To mimic declines seen in an avascular in vivo bone defect, colony forming efficiency by marrow derived nucleated cells was assessed under osteogenic conditions. Variation in the rate of oxygen decline from an oxygen tension of 21% to 0.1% oxygen was explored using an incubator with programmable active control of gas concentrations. The effect of doping cultures with defined concentrations of RBCs was also used to evaluate the potential for RBCs to serve as a natural buffer in the setting of declining oxygen levels. Results: A delay in onset of hypoxia over 96 hours resulted in a 3-fold increase in the relative colony forming efficiency (rCFE) of CTPs as compared to an immediate onset of hypoxia. The presence of RBCs in vitro inhibited the rCFE of CTPs. Given the negative effects of RBCs, methods of RBC removal were evaluated and compared for their effectiveness of RBC removal and retention of colony forming efficiency. Conclusions: These data suggest that conditions of hypoxia compromise colony forming efficiency in marrow derived CTPs. However, slowing the rate of decline of oxygen preserved colony forming efficiency at levels achieved in a stable normoxic (3% O2) environment. These data also suggest that RBCs are detrimental to the rCFE of CTPs and that buffy coat is an effective and preferred method for removing RBCs from marrow aspirates while preserving CTPs. These findings may inform clinical strategies for CTP transplantation

Quantifying proliferative and surface marker heterogeneity in colony‐founding connective tissue progenitors and their progeny using time‐lapse microscopy

Connective tissue progenitors (CTPs) are defined as the heterogeneous population of tissue‐resident stem and progenitor cells that are capable of proliferating and differentiating into connective tissue phenotypes. The prevalence and variation in clonal progeny of CTPs can be characterized using a colony formation assay. However, colony assays do not directly assess the characteristics of the colony‐founding CTP. We performed large, field‐of‐view, time‐lapse microscopy to manually track colonies back to the founding cells. Image processing and analysis was used to characterize the colonies and their founding cells. We found that the traditional colony‐forming unit (CFU) assay underestimates the number of founding cells as colonies can be formed by more than one founding cell. After 6 days in culture, colonies do not completely express CD73, CD90, and CD105. Heterogeneity in colony cells was characterized by two cell populations, proliferative and spread cells. Regression modelling of duration of lag phase and doubling time by cell marker suggests the presence of CD90 and CD105 in CTP subpopulations with different proliferative capabilities. From mathematical modelling of clonal colonies, we quantitatively characterized proliferation, migration, and cell marker expression rates to identify desirable clones for selection. Direct assessment of colony formation parameters led to more accurate assessment of CFU heterogeneity. Furthermore, these parameters can be used to quantify the diversity and hierarchy of stem and progenitor cells from a cell source or tissue for tissue engineering applications

Orion Multi-Purpose Crew Vehicle Active Thermal Control and Environmental Control and Life Support Development Status

The Orion Multi Purpose Crew Vehicle (MPCV) is the first crew transport vehicle to be developed by the National Aeronautics and Space Administration (NASA) in the last thirty years. Orion is currently being developed to transport the crew safely beyond Earth orbit. This year, the vehicle focused on building the Exploration Flight Test 1 (EFT1) vehicle to be launched in September of 2014. The development of the Orion Active Thermal Control (ATCS) and Environmental Control and Life Support (ECLS) System, focused on the integrating the components into the EFT1 vehicle and preparing them for launch. Work also has started on preliminary design reviews for the manned vehicle. Additional development work is underway to keep the remaining component progressing towards implementation on the flight tests of EM1 in 2017 and of EM2 in 2020. This paper covers the Orion ECLS development from April 2013 to April 2014

Post microtextures accelerate cell proliferation and osteogenesis

AbstractThe influence of surface microtexture on osteogenesis was investigated in vitro by examining the proliferation and differentiation characteristics of a class of adult stem cells and their progeny, collectively known as connective tissue progenitor cells (CTPs). Human bone marrow-derived CTPs were cultured for up to 60days on smooth polydimethylsiloxane (PDMS) surfaces and on PDMS with post microtextures that were 10μm in diameter and 6μm in height, with 10μm separation. DNA quantification revealed that the numbers of CTPs initially attached to both substrates were similar. However, cells on microtextured PDMS transitioned from lag phase after 4days of culture, in contrast to 6days for cells on smooth surfaces. By day 9 cells on the smooth surfaces exhibited arbitrary flattened shapes and migrated without any preferred orientation. In contrast, cells on the microtextured PDMS grew along the array of posts in an orthogonal manner. By days 30 and 60 cells grew and covered all surfaces with extracellular matrix. Western blot analysis revealed that the expression of integrin α5 was greater on the microtextured PDMS compared with smooth surfaces. Real time reverse transcription-polymerase chain reaction revealed that gene expression of alkaline phosphatase had decreased by days 30 and 60, compared with that on day 9, for both substrates. Gene expression of collagen I and osteocalcin was consistently greater on post microtextures relative to smooth surfaces at all time points

The influence of tethered epidermal growth factor on connective tissue progenitor colony formation

Strategies to combine aspirated marrow cells with scaffolds to treat connective tissue defects are gaining increasing clinical attention and use. In situations such as large defects where initial survival and proliferation of transplanted connective tissue progenitors (CTPs) are limiting, therapeutic outcomes might be improved by using the scaffold to deliver growth factors that promote the early stages of cell function in the graft. Signaling by the epidermal growth factor receptor (EGFR) plays a role in cell survival and has been implicated in bone development and homeostasis. Providing epidermal growth factor (EGF) in a scaffold-tethered format may sustain local delivery and shift EGFR signaling to pro-survival modes compared to soluble ligand. We therefore examined the effect of tethered EGF on osteogenic colony formation from human bone marrow aspirates in the context of three different adhesion environments using a total of 39 donors. We found that tethered EGF, but not soluble EGF, increased the numbers of colonies formed regardless of adhesion background, and that tethered EGF did not impair early stages of osteogenic differentiation.National Institute of General Medical Sciences (U.S.) (Grant NIH RO1 AR42997)National Institute of General Medical Sciences (U.S.) (Grant NIH RO1 AG024980)National Institute of General Medical Sciences (U.S.) (Grant NIH RO1 GM59870)National Institute of General Medical Sciences (U.S.) (Grant NIH DE019523

Multi Purpose Crew Vehicle Active Thermal Control and Environmental Control and Life Support Development Status

The Orion Multi Purpose Crew Vehicle (MPCV) is the first crew transport vehicle to be developed by the National Aeronautics and Space Administration (NASA) in the last thirty years. Orion is currently being developed to transport the crew safely beyond Earth orbit. This year, the vehicle focused on building the Exploration Flight Test 1 (EFT1) vehicle to be launched in September of 2014. The development of the Orion Active Thermal Control (ATCS) and Environmental Control and Life Support (ECLS) System, focused on the integrating the components into the EFT1 vehicle and preparing them for launch. Work also has started on preliminary design reviews for the manned vehicle. Additional development work is underway to keep the remaining component progressing towards implementation on the flight tests of EM1 in 2017 and of EM2 in 2020. This paper covers the Orion ECLS development from April 2013 to April 201

A Dedicated Promoter Drives Constitutive Expression of the Cell-Autonomous Immune Resistance GTPase, Irga6 (IIGP1) in Mouse Liver

Background: In general, immune effector molecules are induced by infection. Methodology and Principal Findings: However, strong constitutive expression of the cell-autonomous resistance GTPase, Irga6 (IIGP1), was found in mouse liver, contrasting with previous evidence that expression of this protein is exclusively dependent on induction by IFNc. Constitutive and IFNc-inducible expression of Irga6 in the liver were shown to be dependent on transcription initiated from two independent untranslated 59 exons, which splice alternatively into the long exon encoding the full-length protein sequence. Irga6 is expressed constitutively in freshly isolated hepatocytes and is competent in these cells to accumulate on the parasitophorous vacuole membrane of infecting Toxoplasma gondii tachyzoites. Conclusions and Significance: The role of constitutive hepatocyte expression of Irga6 in resistance to parasites invading from the gut via the hepatic portal system is discussed

A formally verified compiler back-end

This article describes the development and formal verification (proof of semantic preservation) of a compiler back-end from Cminor (a simple imperative intermediate language) to PowerPC assembly code, using the Coq proof assistant both for programming the compiler and for proving its correctness. Such a verified compiler is useful in the context of formal methods applied to the certification of critical software: the verification of the compiler guarantees that the safety properties proved on the source code hold for the executable compiled code as well

Standalone vertex finding in the ATLAS muon spectrometer

A dedicated reconstruction algorithm to find decay vertices in the ATLAS muon spectrometer is presented. The algorithm searches the region just upstream of or inside the muon spectrometer volume for multi-particle vertices that originate from the decay of particles with long decay paths. The performance of the algorithm is evaluated using both a sample of simulated Higgs boson events, in which the Higgs boson decays to long-lived neutral particles that in turn decay to bbar b final states, and pp collision data at √s = 7 TeV collected with the ATLAS detector at the LHC during 2011