45 research outputs found
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
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
Quantifying proliferative and surface marker heterogeneity in colonyâfounding connective tissue progenitors and their progeny using timeâlapse microscopy
Connective tissue progenitors (CTPs) are defined as the heterogeneous population of tissueâresident 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 colonyâfounding CTP. We performed large, fieldâofâview, timeâlapse 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 colonyâforming 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
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 10Όm in diameter and 6Όm in height, with 10Όm 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
Satellite observations of banded VLF emissions in conjunction with energyâbanded ions during very large geomagnetic storms
Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/95489/1/jgra22114.pd
Deep-Sequencing Analysis of the Mouse Transcriptome Response to Infection with Brucella melitensis Strains of Differing Virulence
Brucella melitensis is an important zoonotic pathogen that causes brucellosis, a disease that affects sheep, cattle and occasionally humans. B. melitensis strain M5-90, a live attenuated vaccine cultured from B. melitensis strain M28, has been used as an effective tool in the control of brucellosis in goats and sheep in China. However, the molecular changes leading to attenuated virulence and pathogenicity in B. melitensis remain poorly understood. In this study we employed the Illumina Genome Analyzer platform to perform genome-wide digital gene expression (DGE) analysis of mouse peritoneal macrophage responses to B. melitensis infection. Many parallel changes in gene expression profiles were observed in M28- and M5-90-infected macrophages, suggesting that they employ similar survival strategies, notably the induction of anti-inflammatory and antiapoptotic factors. Moreover, 1019 differentially expressed macrophage transcripts were identified 4 h after infection with the different B. melitensis strains, and these differential transcripts notably identified genes involved in the lysosome and mitogen-activated protein kinase (MAPK) pathways. Further analysis employed gene ontology (GO) analysis: high-enrichment GOs identified endocytosis, inflammatory, apoptosis, and transport pathways. Path-Net and Signal-Net analysis highlighted the MAPK pathway as the key regulatory pathway. Moreover, the key differentially expressed genes of the significant pathways were apoptosis-related. These findings demonstrate previously unrecognized changes in gene transcription that are associated with B. melitensis infection of macrophages, and the central signaling pathways identified here merit further investigation. Our data provide new insights into the molecular attenuation mechanism of strain M5-90 and will facilitate the generation of new attenuated vaccine strains with enhanced efficacy
Common Genetic Variation And Age at Onset Of Anorexia Nervosa
Background Genetics and biology may influence the age at onset of anorexia nervosa (AN). The aims of this study were to determine whether common genetic variation contributes to AN age at onset and to investigate the genetic associations between age at onset of AN and age at menarche. Methods A secondary analysis of the Psychiatric Genomics Consortium genome-wide association study (GWAS) of AN was performed which included 9,335 cases and 31,981 screened controls, all from European ancestries. We conducted GWASs of age at onset, early-onset AN (< 13 years), and typical-onset AN, and genetic correlation, genetic risk score, and Mendelian randomization analyses. Results Two loci were genome-wide significant in the typical-onset AN GWAS. Heritability estimates (SNP-h2) were 0.01-0.04 for age at onset, 0.16-0.25 for early-onset AN, and 0.17-0.25 for typical-onset AN. Early- and typical-onset AN showed distinct genetic correlation patterns with putative risk factors for AN. Specifically, early-onset AN was significantly genetically correlated with younger age at menarche, and typical-onset AN was significantly negatively genetically correlated with anthropometric traits. Genetic risk scores for age at onset and early-onset AN estimated from independent GWASs significantly predicted age at onset. Mendelian randomization analysis suggested a causal link between younger age at menarche and early-onset AN. Conclusions Our results provide evidence consistent with a common variant genetic basis for age at onset and implicate biological pathways regulating menarche and reproduction.Peer reviewe
Shared genetic risk between eating disorder- and substance-use-related phenotypes:Evidence from genome-wide association studies
First published: 16 February 202