Eva Kildall Hejbøl

Injectable scaffold materials differ in their cell instructive effects on primary human myoblasts

Scaffolds are materials used for delivery of cells for regeneration of tissues. They support three-dimensional organization and improve cell survival. For the repair of small skeletal muscles, injections of small volumes of cells are attractive, and injectable scaffolds for delivery of cells offer a minimally invasive technique. In this study, we examined in vitro the cell instructive effects of three types of injectable scaffolds, fibrin, alginate, and poly(lactic-co-glycolic acid)-based microparticles on primary human myoblasts. The myoblast morphology and progression in the myogenic program differed, depending on the type of scaffold material. In alginate gel, the cells obtained a round morphology, they ceased to proliferate, and entered quiescence. In the fibrin gels, differentiation was promoted, and myotubes were observed within a few days in culture, while poly(lactic-co-glycolic acid)-based microparticles supported prolonged proliferation. Myoblasts released from the alginate and fibrin gels were studied, and cells released from these scaffolds had retained the ability to proliferate and differentiate. Thus, the study shows that human myogenic cells combined with injectable scaffold materials are guided into different states depending on the choice of scaffold. This opens for in vivo experiments, including testing of the significance of the cell state on regeneration potential of primary human myoblasts.

CL-L1 and CL-K1 Exhibit Widespread Tissue Distribution With High and Co-Localized Expression in Secretory Epithelia and Mucosa

Collectin liver 1 (CL-L1, alias collectin 10) and collectin kidney 1 (CL-K1, alias collectin 11) are oligomeric pattern recognition molecules associated with the complement system, and mutations in either of their genes may lead to deficiency and developmental defects. The two collectins are reportedly localized and synthesized in the liver, kidneys, and adrenals, and can be found in the circulation as heteromeric complexes (CL-LK), which upon binding to microbial high mannose-like glycoconjugates activates the complement system via the lectin activation pathway. The tissue distribution of homo- vs. heteromeric CL-L1 and -K1 complexes, the mechanism of heteromeric complex formation and in which tissues this occurs, is hitherto incompletely described. We have by immunohistochemistry using monoclonal antibodies addressed the precise cellular localization of the two collectins in the main human tissues. We find that the two collectins have widespread and almost identical tissue distribution with a high expression in epithelial cells in endo-/exocrine secretory tissues and mucosa. There is also accordance between localization of mRNA transcripts and detection of proteins, showing that local synthesis likely is responsible for peripheral localization and eventual formation of the CL-LK complexes. The functional implications of the high expression in endo-/exocrine secretory tissue and mucosa is unknown but might be associated with the activity of MASP-3, which has a similar pattern of expression and is known to potentiate the activity of the alternative complement activation pathway.

Mice Knocked Out for the Primary Brain Calcification–Associated Gene Slc20a2 Show Unimpaired Prenatal Survival but Retarded Growth and Nodules in the Brain that Grow and Calcify Over Time

Brain calcification of especially the basal ganglia characterizes primary familial brain calcification (PFBC). PFBC is a rare neurodegenerative disorder with neuropsychiatric and motor symptoms, and only symptomatic treatment is available. Four PFBC-associated genes are known; approximately 40% of patients carry mutations in the gene SLC20A2, which encodes the type III sodium-dependent inorganic phosphate transporter PiT2. To investigate the role of PiT2 in PFBC development, we studied Slc20a2-knockout (KO) mice using histology, microcomputed tomography, electron microscopy, and energy-dispersive X-ray spectroscopy. Slc20a2-KO mice showed histologically detectable nodules in the brain already at 8 weeks of age, which contained organic material and were weakly calcified. In 15-week-old mice, the nodules were increased in size and number and were markedly more calcified. The major minerals in overt calcifications were Ca and P, but Fe, Zn, and Al were also generally present. Electron microscopy suggested that the calcifications initiate intracellularly, mainly in pericytes and astrocytes. As the calcification grew, they incorporated organic material. Furthermore, endogenous IgG was detected around nodules, suggesting local increased blood-brain barrier permeabilities. Nodules were found in all 8-week-old Slc20a2-KO mice, but no prenatal or marked postnatal lethality was observed. Thus, besides allowing for the study of PFBC development, the Slc20a2-KO mouse is a potential solid preclinical model for evaluation of PFBC treatments.

Classification of wooden breast myopathy in chicken pectoralis major by a standardised method and association with conventional quality assessments

1 Department of Food Science, Aarhus University, Blichers All e 20, 8830 Tjele, Denmark 2 Section for Sports Science, Aarhus University, Dalgas Avenue 4, 8000 Aarhus C., Denmark 3 Department of Food Science, Aarhus University, Kirstinebjergvej 10, 5792 Arslev, Denmark 4 Danish Meat Research Institute, Technological Institute, Gregersensvej 9, 2630 Taastrup, Denmark 5 Institute of Clinical Research, University of Southern Denmark, Odense, Denmark 6 Danpo A/S, Vestre Skovvej 3, 9600 Aars, Denmark 7 ISI Food Protection, Agro Food Park 13, 8200 Aarhus N, Denmark

Low Oxygen Tension Enhances Expression of Myogenic Genes When Human Myoblasts Are Activated from G0 Arrest

Objectives Most cell culture studies have been performed at atmospheric oxygen tension of 21%, however the physiological oxygen tension is much lower and is a factor that may affect skeletal muscle myoblasts. In this study we have compared activation of G0 arrested myoblasts in 21% O2 and in 1% O2 in order to see how oxygen tension affects activation and proliferation of human myoblasts. Materials and Methods Human myoblasts were isolated from skeletal muscle tissue and G0 arrested in vitro followed by reactivation at 21% O2 and 1% O2. The effect was assesses by Real-time RT-PCR, immunocytochemistry and western blot. Results and Conclusions We found an increase in proliferation rate of myoblasts when activated at a low oxygen tension (1% O2) compared to 21% O2. In addition, the gene expression studies showed up regulation of the myogenesis related genes PAX3, PAX7, MYOD, MYOG (myogenin), MET, NCAM, DES (desmin), MEF2A, MEF2C and CDH15 (M-cadherin), however, the fraction of DES and MYOD positive cells was not increased by low oxygen tension, indicating that 1% O2 may not have a functional effect on the myogenic response. Furthermore, the expression of genes involved in the TGFβ, Notch and Wnt signaling pathways were also up regulated in low oxygen tension. The differences in gene expression were most pronounced at day one after activation from G0-arrest, thus the initial activation of myoblasts seemed most sensitive to changes in oxygen tension. Protein expression of HES1 and β-catenin indicated that notch signaling may be induced in 21% O2, while the canonical Wnt signaling may be induced in 1% O2 during activation and proliferation of myoblasts.

Circulating CD45+CD34+CD11+ cells are immature fibrocytes and the level correlates with the number of mononuclear cells that in vitro differentiate to mature fibrocytes: A potential new marker for intestinal lung disease in rheumatoid arthritis

Background Circulating fibrocytes express both hematopoietic (CD45+ CD34+), and mesenchymal markers (Collagen+). The circulating “immature” fibrocytes can differentiate to “mature” fibrocytes in the target organ and there produce fibrosis components. Chronic inflammatory stimuli mediate differentiation of fibrocytes in diseased organs as observed in idiopathic pulmonary fibrosis (IPF) and asthma, where the levels of circulating fibrocytes correlated to disease severity. In Rheumatoid Arthritis (RA), interstitial lung disease (ILD) is an fibrotic disease manifestation with limited treatment options. Objectives 1) To demonstrate that circulating CD45+ CD34+ CD11b+ cells can express Procollagen and thereby show that these markers can be used to identify circulating fibrocytes. 2) To measure the levels of CD45+, CD34+, CD11b+ cells in patients with RA, Fibrotic ILDs, severe asthma and in healthy controls (HC). 3) To compare the levels with number of peripheral blood mononuclear cells (PBMC) that differentiate to mature fibrocytes in vitro. Methods CD45+, CD34+, CD11b+ cells from 4 patients were isolated by cell sorting, and stained for Procollagen. 30 patients (10 of each RA, fibrotic ILDs: IPF or non-specific interstitial pneumonitis, severe asthma) and 10 HC were included. Current medication, disease activity, lung function and radiographic data were collected. In 100 μL of lysed blood cells were enumerated by flowcytometry. Further, PBMC were isolated and cultured for 5 days. Coverslips from two wells were stained and mature fibrocytes counted and reported as fibrocytes per 106 PBMC originally added to the well. Results A higher fraction of sorted CD45+ CD34+ CD11b+ cells stained positive for Procollagen, compared to CD45+ CD34+ CD11b- cells. All groups had higher level of CD45+ CD34+ CD11b+ cells compared to HC (all p<0.05). HC had a median of 1.02 cells/μL (IQR 0.71–1.32), RA 2.38 (IQR 1.93–4.39), asthma 3.45 (IQR 2.25–8.08) and fibrotic ILDs 5.89 (IQR 2.86–10.2). The number of CD45+ CD34+ CD11b+ cells correlated to number of PBMC that in vitro differentiated to mature fibrocytes, overall (r=0.62, p=0.0006), notably in the RA group (r=0.92, p=0.0005), the level of CD45+ CD34+ CD11b- cells did not. Conclusions This study supports that the markers CD45+ CD34+ CD11b+ can be used to identify immature fibrocytes, as they can express Procollagen and are more abundant in fibrotic diseases compared to HC. Further, the level correlates to the number of PBMC that differentiate to mature fibrocytes in vitro. The results indicate that circulating CD45+, CD34+, CD11b+ cells could be involved in the development of organ fibrosis, and thereby be a biomarker of early fibrosis development in RA ILD. Associations to inflammatory markers, pulmonary function and disease activity will be presented at the conference. Disclosure of Interest None declared