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Tenocytes, the main cell type of the tendon, require mechanical stimuli for their proper function. When the tenocyte environment changes due to tissue damage or by transferring tenocytes from their native environment into cell culture, the signals from the tenocyte niche are lost, leading towards a decline of phenotypic markers. It is known that micro-topographies can influence cell fate by the physical cues they provide. To identify the optimal topography-induced biomechanical niche in vitro, we seeded tenocytes on the TopoChip, a micro-topographical screening platform, and measured expression of the tendon transcription factor Scleraxis. Through machine learning algorithms, we associated elevated Scleraxis levels with topological design parameters. Fabricating micro-topographies with optimal surface characteristics on larger surfaces allowed finding an improved expression of multiple tenogenic markers. However, long-term confluent culture conditions coincided with osteogenic marker expression and the loss of morphological characteristics. In contrast, passaging tenocytes which migrated from the tendon directly on the topography resulted in prolonged elongated morphology and elevated Scleraxis levels. This research provides new insights into how micro-topographies influence tenocyte cell fate, and supports the notion that micro-topographical design can be implemented in a new generation of tissue culture platforms for supporting the phenotype of tenocytes. Statement of Significance: The challenge in controlling in vitro cell behavior lies in controlling the complex culture environment. Here, we present for the first time the use of micro-topographies as a biomechanical niche to support the phenotype of tenocytes. For this, we applied the TopoChip platform, a screening tool with 2176 unique micro-topographies for identifying feature characteristics associated with elevated Scleraxis expression, a tendon related marker. Large area fabrication of micro-topographies with favorable characteristics allowed us to find a beneficial influence on other tenogenic markers as well. Furthermore, passaging cells is more beneficial for Scleraxis marker expression and tenocyte morphology compared to confluent conditions. This study presents important insights for the understanding of tenocyte behavior in vitro, a necessary step towards tendon engineering.

Macrophages play a central role in orchestrating immune responses to foreign materials, which are often responsible for the failure of implanted medical devices. Material topography is known to influence macrophage attachment and phenotype, providing opportunities for the rational design of “immune‐instructive” topographies to modulate macrophage function and thus foreign body responses to biomaterials. However, no generalizable understanding of the inter‐relationship between topography and cell response exists. A high throughput screening approach is therefore utilized to investigate the relationship between topography and human monocyte–derived macrophage attachment and phenotype, using a diverse library of 2176 micropatterns generated by an algorithm. This reveals that micropillars 5–10 µm in diameter play a dominant role in driving macrophage attachment compared to the many other topographies screened, an observation that aligns with studies of the interaction of macrophages with particles. Combining the pillar size with the micropillar density is found to be key in modulation of cell phenotype from pro to anti‐inflammatory states. Machine learning is used to successfully build a model that correlates cell attachment and phenotype with a selection of descriptors, illustrating that materials can potentially be designed to modulate inflammatory responses for future applications in the fight against foreign body rejection of medical devices., Using an unbiased screen of an algorithm generated topographical library in combination with machine learning, this study identifies topographies which instruct both the attachment and polarization of human macrophages in the absence of exogenous cytokines.

Learning rules by which cell shape impacts cell function would enable control of cell physiology and fate in medical applications, particularly, on the interface of cells and material of the implants. We defined the phenotypic response of human bone marrow-derived mesenchymal stem cells (hMSCs) to 2176 randomly generated surface topographies by probing basic functions such as migration, proliferation, protein synthesis, apoptosis, and differentiation using quantitative image analysis. Clustering the surfaces into 28 archetypical cell shapes, we found a very strict correlation between cell shape and physiological response and selected seven cell shapes to describe the molecular mechanism leading to phenotypic diversity. Transcriptomics analysis revealed a tight link between cell shape, molecular signatures, and phenotype. For instance, proliferation is strongly reduced in cells with limited spreading, resulting in down-regulation of genes involved in the G2/M cycle and subsequent quiescence, whereas cells with large filopodia are related to activation of early response genes and inhibition of the osteogenic process. In this paper we were aiming to identify a universal set of genes that regulate the material-induced phenotypical response of human mesenchymal stem cells. This will allow designing implants that can actively regulate cellular, molecular signalling through cell shape. Here we are proposing an approach to tackle this question.

Chemical and mechanical cues are well-established influencers of in vitro chondrogenic differentiation of ATDC5 cells. Here, we investigate the role of topographical cues in this differentiation process, a study not been explored before. Previously, using a library of surface micro-topographies we found some distinct patterns that induced alkaline phosphatase (ALP) production in human mesenchymal stromal cells. ALP is also a marker for hypertrophy, the end stage of chondrogenic differentiation preceding bone formation. Thus, we hypothesized that these patterns could influence end-stage chondrogenic differentiation of ATDC5 cells. In this study, we randomly selected seven topographies among the ALP influencing hits. Cells grown on these surfaces displayed varying nuclear shape and actin filament structure. When stimulated with insulin-transferrin-selenium (ITS) medium, nodule formation occurred and in some cases showed alignment to the topographical patterns. Gene expression analysis of cells growing on topographical surfaces in the presence of ITS medium revealed a downregulation of early markers and upregulation of late markers of chondrogenic differentiation compared to cells grown on a flat surface. In conclusion, we demonstrated that surface topography in addition to other cues can promote hypertrophic differentiation suitable for bone tissue engineering.

OBJECTIVE: To generate a clinical and pathologic phenotype of patients carrying rare loss-of-function mutations in ABCA7, identified in a Belgian Alzheimer patient cohort and in an autosomal dominant family. METHODS: We performed a retrospective review of available data records, medical records, results of CSF analyses and neuroimaging studies, and neuropathology data. RESULTS: The mean onset age of the mutation carriers (n = 22) was 73.4 ± 8.4 years with a wide age range of 36 (54-90) years, which was independent of APOE genotype and cerebrovascular disease. The mean disease duration was 5.7 ± 3.0 years (range 2-12 years). A positive family history was recorded for 10 carriers (45.5%). All patient carriers except one presented with memory complaints. The 4 autopsied brains showed typical immunohistochemical changes of late-onset Alzheimer disease. CONCLUSIONS: All patients carrying a loss-of-function mutation in ABCA7 exhibited a classical Alzheimer disease phenotype, though with a striking wide onset age range, suggesting the influence of unknown modifying factors. ispartof: Neurology vol:86 issue:23 pages:2126-2133 ispartof: location:United States status: published

Summary Background Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are extremes of a clinically, pathologically, and genetically overlapping disease spectrum. A locus on chromosome 9p21 has been associated with both disorders, and we aimed to identify the causal gene within this region. Methods We studied 305 patients with FTLD, 137 with ALS, and 23 with concomitant FTLD and ALS (FTLD-ALS) and 856 controls from Flanders (Belgium); patients were identified from a hospital-based cohort and were negative for mutations in known FTLD and ALS genes. We also examined the family of one patient with FTLD-ALS previously linked to 9p21 (family DR14). We analysed 130 kbp at 9p21 in association and segregation studies, genomic sequencing, repeat genotyping, and expression studies to identify the causal mutation. We compared genotype-phenotype correlations between mutation carriers and non-carriers. Findings In the patient-control cohort, the single-nucleotide polymorphism rs28140707 within the 130 kbp region of 9p21 was associated with disease (odds ratio [OR] 2·6, 95% CI 1·5–4·7; p=0·001). A GGGGCC repeat expansion in C9orf72 completely co-segregated with disease in family DR14. The association of rs28140707 with disease in the patient-control cohort was abolished when we excluded GGGGCC repeat expansion carriers. In patients with familial disease, six (86%) of seven with FTLD-ALS, seven (47%) of 15 with ALS, and 12 (16%) of 75 with FTLD had the repeat expansion. In patients without known familial disease, one (6%) of 16 with FTLD-ALS, six (5%) of 122 with ALS, and nine (4%) of 230 with FTLD had the repeat expansion. Mutation carriers primarily presented with classic ALS (10 of 11 individuals) or behavioural variant FTLD (14 of 15 individuals). Mean age at onset of FTLD was 55·3 years (SD 8·4) in 21 mutation carriers and 63·2 years (9·6) in 284 non-carriers (p=0·001); mean age at onset of ALS was 54·5 years (9·9) in 13 carriers and 60·4 years (11·4) in 124 non-carriers. Postmortem neuropathological analysis of the brains of three mutation carriers with FTLD showed a notably low TDP-43 load. In brain at postmortem, C9orf72 expression was reduced by nearly 50% in two carriers compared with nine controls (p=0·034). In familial patients, 14% of FTLD-ALS, 50% of ALS, and 62% of FTLD was not accounted for by known disease genes. Interpretation We identified a pathogenic GGGGCC repeat expansion in C9orf72 on chromosome 9p21, as recently also reported in two other studies. The GGGGCC repeat expansion is highly penetrant, explaining all of the contribution of chromosome 9p21 to FTLD and ALS in the Flanders-Belgian cohort. Decreased expression of C9orf72 in brain suggests haploinsufficiency as an underlying disease mechanism. Unidentified genes probably also contribute to the FTLD-ALS disease spectrum. Funding Full funding sources listed at end of paper (see Acknowledgments).

Background The clusterin (CLU) gene has been identified as an important risk locus for Alzheimer’s disease (AD). Although the actual risk–increasing polymorphisms at this locus remain to be identified, we previously observed an increased frequency of rare non-synonymous mutations and small insertion-deletions of CLU in AD patients, which specifically clustered in the β-chain domain of CLU. Nonetheless the pathogenic nature of these variants remained unclear. Here we report a novel non-synonymous CLU mutation (p.I360N) in a Belgian Alzheimer patient and have explored the pathogenic nature of this and 10 additional CLU mutations on protein localization and secretion in vitro using immunocytochemistry, immunodetection and ELISAs. Results Three patient-specific CLU mutations in the β-chain (p.I303NfsX13, p.R338W and p.I360N) caused an alteration of the subcellular CLU localization and diminished CLU transport through the secretory pathway, indicative of possible degradation mechanisms. For these mutations, significantly reduced CLU intensity was observed in the Golgi while almost all CLU protein was exclusively present in the endoplasmic reticulum. This was further confirmed by diminished CLU secretion in HEK293T and HEK293 FLp-In cell lines. Conclusions Our data lend further support to the contribution of rare coding CLU mutations in the pathogenesis of neurodegenerative diseases. Functional analyses suggest reduced secretion of the CLU protein as the mode of action for three of the examined CLU mutations. One of those is a frameshift mutation leading to a loss of secreted protein, and the other two mutations are amino acid substitutions in the disulfide bridge region, possibly interfering with heterodimerization of the α- and β-chain of CLU. Electronic supplementary material The online version of this article (doi:10.1186/s13024-015-0024-9) contains supplementary material, which is available to authorized users.

Summary Background ABCA7 was identified as a risk gene for Alzheimer's disease in genome-wide association studies (GWAS). It was one of the genes most strongly associated with risk of Alzheimer's disease in a Belgian cohort. Using targeted resequencing, we investigated ABCA7 in this cohort with the aim to directly detect rare and common variations in this gene associated with Alzheimer's disease pathogenesis. Methods We did massive parallel resequencing of ABCA7 after HaloPlex target enrichment of the exons, introns, and regulatory regions in 772 unrelated patients with Alzheimer's disease (mean age at onset 74·6 years [SD 8·9]) recruited at two memory clinics in Flanders, Belgium, and 757 geographically matched community-dwelling controls (mean age at inclusion 73·9 years [8·0]). After bioinformatic processing, common variants were analysed with conditional logistic regression and rare variant association analysis was done in Variant Association Tools. To explore an observed founder effect, additional unrelated patients with Alzheimer's disease (n=183, mean age at onset 78·8 years [SD 6·0]) and control individuals (n=265, mean age at inclusion 56·9 years [10·8]) from the same cohort who had not been included in massive parallel resequencing because of insufficient biosamples were screened for the ABCA7 frameshift mutation Glu709fs with Sanger sequencing. The effect of loss-of-function mutations on ABCA7 expression was investigated with quantitative real-time PCR in post-mortem brains of patients (n=3) and control individuals (n=4); nonsense mediated mRNA decay was investigated in lymphoblast cell lines from three predicted loss-of-function mutation carriers from the cohort of 772 patients with Alzheimer's disease. Findings An intronic low-frequency variant rs78117248 (minor allele frequency 3·8% in 58 patients with Alzheimer's disease and in controls 1·8% in 28 controls) showed strongest association with Alzheimer's disease (odds ratio 2·07, 95% CI 1·31–3·27; p=0·0016), and remained significant after conditioning for the GWAS top single nucleotide polymorphisms rs3764650, rs4147929, and rs3752246 (2·00, 1·22–3·26; p=0·006). We identified an increased frequency of predicted loss-of-function mutations in the patients compared with the controls (relative risk 4·03, 95% CI 1·75–9·29; p=0·0002). One frameshift mutation (Glu709fs) showed a founder effect in the study population, and was found to segregate with disease in a family with autosomal dominant inheritance of Alzheimer's disease. Expression of ABCA7 was reduced in the two carriers of loss-of-function mutations found only in patients with Alzheimer's disease (Glu709fs and Trp1214*) compared with four non-carrier controls (relative expression 0·45, 95% CI 0·25–0·84; p=0·002) and in lymphoblast cell lines from three carriers of Glu709fs compared with those from two non-carrier controls. Interpretation We propose that a low-frequency variant can explain the association between ABCA7 and Alzheimer's disease, and the evidence of loss-of-function mutations in this risk gene suggests that partial loss-of-function of ABCA7 could be a potential pathogenetic mechanism of Alzheimer's disease. Funding Belgian Science Policy Office Interuniversity Attraction Poles program P7/16, Alzheimer Research Foundation, King Baudouin Foundation AB Fund, Methusalem Excellence Program initiative of the Flemish Government, Flanders Impulse Program on Networks for Dementia Research, Research Foundation Flanders, Agency for Innovation by Science and Technology Flanders, University of Antwerp Research Fund, and European Union's Seventh Framework Programme for Research, Technological development and Demonstration (AgedBrainSYSBIO).

Background We have followed-up on the recent genome-wide association (GWA) of the clusterin gene (CLU) with increased risk for Alzheimer disease (AD), by performing an unbiased resequencing of all CLU coding exons and regulatory regions in an extended Flanders-Belgian cohort of Caucasian AD patients and control individuals (n = 1930). Moreover, we have replicated genetic findings by targeted resequencing in independent Caucasian cohorts of French (n = 2182) and Canadian (n = 573) origin and by performing meta-analysis combining our data with previous genetic CLU screenings. Results In the Flanders-Belgian cohort, we identified significant clustering in exons 5-8 of rare genetic variations leading to non-synonymous substitutions and a 9-bp insertion/deletion affecting the CLU β-chain (p = 0.02). Replicating this observation by targeted resequencing of CLU exons 5-8 in 2 independent Caucasian cohorts of French and Canadian origin identified identical as well as novel non-synonymous substitutions and small insertion/deletions. A meta-analysis, combining the datasets of the 3 cohorts with published CLU sequencing data, confirmed that rare coding variations in the CLU β-chain were significantly enriched in AD patients (ORMH = 1.96 [95% CI = 1.18-3.25]; p = 0.009). Single nucleotide polymorphisms (SNPs) association analysis indicated the common AD risk association (GWA SNP rs11136000, p = 0.013) in the 3 combined datasets could not be explained by the presence of the rare coding variations we identified. Further, high-density SNP mapping in the CLU locus mapped the common association signal to a more 5' CLU region. Conclusions We identified a new genetic risk association of AD with rare coding CLU variations that is independent of the 5' common association signal identified in the GWA studies. At this stage the role of these coding variations and their likely effect on the β-chain domain and CLU protein functioning remains unclear and requires further studies.

We previously described an intragenic functional copy number variation (CNV) in complement receptor 1 (CR1) that is associated with Alzheimer disease (AD) risk. A recent study, however, reported a rare CR1 coding variant p.Ser1610Thr (rs4844609) associated with AD susceptibility, explaining the effect of genome wide association (GWA) top single nucleotide polymorphism rs6656401. We assessed the role of the Ser1610Thr variant in AD pathogenesis and the effect on AD-related endophenotypes in a Flanders-Belgian cohort. We evaluated whether this rare variant rather than the CR1 CNV could explain the association of CR1 in our population. The Ser1610Thr variant was not associated with AD, memory impairment, total tau, amyloid beta(1-42) or tau phosphorylated at threonine 181 levels. It did not explain (part of) the association of genome wide association top single-nucleotide polymorphisms rs3818361/rs6656401, nor of the CR1 CNV, with AD in our cohort, whereas the CR1 CNV and rs3818361/rs6656401 represented the same association signal. These findings question a role for the Ser1610Thr variant in AD risk and related endophenotypes, and reaffirm our previous observation that the CR1 CNV could be the true functional risk factor explaining the association between CR1 and AD. (C) 2013 Elsevier Inc. All rights reserved.

Development of multicellular organisms is a highly orchestrated process, with cells responding to factors and features present in the extracellular milieu. Changes in the surrounding environment help decide the fate of cells at various stages of development. This review highlights recent research that details the effects of mechanical properties of the surrounding environment and extracellular matrix and the underlying molecular mechanisms that regulate the behavior of embryonic stem cells (ESCs). In this study, we review the role of mechanical properties during embryogenesis and discuss the effect of engineered microtopographies on ESC pluripotency.