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Biomaterials can control cell and nuclear morphology. Since the shape of the nucleus influences chromatin architecture, gene expression and cell identity, surface topography can control cell phenotype. This study provides fundamental insights into how surface topography influences nuclear morphology, histone modifications, and expression of histone-associated proteins through advanced histone mass spectrometry and microarray analysis. The authors find that nuclear confinement is associated with a loss of histone acetylation and nucleoli abundance, while pathway analysis reveals a substantial reduction in gene expression associated with chromosome organization. In light of previous observations where the authors found a decrease in proliferation and metabolism induced by micro-topographies, they connect these findings with a quiescent phenotype in mesenchymal stem cells, as further shown by a reduction of ribosomal proteins and the maintenance of multipotency on micro-topographies after long-term culture conditions. Also, this influence of micro-topographies on nuclear morphology and proliferation is reversible, as shown by a return of proliferation when re-cultured on a flat surface. The findings provide novel insights into how biophysical signaling influences the epigenetic landscape and subsequent cellular phenotype. ispartof: ADVANCED SCIENCE vol:10 issue:1 ispartof: location:Germany status: published

In the field of regenerative medicine, optimization of the parameters leading to a desirable outcome remains a huge challenge. Examples include protocols for the guided differentiation of pluripotent cells towards specialized and functional cell types, phenotypic maintenance of primary cells in cell culture, or engineering of materials for improved tissue interaction with medical implants. This challenge originates from the enormous design space for biomaterials, chemical and biochemical compounds, and incomplete knowledge of the guiding biological principles. To tackle this challenge, high-throughput platforms allow screening of multiple perturbations in one experimental setup. In this review, we provide an overview of screening platforms that are used in regenerative medicine. We discuss their fabrication techniques, and in silico tools to analyze the extensive data sets typically generated by these platforms.

In vivo cells reside in a complex extracellular matrix (ECM) that presents spatially distributed biochemical and ‑physical cues at the nano- to micrometer scales. Chemical micropatterning is successfully used to generate adhesive islands to control where and how cells attach and restore cues of the ECM in vitro. Although chemical micropatterning has become a powerful tool to study cell–material interactions, only a fraction of the possible micropattern designs was covered so far, leaving many other possible designs still unexplored. Here, a high-throughput screening platform called “Galapagos chip” is developed. It contains a library of 2176 distinct subcellular chemical patterns created using mathematical algorithms and a straightforward UV-induced two-step surface modification. This approach enables the immobilization of ligands in geometrically defined regions onto cell culture substrates. To validate the system, binary RGD/polyethylene glycol patterns are prepared on which human mesenchymal stem cells are cultured, and the authors observe how different patterns affect cell and organelle morphology. As proof of concept, the cells are stained for the mechanosensitive YAP protein, and, using a machine-learning algorithm, it is demonstrated that cell shape and YAP nuclear translocation correlate. It is concluded that the Galapagos chip is a versatile platform to screen geometrical aspects of cell–ECM interaction.

Surface topography is a tool to endow biomaterials with bioactive properties. However, the large number of possible designs makes it challenging to find the optimal surface structure to induce a specific cell response. The TopoChip platform is currently the largest collection of topographies with 2176 in silico designed microtopographies. Still, it is exploring only a small part of the design space due to design algorithm limitations and the surface engineering strategy. Inspired by the diversity of natural surfaces, it is assessed as to what extent the topographical design space and consequently the resulting cellular responses can be expanded using natural surfaces. To this end, 26 plant and insect surfaces are replicated in polystyrene and their surface properties are quantified using white light interferometry. Through machine-learning algorithms, it is demonstrated that natural surfaces extend the design space of the TopoChip, which coincides with distinct morphological and focal adhesion profiles in mesenchymal stem cells (MSCs) and Pseudomonas aeruginosa colonization. Furthermore, differentiation experiments reveal the strong potential of the holy lotus to improve osteogenesis in MSCs. In the future, the design algorithms will be trained with the results obtained by natural surface imprint experiments to explore the bioactive properties of novel surface topographies.

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.

The distribution of signaling molecules following mechanical or chemical stimulation of a cell defines cell polarization, with regions of high active Cdc42 at the front and low active Cdc42 at the rear. As reaction-diffusion phenomena between signaling molecules, such as Rho GTPases, define the gradient dynamics, we hypothesize that the cell shape influences the maintenance of the “front-to-back” cell polarization patterns. We investigated the influence of cell shape on the Cdc42 patterns using an established computational polarization model. Our simulation results showed that not only cell shape but also Cdc42 and Rho-related (in)activation parameter values affected the distribution of active Cdc42. Despite an initial Cdc42 gradient, the in silico results showed that the maximal Cdc42 concentration shifts in the opposite direction, a phenomenon we propose to call “reverse polarization”. Additional in silico analyses indicated that “reverse polarization” only occurred in a particular parameter value space that resulted in a balance between inactivation and activation of Rho GTPases. Future work should focus on a mathematical description of the underpinnings of reverse polarization, in combination with experimental validation using, for example, dedicated FRET-probes to spatiotemporally track Rho GTPase patterns in migrating cells. In summary, the findings of this study enhance our understanding of the role of cell shape in intracellular signaling.

We previously found that surface topographies induce the expression of the Scxa gene, encoding Scleraxis in tenocytes. Because Scxa is a TGF-beta responsive gene, we investigated the link between mechanotransduction and TGF-beta signaling. We discovered that mesenchymal stem cells exposed to both micro-topographies and TGF-beta 2 display synergistic induction of SMAD phosphorylation and transcription of the TGF-beta target genes SCX, alpha-SMA, and SOX9. Pharmacological perturbations revealed that Rho/ROCK/SRF signaling is required for this synergistic response. We further found an activation of the early response genes SRF and EGR1 during the early adaptation phase on micro-topographies, which coincided with higher expression of the TGF-beta type II receptor gene. Of interest, PKC activators Prostratin and Ingenol-3, known for inducing actin reorganization and activation of serum response elements, were able to mimic the topography-induced TGF-beta response. These findings provide novel insights into the convergence of mechanobiology and TGF-beta signaling, which can lead to improved culture protocols and therapeutic applications.

The prevention of biofilm development on the surfaces of implanted medical devices is a global challenge for the healthcare sector. Bioinstructive materials that intrinsically prevent bacterial biofilm formation and drive an appropriate host immune response are required to reduce the burden of healthcare associated infections. Although bacterial surface attachment is sensitive to micro- and nano- surface topographies, its exploitation has been limited by the lack of unbiased high throughput biomaterial screens combined with model-based methods capable of generating correlations and predicting generic responses. Consequently, we sought to fill this knowledge gap by using polymer chips (TopoChips) incorporating 2,176 combinatorially generated micro-topographies. Specific surface topographies exerted a profound impact on bacterial pathogen attachment independent of surface chemistry. A strong correlation between local surface landscape, bacterial attachment and biofilm formation was established using machine learning methods to facilitate analysis of specific surface parameters for predicting attachment. In vitro, lead topographies prevented colonization by motile (P. aeruginosa and P. mirabilis) and non-motile ( Staphylococcus aureus and Acinetobacter baumanii bacterial pathogens. In a murine foreign body infection model, specific anti-attachment topographies were shown to be refractory to P. aeruginosa colonization.

Surface topography guides cell behavior and is a tool to endow biomaterials with bioactive properties. The large number of possible designs makes it challenging to find the optimal surface structure to induce a specific cell response. The TopoChip platform is currently the largest collection of topographies with 2176 in silico designed micro-topographies. Still, it is exploring only a small part of the design space due to the boundary conditions of the design algorithm and the surface engineering strategy. Inspired by the diversity of natural surfaces, we assessed to what extend we could expand the topographical design space and consequently the resulting cellular responses using natural surfaces. To this end, we replicated twenty-six plant and insect surfaces in polystyrene and quantified their surface properties using white light interferometry, image analysis and principle component analysis. Next, we quantified mesenchymal stem cell morphology and the pattern of Pseudomonas aeruginosa colonization and compared it to previous data from TopoChip screens. Our data show that natural surfaces extended the TopoChip design space. Moreover, the natural surfaces induced MSC morphologies and bacterial attachment patterns not previously observed on the TopoChip. In the future, we will train our design algorithms with the results obtained by natural surface imprint experiments to further explore the design space and bio-active properties of surface topography.

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.

Human mesenchymal stem cells (hMSCs) are widely represented in ongoing regenerative medicine clinical trials due to their ease of autologous implantation. In bone regeneration, crosstalk between macrophages and hMSCs is critical with macrophages playing a key role in the recruitment and differentiation of hMSCs. However, engineered biomaterials able to both direct hMSC fate and modulate macrophage phenotype have not yet been identified. A novel combinatorial chemistry-microtopography screening platform, the ChemoTopoChip, is used to identify materials suitable for bone regeneration by screening with human immortalized mesenchymal stem cells (hiMSCs) and human macrophages. The osteoinduction achieved in hiMSCs cultured on the “hit” materials in basal media is comparable to that seen when cells are cultured in osteogenic media, illustrating that these materials offer a materials-induced alternative in bone-regenerative applications. These also exhibit immunomodulatory effects, concurrently polarizing macrophages towards a pro-healing phenotype. Control of cell response is achieved when both chemistry and topography are recruited to instruct the required cell phenotype, combining synergistically. The large library of materials reveals that the relative roles of microtopography and material chemistry are similar, and machine learning identifies key material and topographical features for cell-instruction.

We previously found that surface topographies induce the expression of theScxagene, encoding Scleraxis in tenocytes. BecauseScxais a TGF-β responsive gene, we investigated the link between mechanotransduction and TGF-β signaling. We discovered that mesenchymal stem cells exposed to both micro-topographies and TGF-β2 display synergistic induction of SMAD phosphorylation and transcription of the TGF-β target genesSCXA,a-SMA, andSOX9. Pharmacological perturbations revealed that Rho/ROCK/SRF signaling is required for this synergistic response. We further found an activation of the early response genesSRFandEGR1during the early adaptation phase on micro-topographies, which coincided with higher expression of the TGF-β type-II receptor gene. Of interest, PKC activators Prostratin and Ingenol-3, known for inducing actin reorganization and activation of serum response elements, were able to mimic the topography-induced TGF-β response. These findings provide novel insights into the convergence of mechanobiology and TGF-β signaling, which can lead to improved culture protocols and therapeutic applications.

Mesenchymal stromal cells (MSC) secrete factors that contribute to organ homeostasis and repair in a tissue specific manner. For instance, kidney perivascular mesenchymal stromal cells (kPSCs) can facilitate renal epithelial repair through secretion of hepatocyte growth factor (HGF) while the secretome of bone marrow MSCs gives rise to immunosuppression. Stromal cells function in a complex 3-dimensional (3D) connective tissue architecture that induces conformational adaptation. Here we tested the hypothesis that surface topography and associated cell adaptations dictate stromal cell function through tuning of the cytokines released. To this end, we cultured human bone marrow and kidney perivascular stromal cells in the TopoWell plate, a custom-fabricated multi-well plate containing 76 unique bioactive surface topographies. Using fluorescent imaging, we observed profound changes in cell shape, accompanied by major quantitative changes in the secretory capacity of the MSCs. The cytokine secretion profile was closely related to cell morphology and was stromal cell type specific. Our data demonstrate that stromal cell function is determined by microenvironment structure and can be manipulated in an engineered setting. Our data also have implications for the clinical manufacturing of mesenchymal stromal cell therapy, where surface topography during bioreactor expansion should be taken into account to preserve therapeutic properties.

© 2021 The Authors Human mesenchymal stem cells (hMSCs) are widely represented in regenerative medicine clinical strategies due to their compatibility with autologous implantation. Effective bone regeneration involves crosstalk between macrophages and hMSCs, with macrophages playing a key role in the recruitment and differentiation of hMSCs. However, engineered biomaterials able to simultaneously direct hMSC fate and modulate macrophage phenotype have not yet been identified. A novel combinatorial chemistry-topography screening platform, the ChemoTopoChip, is used here to identify materials suitable for bone regeneration by screening 1008 combinations in each experiment for human immortalized mesenchymal stem cell (hiMSCs) and human macrophage response. The osteoinduction achieved in hiMSCs cultured on the “hit” materials in basal media is comparable to that seen when cells are cultured in osteogenic media, illustrating that these materials offer a materials-induced alternative to osteo-inductive supplements in bone-regeneration. Some of these same chemistry-microtopography combinations also exhibit immunomodulatory stimuli, polarizing macrophages towards a pro-healing phenotype. Maximum control of cell response is achieved when both chemistry and topography are recruited to instruct the required cell phenotype, combining synergistically. The large combinatorial library allows us for the first time to probe the relative cell-instructive roles of microtopography and material chemistry which we find to provide similar ranges of cell modulation for both cues. Machine learning is used to generate structure-activity relationships that identify key chemical and topographical features enhancing the response of both cell types, providing a basis for a better understanding of cell response to micro topographically patterned polymers.

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

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).