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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.

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.

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.

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.

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.

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.