Observations that suggest a contribution of altered dermal papilla mitochondrial function to androgenetic alopecia.

Androgenetic alopecia (AGA) is a prevalent hair loss condition in males that develops due to the influence of androgens and genetic predisposition. With the aim of elucidating genes involved in AGA pathogenesis, we modelled AGA with three-dimensional culture of keratinocyte-surrounded dermal papilla (DP) cells. We co-cultured immortalised balding and non-balding human DP cells (DPCs) derived from male AGA patients with epidermal keratinocyte (NHEK) using multi-interfacial polyelectrolyte complexation technique. We observed up-regulated mitochondria-related gene expression in balding compared with non-balding DP aggregates which indicated altered mitochondria metabolism. Further observation of significantly reduced electron transport chain complex activity (complexes I, IV and V), ATP levels and ability to uptake metabolites for ATP generation demonstrated compromised mitochondria function in balding DPC. Balding DP was also found to be under significantly higher oxidative stress than non-balding DP. Our experiments suggest that application of antioxidants lowers oxidative stress levels and improves metabolite uptake in balding DPC. We postulate that the observed up-regulation of mitochondria-related genes in balding DP aggregates resulted from an over-compensatory effort to rescue decreased mitochondrial function in balding DP through the attempted production of new functional mitochondria. In all, our three-dimensional co-culturing revealed mitochondrial dysfunction in balding DPC, suggesting a metabolic component in the aetiology of AGA.

Polysulfone membranes coated with polymerized 3,4-dihydroxy-l-phenylalanine are a versatile and cost-effective synthetic substrate for defined long-term cultures of human pluripotent stem cells.

Clinical and industrial applications of human pluripotent stem cells (hPSC) require large amounts of cells that have been expanded under defined conditions. Labor-intensive techniques and ill-defined or expensive compounds and substrates are not applicable. Here we describe a chemically defined synthetic substrate consisting of polysulfone (PSF) membranes coated with polymerized 3,4-dihydroxy-l-phenylalanine (DOPA). DOPA/PSF is inexpensive and can be easily produced at various shapes and sizes. DOPA/PSF supports long-term self-renewal of undifferentiated human embryonic (hESC) and human induced pluripotent stem cells (hiPSC) under defined conditions. Pluripotency is maintained for at least 10 passages. Adhesion of hPSC to DOPA/PSF is mainly mediated by a specific integrin heterodimer. Proliferation and gene expression patterns on DOPA/PSF and control substrates are comparable. Labor-intensive cultivation methods and use of serum or coating with proteins are not required. Together, these features make DOPA/PSF attractive for applications where large-scale expansion of human pluripotent stem cells under defined conditions is essential.

Fungus-derived protein particles as cell-adhesive matrices for cell-cultivated food.

Cell-adhesive factors mediate adhesion of cells to substrates via peptide motifs such as the Arg-Gly-Asp (RGD) sequence. With the onset of sustainability issues, there is a pressing need to find alternatives to animal-derived cell-adhesive factors, especially for cell-cultivated food applications. In this paper, we show how data mining can be a powerful approach toward identifying fungal-derived cell-adhesive proteins and present a method to isolate and utilize these proteins as extracellular matrices (ECM) to support cell adhesion and culture in 3D. Screening of a protein database for fungal and plant proteins uncovered that ~5.5% of the unique reported proteins contain RGD sequences. A plot of fungi species vs RGD percentage revealed that 98% of the species exhibited an RGD percentage > = 1%. We observed the formation of protein particles in crude extracts isolated from basidiomycete fungi, which could be correlated to their stability towards particle aggregation at different temperatures. These protein particles were incorporated in 3D fiber matrices encapsulating mouse myoblast cells, showing a positive effect on cell alignment. We demonstrated a cell traction stress on the protein particles (from Flammulina velutipes) that was comparable to cells on fibronectin. A snapshot of the RGD-containing proteins in the fungal extracts was obtained by combining SDS-PAGE and mass spectrometry of the peptide fragments obtained by enzymatic cleavage. Therefore, a sustainable source of cell-adhesive proteins is widely available in the fungi kingdom. A method has been developed to identify candidate species and produce cell-adhesive matrices, applicable to the cell-cultivated food and healthcare industries.

Xeno-free self-assembling peptide scaffolds for building 3D organotypic skin cultures.

Organotypic skin cultures represent in vitro models of skin which can be used for disease modeling, tissue engineering, and screening applications. Non-human collagen is currently the gold standard material used for the construction of the supporting matrix, however, its clinical applications are limited due to its xenogeneic origin. We have developed a novel peptide hydrogel-based skin construct that shows a pluristratified epidermis, basement membrane, and dermal compartment after 3 weeks of in vitro culture. Peptide-based constructs were compared to collagen-based constructs and stratification marker expression was histologically higher in peptide constructs than in collagen constructs. Transepithelial electrical resistance also showed mature barrier function in peptide constructs. This study presents a novel application of the self-assembling peptide hydrogel in a defined xeno-free in vitro system.

Delivery of chemotropic proteins and improvement of dopaminergic neuron outgrowth through a thixotropic hybrid nano-gel.

Chemotropic proteins guide neuronal projections to their final target during embryo development and are useful to guide axons of neurons used in transplantation therapies. Site-specific delivery of the proteins however is needed for their application in the brain to avoid degradation and pleiotropic affects. In the present study we report the use of Poly (ethylene glycol)-Silica (PEG-Si) nanocomposite gel with thixotropic properties that make it injectable and suitable for delivery of the chemotropic protein semaphorin 3A. PEG-Si gel forms a functional gradient of semaphorin that enhances axon outgrowth of dopaminergic neurons from rat embryos or differentiated from stem cells in culture. It is not cytotoxic and its properties allowed its injection into the striatum without inflammatory response in the short term. Long term implantation however led to an increase in macrophages and glial cells. The inflammatory response could have resulted from non-degraded silica particles, as observed in biodegradation assays.

Systems for Muscle Cell Differentiation: From Bioengineering to Future Food.

In light of pressing issues, such as sustainability and climate change, future protein sources will increasingly turn from livestock to cell-based production and manufacturing activities. In the case of cell-based or cultured meat a relevant aspect would be the differentiation of muscle cells into mature muscle tissue, as well as how the microsystems that have been developed to date can be developed for larger-scale cultures. To delve into this aspect we review previous research that has been carried out on skeletal muscle tissue engineering and how various biological and physicochemical factors, mechanical and electrical stimuli, affect muscle cell differentiation on an experimental scale. Material aspects such as the different biomaterials used and 3D vs. 2D configurations in the context of muscle cell differentiation will also be discussed. Finally, the ability to translate these systems to more scalable bioreactor configurations and eventually bring them to a commercial scale will be touched upon.

Primitive Cancer Cell States: A Target for Drug Screening?

At present, most drug screening efforts employ bulk cancer cell populations, which may lead to selection of the more drug-resistant cancer stem cells (CSCs). However, drug screening using CSCs has been limited, mainly owing to the difficulty of their isolation. This article discusses how methods of reprogramming cancer cells to primitive cancer cell states, such as transcription factor reprogramming, epithelial-mesenchymal transition (EMT), conditional reprogramming, and hypoxia, may approach the CSC state and thus be relevant for drug screening purposes. This leads to the importance of recapitulating the stem cell niche in drug assays, which is enabled by recent advances in cell culture and tissue engineering. With the advent of these technologies, this article addresses the question of whether the stem cell phenotype should be targeted for drug screening.

Sensitization of Cancer Cells via Non-Viral Delivery of Apoptosis Inducing Proteins Using a Cationic Bolaamphiphile.

Cationic bolaamphiphile polymers had been previously studied as efficient delivery system for the delivery of proteins with relatively low toxicity. Here, the authors investigate the use of a protein delivery system based on a cationic bolaamphiphile to sensitize cancer cells toward apoptosis-inducing drugs as a novel approach for cancer therapy. The authors demonstrates the efficacy of the system by two strategies. The first strategy involves delivery of a survivin antibody to inhibit survivin activity. Sensitization of MCF-7 cells to doxorubicin is observed by survivin inhibition by antibodies. The IC50 of doxorubicin is reduced ≈2.5-fold after delivery of survivin antibodies to breast cancer cells and induction of apoptosis is shown by Western blotting with apoptosis specific antibodies. In a second approach, functional wild type p53 is delivered into p53-null liver cancer (Hep3B) cells, sensitizing the cells toward the p53 pathway drug, Nutlin. Nutlin reduced the viability of Hep3B cells by ≈42% at 15 µM concentration, demonstrating the effectiveness of p53 delivery. The expression of p21, a downstream target of p53 further confirmed the functional status of the delivered protein. In conclusion. The successful delivery of apoptosis inducing proteins and sensitization of cancer cells via cationic bolaamphiphile polymer represents a promising system for cancer therapeutics.

pH-Degradable imidazolium oligomers as antimicrobial materials with tuneable loss of activity.

Antimicrobial resistance (AMR) has become a global public health threat. One of the major causes of AMR development is the accumulation of low levels of antimicrobials in the environment. To tackle this problem, novel antimicrobial agents that do not leave active residues after treatment are needed. In this study, a strategy for synthesizing a series of main-chain imidazolium oligomers that incorporate carbonate, hemiaminal, ester and urea functional groups to serve as degradable linkers is presented. These oligomers exhibit excellent microbicidal activity and kill E. coli at low concentrations in a short time (99% killing efficiency in 2 min). Moreover, the oligomers are self-degradable and biocompatible. The degradation of these oligomers is studied in buffered solutions with different pH. Under basic conditions (pH = 8), carbonate-linked and ester-linked oligomers degrade to inactive and less toxic small molecules within weeks, making it less likely for these oligomers to induce antimicrobial resistance as compared to traditional antibiotics. The application of these oligomers for the in vivo treatment of S. aureus infected wounds is demonstrated in a mouse model. Notably, the oligomers demonstrate antibacterial efficacy and accelerated wound healing comparable to vancomycin, a first-line antibiotic for the treatment of complicated skin infections.

A Chemically Well-Defined, Self-Assembling 3D Substrate for Long-Term Culture of Human Pluripotent Stem Cells.

Clinical applications of human pluripotent stem cells (PSCs) are limited by the lack of chemically well-defined scaffolds for cell expansion, differentiation, and implantation. In this study, we systematically screened various self-assembling hexapeptides to identify the best matrix for long-term 3D PSC culture. Lysine-containing Ac-ILVAGK-NH2 hydrogels maintained best the pluripotency of human embryonic and induced PSCs even after 30 passages. This peptide matrix is also compatible with the use of xeno-free and defined differentiation media. By exploiting its stimuli-responsive sol-gel transition, arrays of encapsulated PSCs can be bioprinted for large-scale cell expansion and derivation of miniaturized organoid cultures for high-throughput screening.

A general synthesis for PEDOT-coated nonconductive materials and PEDOT hollow particles by aqueous chemical polymerization.

A method for coating functionalized poly(3,4-ethylenedioxythiophene) (PEDOT) on nonconductive substrates in aqueous solution allows the deposition of PEDOT thin layers on various substrates, including silica and polystyrene (PS) nanoparticles, siliceous mesocellular foam, and chitosan-alginate fibers. The surface property is tuned by controlling the monomer composition in the aqueous solutions. Using appropriate organic solvents to remove the PS cores of PEDOT-coated PS nanoparticles, hollow PEDOT particles with single holes and PEDOT capsules can be formed.

Lineage-specific exosomes could override extracellular matrix mediated human mesenchymal stem cell differentiation.

Lineage specification is an essential process in stem cell fate, tissue homeostasis and development. Microenvironmental cues provide direct and selective extrinsic signals to regulate lineage specification of stem cells. Microenvironmental milieu consists of two essential components, one being extracellular matrix (ECM) as the substratum, while the other being cell secreted exosomes and growth factors. ECM of differentiated cells modulates phenotypic expression of stem cells, while their exosomes contain phenotype specific instructive factors (miRNA, RNA and proteins) that control stem cell differentiation. This study demonstrates that osteoblasts-derived (Os-Exo) and adipocytes-derived (Ad-Exo) exosomes contain instructive factors that regulate the lineage specification of human mesenchymal stem cells (hMSCs). Analyses of exosomes revealed the presence of transcription factors in the form of RNA and protein for osteoblasts (RUNX2 and OSX) and adipocytes (C/EBPα and PPARγ). In addition, several miRNAs reported to have osteogenic and adipogenic differentiation potentials are also identified in these exosomes. Kinetic and differentiation analyses indicate that both osteoblast and adipocyte exosomes augment ECM-mediated differentiation of hMSCs into the respective lineage. The combination of osteoblast/adipocyte ECM and exosomes turned-on the lineage specific gene expressions at earlier time points of differentiation compared to the respective ECM or exosomes administered individually. Interestingly, the hMSCs differentiated on osteoblast ECM with adipogenic exosomes showed expression of adipogenic lineage genes, while hMSCs differentiated on adipocyte ECM with osteoblast exosomes showed osteogenic lineage genes. Based on these observations, we conclude that exosomes might override the ECM mediated instructive signals during lineage specification of hMSC.

Synthetic Poly(Vinylalcohol)-Based Membranes for Cartilage Surgery and Repair.

Cell-based therapies for cartilage repair are continually being developed to treat osteoarthritis. The cells are either introduced directly by intra-articular injection or via a cell-seeded matrix scaffold. Here, poly(vinylalcohol)-based membranes are developed to be used for mesenchymal stem cell implantation in cartilage repair procedures, having controllable physicochemical properties such as porosity, mechanical strength, and permeability, and a unique self-sealing property. The membranes possess a bilayer structure with a less porous layer providing mechanical strength and selective permeability, exhibit an elastic modulus of between 0.3 and 0.9 MPa, and are permeable to molecules <40 kDa, which is in the range of cartilage permeability. Three different peptide ligands with the sequences Ac-GCGYGRGDSPG, Ac-GCG(OPG)4REGOFG(OPG)4, and Ac-GCG(OPG)7, respectively, are conjugated to the membranes and subject to in vitro cell adhesion and differentiation assays. Col I/Col II gene expression ratios indicated that the collagen-mimetic peptide, Ac-GCG(OPG)7, best supported mesenchymal stem cell differentiation into the chondrogenic lineage. Although low retention of the membrane is observed in vivo in a rabbit knee model, results suggest that the membrane was able to facilitate mesenchymal stem cell implantation and differentiation to chondrocytes. These PVA-based membranes provide a feasible, synthetic, off-the-shelf material for the delivery of stem cells, and can be modified for other surgical applications.

Engineering a functional three-dimensional human cardiac tissue model for drug toxicity screening.

Cardiotoxicity is one of the major reasons for clinical drug attrition. In vitro tissue models that can provide efficient and accurate drug toxicity screening are highly desired for preclinical drug development and personalized therapy. Here, we report the fabrication and characterization of a human cardiac tissue model for high throughput drug toxicity studies. Cardiac tissues were fabricated via cellular self-assembly of human transgene-free induced pluripotent stem cells-derived cardiomyocytes in pre-fabricated polydimethylsiloxane molds. The formed tissue constructs expressed cardiomyocyte-specific proteins, exhibited robust production of extracellular matrix components such as laminin, collagen and fibronectin, aligned sarcomeric organization, and stable spontaneous contractions for up to 2 months. Functional characterization revealed that the cardiac cells cultured in 3D tissues exhibited higher contraction speed and rate, and displayed a significantly different drug response compared to cells cultured in age-matched 2D monolayer. A panel of clinically relevant compounds including antibiotic, antidiabetic and anticancer drugs were tested in this study. Compared to conventional viability assays, our functional contractility-based assays were more sensitive in predicting drug-induced cardiotoxic effects, demonstrating good concordance with clinical observations. Thus, our 3D cardiac tissue model shows great potential to be used for early safety evaluation in drug development and drug efficiency testing for personalized therapy.

Proliferation and differentiation of human mesenchymal stem cell encapsulated in polyelectrolyte complexation fibrous scaffold.

A biofunctional scaffold was constructed with human mesenchymal stem cells (hMSCs) encapsulated in polyelectrolyte complexation (PEC) fibers. Human MSCs were either encapsulated in PEC fibers and constructed into a fibrous scaffold or seeded on PEC fibrous scaffolds. The proliferation, chondrogenic and osteogenic differentiation of the encapsulated and seeded hMSCs were compared for a culture period of 5.5 weeks. Gene expression and extracellular matrix production showed evidences of chondrogenesis and osteogenesis in the cell-encapsulated scaffolds and cell-seeded scaffolds when the samples were cultured in the chondrogenic and osteogenic differentiation media, respectively. However, better cell proliferation and differentiation were observed on the hMSC-encapsulated scaffolds compared to the hMSC-seeded scaffolds. The study demonstrated that the cell-encapsulated PEC fibers could support proliferation and chondrogenic and osteogenic differentiation of the encapsulated-hMSCs. Together with our previous works, which demonstrated the feasibility of PEC fiber in controlled release of drug, protein and gene delivery, the reported PEC fibrous scaffold system will have the potential in composing a multi-component system for various tissue-engineering applications.

Recapitulating Cell-Cell Interactions for Organoid Construction - Are Biomaterials Dispensable?

Cells are spatially patterned in 3D space to allow an intricately orchestrated exchange of signals that regulate their migration, proliferation, differentiation, and death. In recent years cellular self-assembly has emerged as an attractive method to achieve the complexity of organ structures, where the essential cell types co-cultured under carefully defined conditions in vitro have been shown to give rise to organoids such as the optic cup, brain, intestine, liver, and kidney. In view of these developments, what would the revised role of biomaterial-based technologies be, or do they retain any role at all? This Opinion article maintains that biomaterials will not only retain their value but will also synergize with organoid technologies in recapitulating cell-cell interactions.

Targeting Warburg Effect in Cancers with PEGylated Glucose.

In highly proliferative cancer cells, energy is predominantly produced by a high rate of glycolysis, followed by lactic acid fermentation, despite the availability of oxygen - an observation known as the Warburg effect. As a consequence, cells employing this glycolytic pathway require high uptake of glucose and increased metabolic rates to maintain their proliferation. It has been hypothesized that by blocking glucose uptake using modified glucose molecules, apoptosis in the cancer cells can be induced. In this study, it has been showed that several poly(ethylene glycol) (PEG)-modified glucose compounds could reduce cell proliferation in various cancer cell lines by a phenomenon that blocked the availability of the glucose transporters and reduced AKT1 (serine/threonine-specific protein kinase) activation. Xenograft cancer models that are intravenously administered with glucose-conjugated branched PEG (GBrP) daily for 14 d show little tumor development, as compared to the control group without GBrP treatment. The toxicological effects and the pharmacokinetics of the PEGylated glucose are studied in rodents. The PEGylated glucose exerts no systemic toxicity at 40 mg kg(-1) dosage. However, doses above 80 mg kg(-1) show dose-dependent toxicity in all the organs analyzed. The present results suggest PEGylated glucose as a promising "metabolic therapy" approach for the treatment of cancer.

Cell type dependent morphological adaptation in polyelectrolyte hydrogels governs chondrogenic fate.

Repair of critical-size articular cartilage defects typically involves delivery of cells in biodegradable, 3D matrices. Differences in the developmental status of mesenchymal stem cells (MSCs) and terminally differentiated mature chondrocytes might be a critical factor in engineering appropriate 3D matrices for articular cartilage tissue engineering. This study examined the relationship between material-driven early cell morphological adaptations and chondrogenic outcomes, by studying the influence of aligned collagen type I (Col I) presentation on chondrocytes and MSC in interfacial polyelectrolyte complexation (IPC)-based hydrogels. In the absence of Col I, both chondrocytes and MSCs adopted rounded cell morphology and formed clusters, with chondrocyte clusters favoring the maintenance of hyaline phenotype, while MSC clusters differentiated to fibro-superficial zone-like chondrocytes. Encapsulated chondrocytes in IPC-Col I hydrogel adopted a fibroblastic morphology forming fibro-superficial zone-like phenotype, which could be reversed by inhibiting actin polymerization using cytochalasin D (CytD). In contrast, adoption of fibroblastic morphology by encapsulated MSCs in IPC-Col I facilitated superior chondrogenesis, generating a mature, hyaline neocartilage tissue. CytD treatment abrogated the elongation of MSCs and brought about a single cell-like state, resulting in insignificant chondrogenic differentiation, underscoring the essential requirement of providing matrix environments that are amenable to cell-cell interactions for robust MSC chondrogenic differentiation. Our study demonstrates that MSCs and culture-expanded chondrocytes favour differential microenvironmental niches and emphasizes the importance of designing biomaterials that meet cell type-specific requirements, in adopting chondrocyte or MSC-based approaches for regenerating hyaline, articular cartilage.