Publications

3D model of a human epiblast reveals BMP4-driven symmetry breaking | Nature Cell Biology

Breaking the anterior–posterior symmetry in mammals occurs at gastrulation. Much of the signalling network underlying this process has been elucidated in the mouse; however, there is no direct molecular evidence of events driving axis formation in humans. Here, we use human embryonic stem cells to generate an in vitro 3D model of human epiblast whose size, cell polarity and gene expression are similar to a day 10 human epiblast. A defined dose of BMP4 spontaneously breaks axial symmetry, and induces markers of the primitive streak and epithelial-to-mesenchymal transition. We show that WNT sig- nalling and its inhibitor DKK1 play key roles in this process downstream of BMP4. Our work demonstrates that a model human epiblast can break axial symmetry despite the absence of asymmetry in the initial signal and of extra-embryonic tissues or maternal cues. Our three-dimensional model is an assay for the molecular events underlying human axial symmetry breaking.

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Epac2 elevation reverses inhibition by chondroitin sulfate proteoglycans in vitro and transforms post-lesion inhibitory environment to promote axonal outgrowth in an ex vivo model of spinal cord injury | The Journal of Neuroscience

Millions of patients suffer from debilitating spinal cord injury (SCI) without effective treatments. Elevating cAMP promotes CNS neuron growth in the presence of growth-inhibiting molecules. cAMP’s effects on neuron growth is partly mediated by Epac, comprising Epac1 and Epac2 – the latter predominantly expresses in postnatal neural tissue. Here, we hypothesized that Epac2 activation would enhance axonal outgrowth after SCI. Using in vitro assays, we demonstrated for the first time that Epac2 activation using a specific soluble agonist (S-220) significantly enhanced neurite outgrowth of postnatal rat cortical neurons and markedly overcame the inhibition by chondroitin sulphate proteoglycans and mature astrocytes on neuron growth. We further investigated the novel potential of Epac2 activation in promoting axonal outgrowth by an ex vivo rat model of SCI mimicking post-SCI environment in vivo and by delivering S-220 via a self-assembling Fmoc-based hydrogel that has suitable properties for SCI repair. We demonstrated that S-220 significantly enhanced axonal outgrowth across the lesion gaps in the organotypic spinal cord slices, compared with controls. Furthermore, we elucidated for the first time that Epac2 activation profoundly modulated the lesion environment by reducing astrocyte/microglial activation and transforming astrocytes into elongated morphology that guided outgrowing axons. Finally, we showed that S-220, when delivered by the gel at 3 weeks after contusion SCI in male adult rats, resulted in significantly better locomotor performance for up to 4 weeks post-treatment. Our data demonstrate a promising therapeutic potential of S-220 in SCI, via beneficial effects on neurons and glia post-injury to facilitate axonal outgrowth. 


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How some labs put more bio into biomaterials | Nature Methods

Biomaterials researchers straddle disparate worlds as they develop materials to mimic dynamic tissue-based and cellular events and cues. They draw on such fields as materials science, chemistry, physics, nanofabrication, informatics, physiology, cell biology, genomics and immunology1,2,3. Some of this research, and the commercial ventures it inspires, can lead to, for example, enhanced ways to mimic cells and tissue-based processes, improved approaches to tissue regeneration or better modes of delivering drugs into the body. Such applications draw on basic insight about the complex materials–biology interface that is challenging to characterize and for which new assay types are needed. Some biomaterials labs draw those disparate worlds closer by using ‘omics approaches to ‘read out’ a material’s impact on cells and fine-tune the traits of their materials.

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Molecular mechanism of symmetry breaking in a 3D model of a human epiblast

Breaking the anterior-posterior (AP) symmetry in mammals takes place at gastrulation. Much of the signaling network underlying this process has been elucidated in the mouse, however there is no direct molecular evidence of events driving axis formation in humans. Here, we use human embryonic stem cells to generate an in vitro 3D model of a human epiblast whose size, cell po- larity, and gene expression are similar to a 10-day human epiblast. A defined dose of bone mor- phogenetic protein 4 (BMP4) spontaneously breaks axial symmetry, and induces markers of the primitive streak and epithelial to mesenchymal transition. By gene knockouts and live-cell imag- ing we show that, downstream of BMP4, WNT3 and its inhibitor DKK1 play key roles in this process. Our work demonstrates that a model human epiblast can break axial symmetry despite no asymmetry in the initial signal and in the absence of extraembryonic tissues or maternal cues. Our 3D model opens routes to capturing molecular events underlying axial symmetry breaking phenomena, which have largely been unexplored in model human systems.

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Developing a bioink for use in regenerative medicine

The concept of developing a bioink that can be used for all cell types and all printing techniques is at best unrealistic and at worst impossible. What is much more achievable and also more desirable is a modifiable, modular system. A base material in which mechanical properties can be easily adopted for the chosen additive method and then formulated for specific cell type or multiply cell types involved in the end application.

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The role of biomaterials in stem cell based regenerative medicine

Despite their vast therapeutic potential in such areas as cell therapy and tissue engineering, stem cells have yet to live up to their original hype and demonstrate widespread clinical success. There is only one FDA-approved stem cell treatment, that being the use of blood-forming stem cells derived from cord blood, which have been used for the treatment of leukaemia for decades. Despite the recent rapid advances within the field of stem cell research, and an ever-growing population of researchers in academia, biotech, and the pharmaceutical industry focused on the development of stem cell-based treatments for a range of conditions, there has been a limited progression of such research from bench to bedside.

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Controlling cancer cell fate using localized biocatalytic self-assembly of an aromatic carbohydrate amphiphile

This paper describes a simple carbohydrate amphiphile able to self-assemble into nanofibers upon enzymatic dephosphorylation. The self-assembly can be triggered by alkaline phosphatase (ALP) in solution or in situ by the ALP produced by osteosarcoma cell line, SaOs2. In the latter case, assembly and localized gelation occurs mainly on the cell surface. The gelation of the pericellular environment induces a reduction of the SaOs2 metabolic activity at an initial stage (≤7 h) that results in cell death at longer exposure periods (≥24 h). We show that this effect depends on the phosphatase concentration and thus, it is cell-selective with prechondrocytes ATDC5 (that express ~15-20 times lower ALP activity compared to SaOs2) not being affected at concentrations ≤ 1 mM. These results demonstrate that simple carbohydrate derivatives can be used in an anti-osteosarcoma strategy with limited impact on the surrounding healthy cells/tissues.

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Probing the Metabolomics of Stem Cell Differentiation with Biomaterials

In this issue of Chem, Ulijn, Dalby, and co-workers have developed a novel peptide-based hydrogel platform as a screening tool for identifying metabolites that can bias differentiation of mesenchymal stem cells.

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Stem Cell Fate Is a Touchy Subject

Uncoupling synergistic interactions between physio-chemical cues that guide stem cell fate may improve efforts to direct their differentiation in culture. Using supramolecular hydrogels, Alakpa et al. (2016) demonstrate that mesenchymal stem cell differentiation is paired to depletion of bioactive metabolites, which can be utilized to chemically induce osteoblast and chondrocyte fate.

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Self-assembled peptide-based hydrogels as scaffolds for anchorage-dependent cells

We report here the design of a biomimetic nanofibrous hydrogel as a 3D-scaffold for anchorage-dependent cells. The peptide-based bioactive hydrogel is formed through molecular self-assembly and the building blocks are a mixture of two aromatic short peptide derivatives: Fmoc-FF (Fluorenylmethoxycarbonyl-diphenylalanine) and Fmoc-RGD (arginine-glycine-aspartate) as the simplest self-assembling moieties reported so far for the construction of small-molecule-based bioactive hydrogels. This hydrogel provides a highly hydrated, stiff and nanofibrous hydrogel network that uniquely presents bioactive ligands at the fibre surface; therefore it mimics certain essential features of the extracellular matrix. The RGD sequence as part of the Fmoc-RGD building block plays a dual role of a structural component and a biological ligand. Spectroscopic and imaging analysis using CD, FTIR, fluorescence, TEM and AFM confirmed that FF and RGD peptide sequences self-assemble into beta-sheets interlocked by pi-pi stacking of the Fmoc groups. This generates the cylindrical nanofibres interwoven within the hydrogel with the presence of RGDs in tunable densities on the fibre surfaces. This rapid gelling material was observed to promote adhesion of encapsulated dermal fibroblasts through specific RGD-integrin binding, with subsequent cell spreading and proliferation; therefore it may offer an economical model scaffold to 3D-culture other anchorage-dependent cells for in-vitro tissue regeneration.

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Three-dimensional cell culture of chondrocytes on modified di-phenylalanine scaffolds

The design of self-assembled peptide-based structures for three-dimensional cell culture and tissue repair has been a key objective in biomaterials science for decades. In search of the simplest possible peptide system that can self-assemble, we discovered that combinations of di-peptides that are modified with aromatic stacking ligands could form nanometre-sized fibres when exposed to physiological conditions. For example, we demonstrated that a number of Fmoc (fluoren-9-ylmethyloxycarbonyl) modified di- and tri-peptides form highly ordered hydrogels via hydrogen-bonding and pi-pi interactions from the fluorenyl rings. These highly hydrated gels allowed for cell proliferation of chondrocytes in three dimensions [Jayawarna, Ali, Jowitt, Miller, Saiani, Gough and Ulijn (2006) Adv. Mater. 18, 611-614]. We demonstrated that fibrous architecture and physical properties of the resulting materials were dictated by the nature of the amino acid building blocks. Here, we report the self-assembly process of three di-phenylalanine analogues, Fmoc-Phe-Phe-OH, Nap (naphthalene)-Phe-Phe-OH and Cbz (benzyloxycarbonyl)-Phe-Phe-OH, to compare and contrast the self-assembly properties and cell culture conditions attributable to their protecting group difference. Fibre morphology analysis of the three structures using cryo-SEM (scanning electron microscopy) and TEM (transmission electron microscopy) suggested fibrous structures with dramatically varying fibril dimensions, depending on the aromatic ligand used. CD and FTIR (Fourier-transform IR) data confirmed beta-sheet arrangements in all three samples in the gel state. The ability of these three new hydrogels to support cell proliferation of chondrocytes was confirmed for all three materials.

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Fmoc-Diphenylalanine Self Assembles to a Hydrogel via a Novel Architecture Based on pi–pi Interlocked β-Sheets

A number of strategies exist to design molecular materials based on self-assembled peptides and their derivatives. These include soft materials based on a variety of structural motifs including coiled-coils, β-sheets, β-hairpins, and peptide amphiphiles. In these systems, the peptide chains usually contain at least ten amino acids. It has been known for some time that using aromatic components in conjunction with peptides allows the use of much smaller peptides by taking advantage of pi-pi stacking interactions. One system that has been illustrated is that of N-fluorenylmethoxycarbonyl di-phenylalanine (Fmoc-FF) which forms a hydrogel under physiological conditions.

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Nanostructured Hydrogels for Three-Dimensional Cell Culture Through Self-Assembly of Fluorenylmethoxycarbonyl– Dipeptides

Materials that form spontaneously by self-assembly from natural or synthetic building blocks are promising for a variety of applications. In biomedicine, there is significant interest in exploiting self-assembly to construct mimics of the extracellular matrix (ECM) for cell-culture applications. Zhang and co-workers described the use of octapeptides and hexadecapeptides with alternating charged and hydrophobic amino acids to generate highly hydrated gels for cultures of nerve cells, endothelial cells, and chondrocytes. Stupp and co-workers demonstrated that bioactive pentapeptides modified with octapeptide spacers and aliphatic tails spontaneously form nanofibrous scaffolds that support selective differentiation of neural progenitor cells. These successful results clearly illustrate that man-made hydrogels hold promise as scaffold materials for three-dimensional (3D) cell cultures and tissue engineering. This communication details the spontaneous assembly, under physiological conditions, of much smaller amphiphilic building blocks consisting of (mixtures of) dipeptides linked to fluorenylmethoxycarbonyl, and their use as scaffold materials in 3D cell culture.

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TEDD Annual Meeting with 3D Bioprinting Workshop

Bioprinting is the technology of choice for realizing functional tissues such as vascular system, muscle, cartilage and bone. In the future, bioprinting will influence the way we engineer tissues and bring it to a new level of physiological relevance. That was the topic of the 2017 TEDD Annual Meeting at ZHAW Waedenswil on 8th and 9th November. In an exciting workshop and symposium, companies such as regenHU Ltd., CELLINK and Biogelx Ltd. gave us an insight into highly topical applications and collaborations in this domain.

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Improving cartilage phenotype from differentiated pericytes in tunable peptide hydrogels

Differentiation of stem cells to chondrocytes in vitro usually results in a heterogeneous phenotype. This is evident in the often detected over expression of type X collagen which, in hyaline cartilage structure is not characteristic of the mid-zone but of the deep-zone ossifying tissue. Methods to better match cartilage developed in vitro to characteristic in vivo features are therefore highly desirable in regenerative medicine. This study compares phenotype characteristics between pericytes, obtained from human adipose tissue, differentiated using diphenylalanine/serine (F2/S) peptide hydrogels with the more widely used chemical induced method for chondrogenesis. Significantly higher levels of type II collagen were noted when pericytes undergo chondrogenesis in the hydrogel in the absence of induction media. There is also a balanced expression of collagen relative to aggrecan production, a feature which was biased toward collagen production when cells were cultured with induction media. Lastly, metabolic profiles of each system show considerable overlap between both differentiation methods but subtle differences which potentially give rise to their resultant phenotype can be ascertained. The study highlights how material and chemical alterations in the cellular microenvironment have wide ranging effects on resultant tissue type.

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Introducing chemical functionality in Fmoc-peptide gels for cell culture

Aromatic short peptide derivatives, i.e. peptides modified with aromatic groups such as 9-fluorenylmethoxycarbonyl (Fmoc), can self-assemble into self-supporting hydrogels. These hydrogels have some similarities to extracellular matrices due to their high hydration, relative stiffness and nanofibrous architecture. We previously demonstrated that Fmoc-diphenylalanine (Fmoc-F2) provides a suitable matrix for two-dimensional (2D) or three-dimensional (3D) culture of primary bovine chondrocytes. In this paper we investigate whether the introduction of chemical functionality, such as NH2, COOH or OH, enhances compatibility with different cell types. A series of hydrogel compositions consisting of combinations of Fmoc-F2 and N-protected Fmoc amino acids, lysine (K), glutamic acid (D), and serine (S) were studied. All compositions produced fibrous scaffolds with fibre diameters in the range of 32–65 nm as assessed by cryo-scanning electron microscopy and atomic force microscopy. Fourier transform infrared spectroscopy analysis suggested that peptide segments adopt a predominantly antiparallel β-sheet conformation. Oscillatory rheology results show that all four hydrogels have mechanical profiles of soft viscoelastic materials with elastic moduli dependent on the chemical composition, ranging from 502 Pa (Fmoc-F2/D) to 21.2 KPa (Fmoc-F2). All gels supported the viability of bovine chondrocytes as assessed by a live–dead staining assay. Fmoc-F2/S and Fmoc-F2/D hydrogels in addition supported viability for human dermal fibroblasts (HDF) while Fmoc-F2/S hydrogel was the only gel type that supported viability for all three cell types tested. Fmoc-F2/S was therefore investigated further by studying cell proliferation, cytoskeletal organization and histological analysis in 2D culture. In addition, the Fmoc-F2/S gel was shown to support retention of cell morphology in 3D culture of bovine chondrocytes. These results demonstrate that introduction of chemical functionality into Fmoc-peptide scaffolds may provide gels with tunable chemical and mechanical properties for in vitro cell culture.

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Tunable Supramolecular Hydrogels for Selection of Lineage-Guiding Metabolites in Stem Cell Cultures

Stem cells are known to differentiate in response to the chemical and mechanical properties of the substrates on which they are cultured. Thus, supramolecular biomaterials with tunable properties are well suited for the study of stem cell differentiation. In this report, we exploited this phenomenon by combining stem cell differentiation in hydrogels with variable stiffness and metabolomics analysis to identify specific bioactive lipids that are uniquely used up during differentiation. To achieve this, we cultured perivascular stem cells on supramolecular peptide gels of different stiffness, and metabolite depletion followed. On soft (1 kPa), stiff (13 kPa), and rigid (32 kPa) gels, we observed neuronal, chondrogenic, and osteogenic differentiation, respectively, showing that these stem cells undergo stiffness-directed fate selection. By analyzing concentration variances of >600 metabolites during differentiation on the stiff and rigid gels (and focusing on chondrogenesis and osteogenesis as regenerative targets, respectively), we identified that specific lipids (lysophosphatidic acid and cholesterol sulfate, respectively), were significantly depleted. We propose that these metabolites are therefore involved in the differentiation process. In order to unequivocally demonstrate that the lipid metabolites that we identified play key roles in driving differentiation, we subsequently demonstrated that these individual lipids can, when fed to standard stem cell cultures, induce differentiation toward chondrocyte and osteoblast phenotypes. Our concept exploits the design of supramolecular biomaterials as a strategy for discovering cell-directing bioactive metabolites of therapeutic relevance.

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Bioresponsive hydrogels

The study highlights developments in hydrogel materials with biological responsiveness built in. These ‘smart’ biomaterials change properties in response to selective biological recognition events. When exposed to a biological target (nutrient, growth factor, receptor, antibody, enzyme, or whole cell), molecular recognition events trigger changes in molecular interactions that translate into macroscopic responses, such as swelling/collapse or solution-to-gel transitions. The hydrogel transitions may be used directly as optical readouts for biosensing, linked to the release of actives for drug delivery, or instigate biochemical signaling events that control or direct cellular behavior. Accordingly, bioresponsive hydrogels have gained significant interest for application in diagnostics, drug delivery, and tissue regeneration/wound healing.

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