Tristan I. Croll

Transplantation of 3D scaffolds seeded with human embryonic stem cells: biological features of surrogate tissue and teratoma-forming potential

AIM To generate complex surrogate tissue by transplanting 3D scaffolds seeded with human embryonic stem cells (hESCs) between the liver lobules of severe combined immunodeficient (SCID) mice and to assess the teratoma-forming potential. MATERIALS & METHODS 3D poly-(lactic-co-glycolic acid) (PLGA) scaffolds coated with laminin were seeded with hESCs and then transplanted between the liver lobules of SCID mice. After a period of in vivo differentiation, the scaffolds were retrieved and analyzed using reverse transcription polymerase chain reaction, immunofluorescent staining and scanning electron microscopy. RESULTS A proportion of the hESCs within the scaffolds differentiated into cells that produced proteins characteristic of specific tissues, including endoderm and pancreatic markers glucogon-like peptide-1 receptor, islet amyloid polypeptide and Insulin. Markers of hepatic and neuronal lineages were also investigated. Major matrix proteins abundant in multiple tissue types, including collagen I, laminin and collagen IV, were found to be profuse within the scaffold pores. Transplantation of the seeded scaffolds between liver lobules also resulted in extensive vascularization both from host blood vessel incursion and the differentiation of hESCs into endothelial progenitor cells. An investigation of teratoma-forming potential demonstrated that transplantation of 3D scaffolds seeded with hESCs will, under certain conditions, lead to the growth of teratomas. DISCUSSION Transplantation of 3D scaffolds seeded with hESCs between liver lobules resulted in the development of surrogate tissue containing cells that produced proteins representing the pancreatic, hepatic and neuronal lineages, the assembly of an extracellular matrix structure and the formation of a vasculature. hESCs seeded within 3D scaffolds and transplanted into SCID mice were capable of forming teratomas. However, the formation and progression of teratoma growth is shown to be dependant on both the site of transplantation and the treatment of cells prior to transplantation.

Nanoscale presentation of cell adhesive molecules via block copolymer self-assembly.

Precise control over the nanoscale presentation of adhesion molecules and other biological factors represents a new frontier for biomaterials science. Recently, the control of integrin spacing and cellular shape has been shown to affect fundamental biological processes, such as differentiation and apoptosis. Here, we present the self-assembly of maleimide functionalised polystyrene-block-poly (ethylene oxide) copolymers as a simple, yet highly precise method for controlling the position of cellular adhesion molecules. By manipulating the phase separation of the functional PS-PEO block copolymer used in this study, via a simple blending technique, we alter the nanoscale (on PEO domains of 8-14 nm in size) presentation of the adhesion peptide, GRGDS, decreasing lateral spacing from 62 nm to 44 nm and increasing the number density from approximately 450 to approximately 900 islands per microm2. The results indicate that the spreading of NIH-3T3 fibroblasts increases as the spacing between domains of RGD binding peptides decreases. Further, the same functional PS-PEO surfaces have been utilised to immobilise, via a zinc chelating peptide sequence, poly-histidine tagged proteins and extracellular matrix (ECM) fragments. This method is seen as an ideal platform for investigations into the role of spatial arrangements of cell adhesion molecules and ECM molecules on cell function and, in particular, control of cell phenotype.

Higher-Resolution Structure of the Human Insulin Receptor Ectodomain: Multi-Modal Inclusion of the Insert Domain.

Insulin receptor (IR) signaling is critical to controlling nutrient uptake and metabolism. However, only a low-resolution (3.8 Å) structure currently exists for the IR ectodomain, with some segments ill-defined or unmodeled due to disorder. Here, we revise this structure using new diffraction data to 3.3 Å resolution that allow improved modeling of the N-linked glycans, the first and third fibronectin type III domains, and the insert domain. A novel haptic interactive molecular dynamics strategy was used to aid fitting to low-resolution electron density maps. The resulting model provides a foundation for investigation of structural transitions in IR upon ligand binding.

Vitronectin—Master controller or micromanager?

The concept that the mammalian glycoprotein vitronectin acts as a biological ‘glue’ and key controller of mammalian tissue repair and remodelling activity is emerging from nearly 50 years of experimental in vitro and in vivo data. Unexpectedly, the vitronectin‐knockout (VN‐KO) mouse was found to be viable and to have largely normal phenotype. However, diligent observation revealed that the VN‐KO animal exhibits delayed coagulation and poor wound healing. This is interpreted to indicate that VN occupies a role in the earliest events of thrombogenesis and tissue repair. VN is the foundation upon which the thrombus grows in an organised structure. In addition to sealing the wound, the thrombus also serves to protect the underlying tissue from oxidation, is a reservoir of mitogens and tissue repair mediators, and provides a provisional scaffold for the repairing tissue. In the absence of VN (e.g., VN‐KO animal), this cascade is disrupted before it begins. A wide variety of biologically active species associate with VN. Although initial studies were focused on mitogens, other classes of bioactives (e.g., glycosaminoglycans and metalloproteinases) are now also known to specifically interact with VN. Although some interactions are transient, others are long‐lived and often result in multi‐protein complexes. Multi‐protein complexes provide several advantages: prolonging molecular interactions, sustaining local concentrations, facilitating co‐stimulation of cell surface receptors and thereby enhancing cellular/biological responses. We contend that these, or equivalent, multi‐protein complexes facilitate VN polyfunctionality in vivo. It is also likely that many of the species demonstrated to associate with VN in vitro, also associate with VN in vivo in similar multi‐protein complexes. Thus, the predominant biological function of VN is that of a master controller of the extracellular environment; informing, and possibly instructing cells ‘where’ to behave, ‘when’ to behave and ‘how’ to behave (i.e., appropriately for the current circumstance).

The CC domain structure from the wheat stem rust resistance protein Sr33 challenges paradigms for dimerization in plant NLR proteins

Significance Plants and animals use intracellular immunity receptors, known as nucleotide-binding oligomerization domain-like receptors (NLRs), to defend themselves against invading microbes. In this study, we report the solution structure of the N-terminal coiled-coil (CC) domain from the wheat stem rust resistance protein Sr33. Remarkably, this structure differs substantially from the published crystal structure of the equivalent region from the orthologous barley powdery mildew resistance protein MLA10. Using a structural, biophysical, and functional approach, we compare the Sr33 CC domain with other structurally defined NLR CC domains. Collectively, this work redefines our current understanding of the structure and function of plant NLR CC domains, which has significant implications for future studies into this important class of defense receptors. Plants use intracellular immunity receptors, known as nucleotide-binding oligomerization domain-like receptors (NLRs), to recognize specific pathogen effector proteins and induce immune responses. These proteins provide resistance to many of the world’s most destructive plant pathogens, yet we have a limited understanding of the molecular mechanisms that lead to defense signaling. We examined the wheat NLR protein, Sr33, which is responsible for strain-specific resistance to the wheat stem rust pathogen, Puccinia graminis f. sp. tritici. We present the solution structure of a coiled-coil (CC) fragment from Sr33, which adopts a four-helix bundle conformation. Unexpectedly, this structure differs from the published dimeric crystal structure of the equivalent region from the orthologous barley powdery mildew resistance protein, MLA10, but is similar to the structure of the distantly related potato NLR protein, Rx. We demonstrate that these regions are, in fact, largely monomeric and adopt similar folds in solution in all three proteins, suggesting that the CC domains from plant NLRs adopt a conserved fold. However, larger C-terminal fragments of Sr33 and MLA10 can self-associate both in vitro and in planta, and this self-association correlates with their cell death signaling activity. The minimal region of the CC domain required for both cell death signaling and self-association extends to amino acid 142, thus including 22 residues absent from previous biochemical and structural protein studies. These data suggest that self-association of the minimal CC domain is necessary for signaling but is likely to involve a different structural basis than previously suggested by the MLA10 crystallographic dimer.

Scaffolds, stem cells, and tissue engineering: A potent combination!

Stem cells, either from embryonic or adult sources, have demonstrated the potential to differentiate into a wide range of tissues depending on culture conditions. This makes them prime candidates for use in tissue engineering applications. Current technology allows us to process biocompatible and biodegradable polymers into three-dimensional (3D) configurations, either as solid porous scaffolds or hydrogels, with controlled macro and/or micro spatial geometry and surface chemistry. Such control provides us with the ability to present highly controlled microenvironments to a chosen cell type. However, the precise microenvironments required for optimal expansion and/or differentiation of stem cells are only now being elucidated, and hence the controlled use of stem cells in tissue engineering remains a very young field. We present here a brief review of the current literature detailing interactions between stem cells and 3D scaffolds of varying morphology and chemical properties, concluding with remaining challenges for those interested in tissue engineering using tailored scaffolds and stem cells.

Engineering tissue tubes using novel multilayered scaffolds in the rat peritoneal cavity

Our aim was to develop novel scaffolds to engineer tissue tubes of smooth muscle-like cells for autologous grafting. Small diameter tubular poly(lactic acid) scaffolds with randomly distributed, interconnected pores up to 100 mum were produced using a thermally induced phase separation method. The scaffolds were surface modified using various biomolecules via a layer-by-layer deposition technique, and implanted in the peritoneal cavities of rats. Histological analysis of scaffolds 3 weeks after implantation showed fully-developed tissue capsules on their outer surfaces, with macrophage-like cells present throughout the internal spaces. Surfaces coated in Matrigel supported the strongest cellular response whereas multilayer coatings with elastin, collagen I, collagen III, or chitosan outermost showed the lowest levels of cellular interaction. Although differences in capsule thickness and the presence or absence of cellularized layers on the inside and outside surfaces of the scaffolds were observed, none of these biomolecule coatings was able to overcome the foreign body response within the peritoneal cavity, even in the presence of a nonadsorptive HA undercoat.

Multiple functional self-association interfaces in plant TIR domains

Significance Toll/interleukin-1 receptor/resistance protein (TIR) domains are present in plant and animal innate immunity receptors and appear to play a scaffold function in defense signaling. In both systems, self-association of TIR domains is crucial for their function. In plants, the TIR domain is associated with intracellular immunity receptors, known as nucleotide-binding oligomerization domain-like receptors (NLRs). Previous studies from several plant NLRs have identified two distinct interfaces that are required for TIR:TIR dimerization in different NLRs. We show that the two interfaces previously identified are both important for self-association and defense signaling of multiple TIR–NLR proteins. Collectively, this work suggests that there is a common mechanism of TIR domain self-association in signaling across the TIR–NLR class of receptor proteins. The self-association of Toll/interleukin-1 receptor/resistance protein (TIR) domains has been implicated in signaling in plant and animal immunity receptors. Structure-based studies identified different TIR-domain dimerization interfaces required for signaling of the plant nucleotide-binding oligomerization domain-like receptors (NLRs) L6 from flax and disease resistance protein RPS4 from Arabidopsis. Here we show that the crystal structure of the TIR domain from the Arabidopsis NLR suppressor of npr1-1, constitutive 1 (SNC1) contains both an L6-like interface involving helices αD and αE (DE interface) and an RPS4-like interface involving helices αA and αE (AE interface). Mutations in either the AE- or DE-interface region disrupt cell-death signaling activity of SNC1, L6, and RPS4 TIR domains and full-length L6 and RPS4. Self-association of L6 and RPS4 TIR domains is affected by mutations in either region, whereas only AE-interface mutations affect SNC1 TIR-domain self-association. We further show two similar interfaces in the crystal structure of the TIR domain from the Arabidopsis NLR recognition of Peronospora parasitica 1 (RPP1). These data demonstrate that both the AE and DE self-association interfaces are simultaneously required for self-association and cell-death signaling in diverse plant NLRs.

Modeling the adhesion of human embryonic stem cells to poly(lactic-co-glycolic acid) surfaces in a 3D environment

Human embryonic stem cells (hESCs) have previously been cultured on three dimensional (3D) biodegradable polymer scaffolds. Although complex structures were formed from the hESCs, very little is known about the mechanism of adhesion of these cells to the surfaces of the scaffolds. In this study, we achieved the efficient adhesion of pluripotent hESCs to 3D poly(lactic-co-glycolic acid) (PLGA) scaffolds based on our data from a novel two dimensional (2D) model that imitates the surface properties of the scaffolds. In the 2D model, single cell preparations of pluripotent hESCs adhered efficiently and predominantly to PLGA surfaces coated with laminin in comparison to collagen I, collagen IV, or fibronectin-coated surfaces. Flow cytometry analysis revealed that almost all of the pluripotent single cells expressed the integrin alpha 6, with a small percentage also expressing alpha 3ss1, which facilitates adhesion to laminin. This data was then translated into the 3D environment, with the efficient binding of single pluripotent hESCs to PLGA scaffolds coated with laminin. The utility of this system was shown by the directed differentiation of single hESCs seeded within laminin-coated scaffolds toward the endoderm lineage.

Structural basis of TIR-domain-assembly formation in MAL- and MyD88-dependent TLR4 signaling

Toll-like receptor (TLR) signaling is a key innate immunity response to pathogens. Recruitment of signaling adapters such as MAL (TIRAP) and MyD88 to the TLRs requires Toll/interleukin-1 receptor (TIR)-domain interactions, which remain structurally elusive. Here we show that MAL TIR domains spontaneously and reversibly form filaments in vitro. They also form cofilaments with TLR4 TIR domains and induce formation of MyD88 assemblies. A 7-Å-resolution cryo-EM structure reveals a stable MAL protofilament consisting of two parallel strands of TIR-domain subunits in a BB-loop-mediated head-to-tail arrangement. Interface residues that are important for the interaction are conserved among different TIR domains. Although large filaments of TLR4, MAL or MyD88 are unlikely to form during cellular signaling, structure-guided mutagenesis, combined with in vivo interaction assays, demonstrated that the MAL interactions defined within the filament represent a template for a conserved mode of TIR-domain interaction involved in both TLR and interleukin-1 receptor signaling.