Wound healing properties of a fibrin-based dermal replacement scaffold.

When serious cutaneous injury occurs, the innate wound healing process attempts to restore the skin's appearance and function. Wound healing outcome is affected by factors such as contraction, revascularisation, regeneration versus fibrosis and re-epithelialisation and is also strongly influenced by the pattern and extent of damage to the dermal layer. Dermal replacement scaffolds have been designed to substitute for lost tissue, provide a structure to promote dermal regeneration, and aid skin grafting, resulting in a superior healing outcome. In this study the wound healing properties of a novel fibrin-alginate dermal scaffold were assessed in the porcine wound healing model and also compared to two widely used dermal scaffolds and grafting alone. The fibrin-alginate scaffold, unlike the other scaffolds tested, is not used in combination with an overlying skin graft. Fibrin scaffold treated wounds showed increased, sustained superficial blood flow and reduced contraction during early healing while showing comparable wound closure, re-epithelialisation and final wound outcome to other treatments. The increase in early wound vascularisation coupled with a decrease in contraction and no requirement for a skin graft suggest that the fibrin-based scaffold could provide an effective, distinctive treatment option to improve healing outcomes in human patients.

Pre-screening the intrinsic angiogenic capacity of biomaterials in an optimised ex ovo chorioallantoic membrane model.

Biomaterial development for clinical applications is currently on the rise. This necessitates adequate in vitro testing, where the structure and composition of biomaterials must be specifically tailored to withstand in situ repair and regeneration responses for a successful clinical outcome. The chorioallantoic membrane of chicken embryos has been previously used to study angiogenesis, a prerequisite for most tissue repair and regeneration. In this study, we report an optimised ex ovo method using a glass-cling film set-up that yields increased embryo survival rates and has an improved protocol for harvesting biomaterials. Furthermore, we used this method to examine the intrinsic angiogenic capacity of a variety of biomaterials categorised as natural, synthetic, natural/synthetic and natural/natural composites with varying porosities. We detected significant differences in biomaterials' angiogenesis with natural polymers and polymers with a high overall porosity showing a greater vascularisation compared to synthetic polymers. Therefore, our proposed ex ovo chorioallantoic membrane method can be effectively used to pre-screen biomaterials intended for clinical application.

Single-cell assessment of transcriptome alterations induced by Scriptaid in early differentiated human haematopoietic progenitors during ex vivo expansion.

Priming haematopoietic stem/progenitor cells (HSPCs) in vitro with specific chromatin modifying agents and cytokines under serum-free-conditions significantly enhances engraftable HSC numbers. We extend these studies by culturing human CD133+ HSPCs on nanofibre scaffolds to mimic the niche for 5-days with the HDAC inhibitor Scriptaid and cytokines. Scriptaid increases absolute Lin-CD34+CD38-CD45RA-CD90+CD49f+ HSPC numbers, while concomitantly decreasing the Lin-CD38-CD34+CD45RA-CD90- subset. Hypothesising that Scriptaid plus cytokines expands the CD90+ subset without differentiation and upregulates CD90 on CD90- cells, we sorted, then cultured Lin-CD34+CD38-CD45RA-CD90- cells with Scriptaid and cytokines. Within 2-days and for at least 5-days, most CD90- cells became CD90+. There was no significant difference in the transcriptomic profile, by RNAsequencing, between cytokine-expanded and purified Lin-CD34+CD38-CD45RA-CD49f+CD90+ cells in the presence or absence of Scriptaid, suggesting that Scriptaid maintains stem cell gene expression programs despite expansion in HSC numbers. Supporting this, 50 genes were significantly differentially expressed between CD90+ and CD90- Lin-CD34+CD38-CD45RA-CD49f+ subsets in Scriptaid-cytokine- and cytokine only-expansion conditions. Thus, Scriptaid treatment of CD133+ cells may be a useful approach to expanding the absolute number of CD90+ HSC, without losing their stem cell characteristics, both through direct effects on HSC and potentially also conversion of their immediate CD90- progeny into CD90+ HSC.

Engineering the migration and attachment behaviour of primary dermal fibroblasts.

The availability of primary cells present in pathological conditions is often very limited due to stringent ethical regulation and patient consent. One such condition is chronic wounds, where dermal fibroblasts show a deficient migration. In vitro models with cellular tools that mimic the in vivo scenario would be advantageous to test new therapies for these challenging wounds. Since the availability of primary dermal fibroblasts present in chronic wounds is restricted and their "shelf-life" limited due to the increased senescence, our aim was to engineer human dermal fibroblasts with impaired migration using synthetic Arg-Gly-Asp (RGD) peptides. We studied fibroblast behaviour on three different two dimensional (2D) surfaces, representative of the dermal extracellular matrix and the materials used in the development of dermal scaffolds, in addition to commercially available, collagen-based 3D dermal scaffolds, demonstrating that the concentration of synthetic RGD peptides necessary to impair migration of dermal fibroblasts should be tailored to the particular surface/material and cell population used. The described technology could be translated to other cell types including established cell lines. A wide range of synthetic peptides exists, which differ in the amino acid sequence, thus increasing the possibilities of this technology.

The importance of factorial design in tissue engineering and biomaterials science: Optimisation of cell seeding efficiency on dermal scaffolds as a case study.

This article presents a case study to show the usefulness and importance of using factorial design in tissue engineering and biomaterials science. We used a full factorial experimental design (2 × 2 × 2 × 3) to solve a routine query in every biomaterial research project: the optimisation of cell seeding efficiency for pre-clinical in vitro cell studies, the importance of which is often overlooked. In addition, tissue-engineered scaffolds can be cellularised with relevant cell type(s) to form implantable tissue constructs, where the cell seeding method must be reliable and robust. Our results show the complex relationship between cells and scaffolds and suggest that the optimum seeding conditions for each material may be different due to different material properties, and therefore, should be investigated for individual scaffolds. Our factorial experimental design can be easily translated to other cell types and three-dimensional biomaterials, where multiple interacting variables can be thoroughly investigated for better understanding of cell-biomaterial interactions.

Design of a Novel Two-Component Hybrid Dermal Scaffold for the Treatment of Pressure Sores.

The aim of this study is to design a novel two-component hybrid scaffold using the fibrin/alginate porous hydrogel Smart Matrix combined to a backing layer of plasma polymerized polydimethylsiloxane (Sil) membrane to make the fibrin-based dermal scaffold more robust for the treatment of the clinically challenging pressure sores. A design criteria are established, according to which the Sil membranes are punched to avoid collection of fluid underneath. Manual peel test shows that native silicone does not attach to the fibrin/alginate component while the plasma polymerized silicone membranes are firmly bound to fibrin/alginate. Structural characterization shows that the fibrin/alginate matrix is intact after the addition of the Sil membrane. By adding a Sil membrane to the original fibrin/alginate scaffold, the resulting two-component scaffolds have a significantly higher shear or storage modulus G'. In vitro cell studies show that dermal fibroblasts remain viable, proliferate, and infiltrate the two-component hybrid scaffolds during the culture period. These results show that the design of a novel two-component hybrid dermal scaffold is successful according to the proposed design criteria. To the best of the authors' knowledge, this is the first study that reports the combination of a fibrin-based scaffold with a plasma-polymerized silicone membrane.

Viscoelastic, physical, and bio-degradable properties of dermal scaffolds and related cell behaviour.

Dermal scaffolds promote healing of debilitating skin injuries caused by burns and chronic skin conditions. Currently available products present disadvantages and therefore, there is still a clinical need for developing new dermal substitutes. This study aimed at comparing the viscoelastic, physical and bio-degradable properties of two dermal scaffolds, the collagen-based and clinically well established Integra(®) and a novel fibrin-based dermal scaffold developed at our laboratory called Smart Matrix(®), to further evaluate our previous published findings that suggested a higher influx of cells, reduced wound contraction and less scarring for Smart Matrix(®) when used in vivo. Rheological results showed that Integra(®) (G' = 313.74 kPa) is mechanically stronger than Smart Matrix(®) (G' = 8.26 kPa), due to the presence of the silicone backing layer in Integra(®). Micro-pores were observed on both dermal scaffolds, although nano-pores as well as densely packed nano-fibres were only observed for Smart Matrix(®). Average surface roughness was higher for Smart Matrix(®) (Sa = 114.776 nm) than for Integra(®) (Sa = 75.565 nm). Both scaffolds possess a highly porous structure (80-90%) and display a range of pore micro-sizes that represent the actual in vivo scenario. In vitro proteolytic bio-degradation suggested that Smart Matrix(®) would degrade faster upon implantation in vivo than Integra(®). For both scaffolds, the enzymatic digestion occurs via bulk degradation. These observed differences could affect cell behaviour on both scaffolds. Our results suggest that fine-tuning of scaffolds' viscoelastic, physical and bio-degradable properties can maximise cell behaviour in terms of attachment, proliferation and infiltration, which are essential for tissue repair.

A Novel High-Throughput Screening Platform Reveals an Optimized Cytokine Formulation for Human Hematopoietic Progenitor Cell Expansion.

The main limitations of hematopoietic cord blood (CB) transplantation, viz, low cell dosage and delayed reconstitution, can be overcome by ex vivo expansion. CB expansion under conventional culture causes rapid cell differentiation and depletion of hematopoietic stem and progenitor cells (HSPCs) responsible for engraftment. In this study, we use combinatorial cell culture technology (CombiCult®) to identify medium formulations that promote CD133+ CB HSPC proliferation while maintaining their phenotypic characteristics. We employed second-generation CombiCult screens that use electrospraying technology to encapsulate CB cells in alginate beads. Our results suggest that not only the combination but also the order of addition of individual components has a profound influence on expansion of specific HSPC populations. Top protocols identified by the CombiCult screen were used to culture human CD133+ CB HSPCs on nanofiber scaffolds and validate the expansion of the phenotypically defined CD34+CD38lo/-CD45RA-CD90+CD49f+ population of hematopoietic stem cells and their differentiation into defined progeny.

Albumin removal from human fibrinogen preparations for manufacturing human fibrin-based biomaterials.

Commercially available two component human fibrin sealants are commonly used to manufacture human fibrin-based biomaterials. However, this method is costly and allows little room for further tuning of the biomaterial. Human fibrinogen solutions offer a more cost-effective and versatile alternative to manufacture human fibrin-based biomaterials. Yet, human fibrinogen is highly unstable and contains certain impurities like human albumin. Within the context of biomaterials and tissue engineering we offer a simple yet novel solution based on classical biochemical techniques to significantly reduce albumin in human fibrinogen solutions. This method can be used for various tissue engineering and biomedical applications as an initial step in the manufacturing of human fibrin-based biomaterials to optimise their regenerative application.

Method for estimating protein binding capacity of polymeric systems.

Composite biomaterials made from synthetic and protein-based polymers are extensively researched in tissue engineering. To successfully fabricate a protein-polymer composite, it is critical to understand how strongly the protein binds to the synthetic polymer, which occurs through protein adsorption. Currently, there is no cost-effective and simple method for characterizing this interfacial binding. To characterize this interfacial binding, we introduce a simple three-step method that involves: 1) synthetic polymer surface characterisation, 2) a quick, inexpensive and robust novel immuno-based assay that uses protein extraction compounds to characterize protein binding strength followed by 3) an in vitro 2D model of cell culture to confirm the results of the immuno-based assay. Fibrinogen, precursor of fibrin, was adsorbed (test protein) on three different polymeric surfaces: silicone, poly(acrylic acid)-coated silicone and poly(allylamine)-coated silicone. Polystyrene surface was used as a reference. Characterisation of the different surfaces revealed different chemistry and roughness. The novel immuno-based assay showed significantly stronger binding of fibrinogen to both poly(acrylic acid) and poly(allylamine) coated silicone. Finally, cell studies showed that the strength of the interaction between the protein and the polymer had an effect on cell growth. This novel immuno-based assay is a valuable tool in developing composite biomaterials of synthetic and protein-based polymers with the potential to be applied in other fields of research where protein adsorption onto surfaces plays an important role.

Directed differentiation of embryonic stem cells using a bead-based combinatorial screening method.

We have developed a rapid, bead-based combinatorial screening method to determine optimal combinations of variables that direct stem cell differentiation to produce known or novel cell types having pre-determined characteristics. Here we describe three experiments comprising stepwise exposure of mouse or human embryonic cells to 10,000 combinations of serum-free differentiation media, through which we discovered multiple novel, efficient and robust protocols to generate a number of specific hematopoietic and neural lineages. We further demonstrate that the technology can be used to optimize existing protocols in order to substitute costly growth factors with bioactive small molecules and/or increase cell yield, and to identify in vitro conditions for the production of rare developmental intermediates such as an embryonic lymphoid progenitor cell that has not previously been reported.

The implementation of novel collaborative structures for the identification and resolution of barriers to pluripotent stem cell translation.

Increased global connectivity has catalyzed technological development in almost all industries, in part through the facilitation of novel collaborative structures. Notably, open innovation and crowd-sourcing-of expertise and/or funding-has tremendous potential to increase the efficiency with which biomedical ecosystems interact to deliver safe, efficacious and affordable therapies to patients. Consequently, such practices offer tremendous potential in advancing development of cellular therapies. In this vein, the CASMI Translational Stem Cell Consortium (CTSCC) was formed to unite global thought-leaders, producing academically rigorous and commercially practicable solutions to a range of challenges in pluripotent stem cell translation. Critically, the CTSCC research agenda is defined through continuous consultation with its international funding and research partners. Herein, initial findings for all research focus areas are presented to inform global product development strategies, and to stimulate continued industry interaction around biomanufacturing, strategic partnerships, standards, regulation and intellectual property and clinical adoption.

Stem cell technology for drug discovery and development.

Stem cells have enormous potential to revolutionise the drug discovery process at all stages, from target identification through to toxicology studies. Their ability to generate physiologically relevant cells in limitless supply makes them an attractive alternative to currently used recombinant cell lines or primary cells. However, realisation of the full potential of stem cells is currently hampered by the difficulty in routinely directing stem cell differentiation to reproducibly and cost effectively generate pure populations of specific cell types. In this article we discuss how stem cells have already been used in the drug discovery process and how novel technologies, particularly in relation to stem cell differentiation, can be applied to attain widespread adoption of stem cell technology by the pharmaceutical industry.

Non-immortalized human neural stem (NS) cells as a scalable platform for cellular assays.

The utilization of neural stem cells and their progeny in applications such as disease modelling, drug screening or safety assessment will require the development of robust methods for consistent, high quality uniform cell production. Previously, we described the generation of adherent, homogeneous, non-immortalized mouse and human neural stem cells derived from both brain tissue and pluripotent embryonic stem cells (Conti et al., 2005; Sun et al., 2008). In this study, we report the isolation or derivation of stable neurogenic human NS (hNS) lines from different regions of the 8-9 gestational week fetal human central nervous system (CNS) using new serum-free media formulations including animal component-free conditions. We generated more than 20 adherent hNS lines from whole brain, cortex, lobe, midbrain, hindbrain and spinal cord. We also compared the adherent hNS to some aspects of the human CNS-stem cells grown as neurospheres (hCNS-SCns), which were derived from prospectively isolated CD133(+)CD24(-/lo) cells from 16 to 20 gestational week fetal brain. We found, by RT-PCR and Taqman low-density array, that some of the regionally isolated lines maintained their regional identity along the anteroposterior axis. These NS cells exhibit the signature marker profile of neurogenic radial glia and maintain neurogenic and multipotential differentiation ability after extensive long-term expansion. Similarly, hCNS-SC can be expanded either as neurospheres or in extended adherent monolayer with a morphology and marker expression profile consistent with radial glia NS cells. We demonstrate that these lines can be efficiently genetically modified with standard nucleofection protocols for both protein overexpression and siRNA knockdown of exogenously expressed and endogenous genes exemplified with GFP and Nestin. To investigate the functional maturation of neuronal progeny derived from hNS we (a) performed Agilent whole genome microarray gene expression analysis from cultures undergoing neuronal differentiation for up to 32 days and found increased expression over time for a number of drugable target genes including neurotransmitter receptors and ion channels and (b) conducted a neuropharmacology study utilizing Fura-2 Ca(2+) imaging which revealed a clear shift from an initial glial reaction to carbachol to mature neuron-specific responses to glutamate and potassium after prolonged neuronal differentiation. Fully automated culture and scale-up of select hNS was achieved; cells supplied by the robot maintained the molecular profile of multipotent NS cells and performed faithfully in neuronal differentiation experiments. Here, we present validation and utility of a human neural lineage-restricted stem cell-based assay platform, including scale-up and automation, genetic engineering and functional characterization of differentiated progeny.

Embryonic stem cell technology: applications and uses in functional genomic studies.

In this postgenomic era, the role of functional genomics is becoming increasingly important and playing a key role in this field are embryonic stem cells. These cells are capable of proliferating indefinitely in a pluripotent state and have the potential to differentiate into all somatic cell types. Through a combination of their ease of genetic manipulation and directed in vitro differentiation they have proved themselves to be an extremely valuable tool in functional genomics. Here, some of their applications in functional genomic studies are discussed.

The differentiation program of embryonic definitive hematopoietic stem cells is largely alpha4 integrin independent.

Previous analyses of the roles of alpha4 integrins in hematopoiesis by other groups have led to conflicting evidence. alpha4 integrin mutant cells developing in [alpha4 integrin(-/-): wt] chimeric mice are not capable of completing lymphomyeloid differentiation, whereas conditional inactivation of alpha4 integrin in adult mice has only subtle effects. We show here that circumventing the fetal stage of hematopoietic stem cell (HSC) development by transplantation of embryonic alpha4 integrin(-/-) cells into the adult microenvironment results in robust and stable long-term generation of alpha4 integrin(-/-) lymphoid and myeloid cells, although colonization of Peyer patches and the peritoneal cavity is significantly impaired. We argue here that collectively, our data and the data from other groups suggest a specific requirement for alpha4 integrin during the fetal/neonatal stages of HSC development that is essential for normal execution of the lymphomyeloid differentiation program.

ES cell technology: an introduction to genetic manipulation, differentiation and therapeutic cloning.

ES cells are extraordinary cells, capable of proliferating in a pluripotent state indefinitely and of differentiating spontaneously into all cell types in vivo and many in vitro. However, the manipulation and modification of ES cells by processes such as directed differentiation and genetic modification have placed ES cells at the forefront of many biological studies and could lead to their application in biopharmaceutical areas such as cellular therapy and drug screening. Here we describe some of the ES cell based technologies that have lead to this realisation of ES cell potential.

Labeling of hematopoietic stem and progenitor cells in novel activatable EGFP reporter mice.

Conditional activation and inactivation of genes using the Cre/loxP recombination system is a powerful tool for the analysis of gene function and for tracking cell fate. Here we report a novel silent EGFP reporter mouse line generated by enhancer trap technology using embryonic stem (ES) cells. Following transfection with the silent EGFP reporter construct, positive ES cell clones were treated with Cre recombinase. These "activated clones" were then further selected on the basis of ubiquitous EGFP expression during in vitro differentiation. The parental "silent" clones were then used for generating mice. Upon Cre-mediated activation in ovo tissues tested from these mice express EGFP. Long-term, strong and sustainable expression of EGFP is observed in most myeloid and lymphoid cells. As shown by in vivo transplantation assays, the majority of hematopoietic stem cells (HSCs) and spleen colony-forming units (CFU-S) reside within the EGFP positive fraction. Most in vitro colony-forming units (CFU-Cs) isolated from bone marrow also express EGFP. Thus, these reporter mice are useful for the analysis of Cre-mediated recombination in HSCs and hematopoietic progenitor cells. This, in combination with the high accessibility of the loxP sites, makes these mice a valuable tool for testing cell/tissue-specific Cre-expressing mice. .

Quantitative developmental anatomy of definitive haematopoietic stem cells/long-term repopulating units (HSC/RUs): role of the aorta-gonad-mesonephros (AGM) region and the yolk sac in colonisation of the mouse embryonic liver.

In the developing mouse embryo the first definitive (transplantable-into-the-adult) haematopoietic stem cells/long-term repopulating units (HSC/RUs) emerge in the AGM region and umbilical vessels on 10-11 days post coitum (d.p.c.). Here, by limiting dilution analysis, we anatomically map the development of definitive HSC/RUs in different embryonic tissues during early colonisation of the liver. We show that by day 12 p.c. the mouse embryo contains about 66 definitive HSC/RUs (53 in the liver, 13 in other tissues), whereas on the previous day the total number of definitive HSC/RUs in the entire conceptus is only about 3. Owing to the length of the cell cycle this dramatic increase in the number of definitive HSC/RUs in only 24 hours is unlikely to be explained purely by cell division. Therefore, extensive maturation of pre-definitive HSCs to a state when they become definitive must take place in the day 11-12 embryo. Here we firstly identify the numbers of HSCs in various organs at 11-13 d.p.c. and secondly, using an organ culture approach, we quantitatively assess the potential of the aorta-gonadmesonephros (AGM) region and the yolk sac to produce/expand definitive HSC/RUs during days 11-12 of embryogenesis. We show that the capacity of the AGM region to generate definitive HSC/RUs is high on 11 d.p.c. but significantly reduced by 12 d.p.c. Conversely, at 12 d.p.c. the YS acquires the capacity to expand and/or generate definitive HSCs/RUs, whereas it is unable to do so on 11 d.p.c. Thus, the final steps in development of definitive HSC/RUs may occur not only within the AGM region, as was previously thought, but also in the yolk sac microenvironment. Our estimates indicate that the cumulative activity of the AGM region and the yolk sac is sufficient to provide the day 12 liver with a large number of definitive HSC/RUs, suggesting that the large pool of definitive HSC/RUs in day 12 foetal liver is formed predominantly by recruiting 'ready-to-use' definitive HSC/RUs from extra-hepatic sources. In accordance with this we observe growing numbers of definitive HSC/RUs in the circulation during days 11-13 of gestation, suggesting a route via which these HSCs migrate.