Bioengineering the microanatomy of human skin.

Recreating the structure of human tissues in the laboratory is valuable for fundamental research, testing interventions, and reducing the use of animals. Critical to the use of such technology is the ability to produce tissue models that accurately reproduce the microanatomy of the native tissue. Current artificial cell-based skin systems lack thorough characterisation, are not representative of human skin, and can show variation. In this study, we have developed a novel full thickness model of human skin comprised of epidermal and dermal compartments. Using an inert porous scaffold, we created a dermal construct using human fibroblasts that secrete their own extracellular matrix proteins, which avoids the use of animal-derived materials. The dermal construct acts as a foundation upon which epidermal keratinocytes were seeded and differentiated into a stratified keratinised epithelium. In-depth morphological analyses of the model demonstrated very close similarities with native human skin. Extensive immunostaining and electron microscopy analysis revealed ultrastructural details such as keratohyalin granules and lamellar bodies within the stratum granulosum, specialised junctional complexes, and the presence of a basal lamina. These features reflect the functional characteristics and barrier properties of the skin equivalent. Robustness and reproducibility of in vitro models are important attributes in experimental practice, and we demonstrate the consistency of the skin construct between different users. In summary, a new model of full thickness human skin has been developed that possesses microanatomical features reminiscent of native tissue. This skin model platform will be of significant interest to scientists researching the structure and function of human skin.

MicroRNAs in the skin: role in development, homoeostasis and regeneration.

The skin is the largest organ of the integumentary system and possesses a vast number of functions. Due to the distinct layers of the skin and the variety of cells which populate each, a tightly regulated network of molecular signals control development and regeneration, whether due to programmed cell termination or injury. MicroRNAs (miRs) are a relatively recent discovery; they are a class of small non-coding RNAs which possess a multitude of biological functions due to their ability to regulate gene expression via post-transcriptional gene silencing. Of interest, is that a plethora of data demonstrates that a number of miRs are highly expressed within the skin, and are evidently key regulators of numerous vital processes to maintain non-aberrant functioning. Recently, miRs have been targeted as therapeutic interventions due to the ability of synthetic 'antagomiRs' to down-regulate abnormal miR expression, thereby potentiating wound healing and attenuating fibrotic processes which can contribute to disease such as systemic sclerosis (SSc). This review will provide an introduction to the structure and function of the skin and miR biogenesis, before summarizing the literature pertaining to the role of miRs. Finally, miR therapies will also be discussed, highlighting important future areas of research.

Engineering a Multilayered Skin Equivalent: The Importance of Endogenous Extracellular Matrix Maturation to Provide Robustness and Reproducibility.

Human skin equivalents (HSEs) are a valuable tool for both academic and industrial laboratories to further the understanding of skin physiology and associated diseases. Over the last few decades, there have been many advances in the development of HSEs that successfully recapitulate the structure of human skin in vitro; however a main limitation is variability due to the use of complex protocols and exogenous extracellular matrix (ECM) proteins. We have developed a robust and unique full-thickness skin equivalent that is highly reproducible due to the use of a consistent scaffold, commercially available cells, and defined low-serum media. The Alvetex® scaffold technology allows fibroblasts to produce their own endogenous ECM proteins within the scaffold, which alleviates the need for exogenous collagen, and supports the differentiation and stratification of the epidermis. Our full-thickness skin equivalent is generated using a detailed step-by-step protocol, which sequentially forms the multilayered structure of human skin in vitro. This model can be adapted for many downstream applications such as disease modeling and testing of active compounds for cosmetics.

A robust and reproducible human pluripotent stem cell derived model of neurite outgrowth in a three-dimensional culture system and its application to study neurite inhibition.

The inability of neurites to grow and restore neural connections is common to many neurological disorders, including trauma to the central nervous system and neurodegenerative diseases. Therefore, there is need for a robust and reproducible model of neurite outgrowth, to provide a tool to study the molecular mechanisms that underpin the process of neurite inhibition and to screen molecules that may be able to overcome such inhibition. In this study a novel in vitro pluripotent stem cell based model of human neuritogenesis was developed. This was achieved by incorporating additional technologies, notably a stable synthetic inducer of neural differentiation, and the application of three-dimensional (3D) cell culture techniques. We have evaluated the use of photostable, synthetic retinoid molecules to promote neural differentiation and found that 0.01 µM EC23 was the optimal concentration to promote differentiation and neurite outgrowth from human pluripotent stem cells within our model. We have also developed a methodology to enable quick and accurate quantification of neurite outgrowth derived from such a model. Furthermore, we have obtained significant neurite outgrowth within a 3D culture system enhancing the level of neuritogenesis observed and providing a more physiological microenvironment to investigate the molecular mechanisms that underpin neurite outgrowth and inhibition within the nervous system. We have demonstrated a potential application of our model in co-culture with glioma cells, to recapitulate aspects of the process of neurite inhibition that may also occur in the injured spinal cord. We propose that such a system that can be utilised to investigate the molecular mechanisms that underpin neurite inhibition mediated via glial and neuron interactions.

Systems modelling ageing: from single senescent cells to simple multi-cellular models.

Systems modelling has been successfully used to investigate several key molecular mechanisms of ageing. Modelling frameworks to allow integration of models and methods to enhance confidence in models are now well established. In this article, we discuss these issues and work through the process of building an integrated model for cellular senescence as a single cell and in a simple tissue context.

Ferrociphenol lipid nanocapsule delivery by mesenchymal stromal cells in brain tumor therapy.

The prognosis of patients with malignant glioma remains extremely poor despite surgery and improvements in radio- and chemo-therapies. Thus, treatment strategies that specifically target these tumors have the potential to greatly improve therapeutic outcomes. "Marrow-isolated adult multilineage inducible" cells (MIAMI cells) are a subpopulation of mesenchymal stromal cells (MSCs) which possess the ability to migrate to brain tumors. We have previously shown that MIAMI cells were able to efficiently incorporate lipid nanocapsules (LNCs) without altering either their stem cell properties or their migration capacity. In this study, we assessed whether the cytotoxic effects of MIAMI cells loaded with LNCs containing an organometallic complex (ferrociphenol or Fc-diOH) could be used to treat brain tumors. The results showed that MIAMI cells internalized Fc-diOH-LNCs and that this internalization did not induce MIAMI cell death. Furthermore, Fc-diOH-LNC-loaded MIAMI cells produced a cytotoxic effect on U87MG glioma cells in vitro. This cytotoxic effect was validated in vivo after intratumoral injection of Fc-diOH-LNC-loaded MIAMI cells in a heterotopic U87MG glioma model in nude mice. These promising results open up a new field of treatment in which cellular vehicles and nanoparticles can be combined to treat brain tumors.

In vitro and in vivo interactions between glioma and marrow-isolated adult multilineage inducible (MIAMI) cells.

The prognosis of patients with malignant glioma remains extremely poor despite surgery and improvements in radio- and chemo-therapies. We recently showed that marrow-isolated adult mutilineage inducible (MIAMI) cells, a subpopulation of human mesenchymal stromal cells (MSCs), can serve as cellular carriers of drug-loaded nanoparticles to brain tumors. However, the safety of MIAMI cells as cellular treatment vectors in glioma therapy must be evaluated, in particular their effect on glioma growth and their fate in a tumor environment. In this study, we showed that MIAMI cells were able to specifically migrate toward the orthotopic U87MG tumor model and did not influence its growth. In this model, MIAMI cells did not give rise to cells resembling endothelial cells, pericytes, cancer-associated fibroblasts (CAFs), or astrocytes. Despite these encouraging results, the effects of MIAMI cells may be glioma-dependent. MIAMI cells did not migrate toward the orthotopic Lab1 GB and they can induce the proliferation of other glioma cell lines in vitro. Furthermore, a fraction of MIAMI cells was found to be in a state of proliferation in the U87MG tumor environment. These findings indicate that the use of MIAMI cells as cellular treatment vectors for malignant tumors must be controlled. These cells may be used as "suicide vectors": vectors for killing not only tumor cells but themselves.

The potential of combinations of drug-loaded nanoparticle systems and adult stem cells for glioma therapy.

The prognosis of patients with malignant glioma remains extremely poor, despite surgery and improvements in radio- and chemo-therapies. Nanotechnologies hold great promise in glioma therapy as they protect the therapeutic agent and allow its sustained release. However, new paradigms permitting tumor-specific targeting and extensive intratumoral distribution must be developed to efficiently deliver nanoparticles. Modifications and functionalizations of nanoparticles have been developed to specifically track tumor cells. However, these nanoparticles have yielded few clinical results due to intra-patient heterogeneity and inter-patient variability. Stem cells with a specific tropism for brain tumors could be used as delivery vehicles for nanoparticles. Indeed, these cells have a natural tendency to migrate and distribute within the tumor mass and they can also incorporate nanoparticles. Stem cell therapy combined with nanotechnology could be a promising tool to efficiently deliver drugs to brain tumors.

Mesenchymal stem cells as cellular vehicles for delivery of nanoparticles to brain tumors.

The prognosis of patients with malignant glioma remains extremely poor, despite surgery and improvements in radio- and chemo-therapies. Nanotechnologies represent great promise in glioma therapy as they protect therapeutic agent and allow its sustained release. However, new paradigms allowing tumor specific targeting and extensive intratumoral distribution must be developed to efficiently deliver nanoparticles (NPs). Knowing the tropism of mesenchymal stem cells (MSCs) for brain tumors, the aim of this study was to obtain the proof of concept that these cells can be used as NP delivery vehicles. Two types of NPs loaded with coumarin-6 were investigated: poly-lactic acid NPs (PLA-NPs) and lipid nanocapsules (LNCs). The results show that these NPs can be efficiently internalized into MSCs while cell viability and differentiation are not affected. Furthermore, these NP-loaded cells were able to migrate toward an experimental human glioma model. These data suggest that MSCs can serve as cellular carriers for NPs in brain tumors.