Human reconstructed skin xenografts on mice to model skin physiology

Author’s Accepted Manuscript Human reconstructed skin xenografts on mice to model skin physiology Giorgiana Salgado, Yi Zhen Ng, Li Fang Koh, Christabelle S.M. Goh, John E. Common www.elsevier.com/locate/diff

PII: DOI: Reference:

S0301-4681(17)30102-0 http://dx.doi.org/10.1016/j.diff.2017.09.004 DIFF507

To appear in: Differentiation Received date: 1 September 2017 Revised date: 11 September 2017 Accepted date: 12 September 2017 Cite this article as: Giorgiana Salgado, Yi Zhen Ng, Li Fang Koh, Christabelle S.M. Goh and John E. Common, Human reconstructed skin xenografts on mice to model skin physiology, Differentiation, http://dx.doi.org/10.1016/j.diff.2017.09.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

TITLE PAGE

Human reconstructed skin xenografts on mice to model skin physiology

Giorgiana Salgado1, Yi Zhen Ng1, Li Fang Koh1, Christabelle S. M. Goh1, John E. Common1,2,#

1Institute 2Skin

of Medical Biology, A*STAR, Singapore 138648, Singapore

Research Institute of Singapore

#Corresponding

author

Key words Human skin xenografts, humanized mouse, skin transplantation, human-mouse chimera, human immune system

Abbreviations used ECM, Extracellular matrix; EGFR, Epidermal growth factor receptor; GvHD, Graft versus host disease; HLA, Human leukocyte antigen; HSC, hematopoietic stem cells; LAB, Laser-assisted bioprinting; NOD, Non-obese diabetic; NSG, NOD SCID gamma; PBMC, Peripheral blood mononuclear cells; SCID, Severe combined immune

deficiency;

SCC,

dimethyl[a]benzanthracene

Corresponding author John E A Common, Ph.D.

squamous

cell

carcinoma;

DMBA,

7,12-

Institute of Medical Biology, A*STAR (Agency for Science, Technology and Research), 8A Biomedical Grove, Immunos #06-08 Singapore 138648 Telephone: (65) 6407 0508 Fax (65) 64642049 Email: [email protected]

ABSTRACT

Xenograft models to study skin physiology have been popular for scientific use since the 1970s, with various developments and improvements to the techniques over the decades. Xenograft models are particularly useful and sought after due to the lack of clinically relevant animal models in predicting drug effectiveness in humans. Such predictions could in turn boost the process of drug discovery, since novel drug compounds have an estimated 8% chance of FDA approval despite years of rigorous preclinical testing and evaluation, albeit mostly in nonhuman models. In the case of skin research, the mouse persists as the most popular animal model of choice, despite its well-known anatomical differences with human skin. Differences in skin biology are especially evident when trying to dissect more complex skin conditions, such as psoriasis and eczema, where interactions between the immune system, epidermis and the environment likely occur. While the use of animal models are still considered the gold standard for systemic toxicity studies under controlled environments, there are now alternative models that have been approved for certain applications. To overcome the biological limitations of the mouse model, research efforts have also focused on “humanizing” the mice model to better recapitulate human skin physiology. In this review, we outline the different approaches undertaken thus far to study skin biology using human tissue xenografts in mice and the technical challenges involved. We also describe more recent developments to generate humanized multi-tissue compartment mice that carry both a functioning human

immune system and skin xenografts. Such composite animal models provide promising opportunities to study drugs, disease and differentiation with greater clinical relevance.

INTRODUCTION

The use of the laboratory mouse, Mus musculus, has emerged as the cornerstone of in vivo preclinical research. The mouse genome has been sequenced for numerous extensively-used laboratory mouse strains, and approximately 99% of mouse genes have a corresponding human homolog (and vice versa) (Mouse Genome Sequencing et al., 2002). Due to their genetic similarity to humans and relative ease of handling, they are the foremost mammalian model used to gain insights into human development, disease and drug testing. With the advent of genetic engineering in the 1970s, it was not long before transgenic mice became a reality (Palmiter and Brinster, 1986). The mouse model became an evolving paradigm to investigate the systemic biological functions of genes, and consequently, human pathology. There are now more than 5000 mouse genotypes modeling more than 1000 human diseases (Blake et al., 2017), including many inherited human skin diseases, skin cancers and inflammatory skin disorders (Chen and Roop, 2008; DiTommaso et al., 2014; Haase et al., 2004; Liakath-Ali et al., 2014). As an important preclinical model, the laboratory mouse is used to test for drug efficacy and toxicity. This is often a compulsory drug regulatory requirement, where the systemic effects of the drugs are studied under controlled conditions in a model organism. However, major challenges still remain when translating and applying the results from mouse experiments to humans, as evidenced by the majority of novel drugs being unsuccessful despite promising preclinical animal trials (Adams and Brantner, 2006; FDA, 2004; Hackam and Redelmeier, 2006). Unsatisfactory translation has been attributed

to several reasons, including suboptimal experimental design, lack of methodological rigour and poor matching of disease phenotypes in mice and humans. The last factor is more apparent in more complex human diseases that are difficult to mimic fully in a mouse model. A classic example is the amytrophic lateral sclerosis (ALS) mouse model with mutations in the RNA binding protein TDP-43 that was purported to display neurodegenerative traits similar to human ALS (Wegorzewska et al., 2009). Drugs developed and tested in human trials ultimately failed due to the model more closely representing gastrointestinal neurodegenerative disease than ALS (Hatzipetros et al., 2014; Perrin, 2014). This example highlights the need for scientists to proceed with caution and due diligence when using mouse models for disease modeling. Comparison of human and mouse skin tissue reveals that significant species differences exist that are important to consider when using the mouse as a substitute for human physiology and disease (Gerber et al., 2014; Khavari, 2006). The skin comprises at least three specialized tissue compartments in both human and mouse: (i) The epithelial compartment that includes the stratified epidermis, hair follicles and sweat glands, consisting mainly of keratinocytes and home to the pigment producing melanocytes and resident specialist immune cells such as Langerhans cells and CD8+ T cells (Pasparakis et al., 2014). (ii) The underlying dermal layer, which is a complex network of extracellular matrix (ECM) with its own specialized cells and structures, including the mesenchymal fibroblasts, various immune cell subsets and both vascular and lymphatic structures. (iii) The subcutaneous adipose tissue, which contains mainly adipocytes but also fibroblasts, endothelial cells, and immune cells, and has now been recognized as being rich in stem cells with various regenerative capacities

(Klar et al., 2017). A key difference between humans and mice is the detailed composition of these compartments and the functions that they deliver. For example, the most visually obvious difference is dense fur coverage on murine skin, with a hair cycle that undergoes a defined synchronous cycle of hair growth that is different from the asynchronous human hair growth cycle (Paus et al., 1999). Histologically, there are structural differences in the skin of the mouse compared to the human, such as a significantly thinner epidermis and dermis in mice, lack of sweat glands (except on the paws) and looser skin attachment (Figure 1). Additionally, murine skin has a thinner subcutaneous adipose layer with a distinct panniculus carnosus, a thin skeletal muscle layer, below the adipose tissue. This layer is limited in humans to the platysma muscle of the neck. In relation to this, in terms of wound healing, unlike human skin which heals by re-epithelization, murine skin heals with a major contraction component (Wong et al., 2011). Moreover, other systemic differences between the human and mouse immune system exist that impact on the skin; these have been reviewed in great detail elsewhere (Mestas and Hughes, 2004; Pasparakis et al., 2014; Sellers, 2017). Due to these differences, the murine genomic response is unrepresentative of human inflammatory diseases (Seok et al., 2013). One clear example for skin is that the major T cell type found in human dermis is the conventional αβ T cells, however, in mice the γδ T cell predominates in the dermis (Bos et al., 1990; Cai et al., 2011; Pasparakis et al., 2014). Due to these highlighted differences between mouse and human skin, it is understandable that many transgenic animal models to study skin physiology or mimic skin diseases are unable to fully recapitulate the corresponding human condition.

As the outermost barrier under constant environmental exposure, it is perhaps not surprising that the skin is under a stronger evolutionary pressure than other tissues. This is reflected in the comparison of 30 tissue-specific gene sets between mouse and human, which found skin to be the one of the topmost evolutionally divergent tissues, along with the eye and testis (Monaco et al., 2015). Only 30% identity exists between human and mouse skin associated genes (Gerber et al., 2014). For example, epidermal growth factor receptor (EGFR) was found to be elevated in human skin compared to murine skin, leading the authors to postulate this underlies the lack of harmful side effects in mice in response to EGFR inhibition, unlike what was observed in humans. In relation to the abovementioned predominance of γδ T cells in mice, the authors also report Skint proteins, which have been linked to maintenance of γδ T cells, as being exclusive to mice (Gerber et al., 2014). Given the concerns regarding the differences and deficiencies in traditional mouse model, efforts have been directed at tackling these problems by engineering “human-like” or “humanized” mouse models. These humanized mouse models express human transgenes or are engrafted with functional human cells or tissues. Instead of relying on mouse skin as a surrogate tissue to understand human responses, such models can more accurately reflect human physiology and better predict relevant clinical response. The scope of this chapter focuses on recent progress in the development of such humanized animals for use in skin research and is divided into 2 major approaches: (1) human skin xenografts, and (2) human immune-responsive, humanized skin xenografts. While such complex animal models may be technically challenging,

they provide great opportunities to study drugs, disease pathology and tissue differentiation with greater clinical relevance.

HUMAN SKIN XENOGRAFTS

Human skin xenografts were made possible in the early 1970s with the advent of immune-deficient mice, reducing host-graft rejection and thereby allowing grafted skin to potentially remain in situ for the lifetime of the mouse (Reed and Manning, 1973; Rygaard, 1974). Today there are a large number of immunedeficient mouse strains, sub-strains and gene-deficient transgenic models that are amenable as recipients for human skin xenografts; a number of these have now been assayed for this purpose. From the numerous strains and specific immune-deficient models that have been generated, the majority of human skin xenograft studies have utilized single-gene mutation models, such as nude-mice (nu) strains presenting a lack of thymus-derived T cells, the severe combined immunodeficiency (SCID) strains, with a deficiency of T cells and B cells, and the NOD/SCID mice that are generated by transferring the SCID mutation onto nonobese diabetic (NOD) mouse strains (Flurkey et al., 2007). These mouse strains are able to accept long term human skin xenografts, have been in existence for many years and are widely available and well used in most animal facilities (Flurkey et al., 2007; Shultz et al., 2007). Various skin xenograft models and techniques have been developed to study human skin on mice. Each has their own challenges but also many advantages, which will be described in detail below.

1)

Full-thickness human skin xenograft

Whole human intact skin xenografting is particularly valuable for studying human skin biology since in addition to keratinocytes and fibroblasts, it contains all cell types residing in the skin tissue at that point in time, including multiple myeloid and lymphoid immune cells, melanocytes and endothelial cells. Xenografts with full thickness skin from human donor material were first successful when nude mice were used as the recipient animal. The xenografts were maintained intact long term before the mice were sacrificed for analysis (Manning et al., 1973; Reed and Manning, 1973; Rygaard, 1974). This method is not overtly destructive to the major compartments of the human skin tissue at the time of grafting and therefore, importantly, the skin architecture remains intact. Additionally, a fully developed human dermal ECM is present, a mature basement membrane between the dermis and epidermis is complete with rete ridge undulations, sweat glands, and hair follicles structures contained in the human biopsy site are also retained. The presence of human vasculature structures also likely aids the long term health of the tissue, however, the impact of nerve components and other structural and cellular factors have not been studied in any great depth. To prepare the recipient mouse, a full thickness wound is made on the dorsal back skin between the shoulder blades that matches the exact size of the donor skin biopsy. The human biopsy is then secured with single-type sutures around the wound margins and bandaged. After 4-6 weeks, the healed transplanted skin graft closely resembles normal human skin histologically and maintains human vasculature, with minimal ingrowth of murine vascular endothelium into the graft bed (Manning et al., 1973; Rygaard, 1974).

This basic technique and concept described has been refined and used for various experimental endpoints, including studying endothelium during inflammation (Yan et al., 1993), interactions between melanoma cells and human stromal components (Juhasz et al., 1993), and psoriasis for analyzing human T cells and the epidermal defects at xenograft site (Boyman et al., 2004; Briggaman, 1980; Krueger et al., 1975). Multiple studies have used this model to study human immunology of the skin, particularly focusing on resident T cell populations that play an essential role in maintaining immune homeostasis in skin tissue (Gratz et al., 2013; Nosbaum et al., 2016; Sanchez Rodriguez et al., 2014).

The full-thickness skin

xenograft

is

also ideal for

studying

photocarcinogenesis to induce non-melanoma and melanoma skin cancer and therefore to develop therapeutic strategies (Berking et al., 2002; Sanchez Rodriguez et al., 2014). This method has also been used extensively to study skin diseases (Briggaman, 1980; Fraki et al., 1983; Kim et al., 1992). Further technical improvements include using a thinner 0.4 mM thickness skin instead of fullthickness skin to improve the graft take rate (Sanchez Rodriguez et al., 2014). This method is technically the simplest skin xenograft technique to implement in the laboratory as it does not require any specialized cell culture or tissue reconstruction steps. However, the collection of multiple skin biopsies from volunteers presents its own unique challenges and is generally not amenable to large scale experimentation. Additionally there would likely be biopsy-to-biopsy variability relating to donor genetic background, body site, age, gender and sun exposure history that introduces heterogeneity between experiments (Berking et al., 2002; Garcia et al., 2007). These particular challenges are why alternative methods, such as those described below, also

provide attractive options for improved experimental design and more refined human skin models.

2)

Reconstructed three-dimensional human skin graft

In vitro reconstructed three-dimensional (3D) skin is an organoid tissue culture system that mimics organizational and cellular differentiation status similar to in vivo skin. Coupling reconstructed 3D skin with xenograft techniques allow for manipulation of human cellular material in vitro before incorporation of the xenograft onto the mouse. This provides extensive potential to use molecular and tissue engineering methodologies to generate reconstructed skin and improve the study of human skin function (Gache et al., 2004; Garcia et al., 2007; Khavari, 2006; Spirito et al., 2006). The predecessor to xenografting reconstructed 3D skin was the autograft of epidermal sheets or cell suspensions on to the back of rabbits to regenerate epidermis (Billingham and Reynolds, 1952). Xenograft experiements with human cells on athymic mice using these methods provided the proof of principle evidence required prior to the first autografts in human burn patients (Banks-Schlegel and Green, 1980; Barrandon et al., 1988; Green et al., 1979; O'Connor et al., 1981). The starting point for generating keratinocyte sheets and reconstructed skin is the isolation of desired primary cell types into pure cultures. In vitro 3D human skin cultures would not be possible without pioneering work to isolate and efficiently culture human keratinocytes, the major cell of the epidermis (Green, 2008; Rheinwald and Green, 1975). Primary cell culture enables the expansion and manipulation of cells in vitro and represents a major advantage to gain experimental flexibility, cell source reproducibility and standardization, as

well as potential for increased throughput without relying on regular skin donations and potential donor variability as described above. Pure primary cell cultures of keratinocytes, fibroblasts or melanocytes, can be isolated from donors with a particular trait of interest or can be selectively engineered in vitro to alter the genome directly or by altering gene expression by targeting RNA. The manipulated cells are then combined with other cellular and matrix elements to regenerate the skin construct and study biological consequences. The caveat to all these advantages is that it is not yet technically possible to reconstruct the entire complexity that is inherently present during in vivo human skin development. Thus reconstructed skin models used for xenograft models only contain a portion of the cellular and extracellular components of the human skin. Multiple methods have been proposed, developed and used to generate reconstructed human xenografts: a) Reconstructed 3D skin using human acellular dermal scaffold Human acellular dermal scaffolds provide a suitable microenvironment to support the stratification, growth and differentiation of a reconstructed epidermis. Cadaveric skin is decellularized leaving an acellular dermal scaffold matrix that can be used for in vitro skin reconstruction and grafting. Freezedrying is often used to ensure all cellular material is destroyed, however the basement membrane, dermal ECM composition and architecture are preserved (Cuono et al., 1986). The papillary dermal surface, which retains the basement membrane, is populated with keratinocytes and the reticular dermal surface is re-populated with human dermal fibroblasts. This reconstruction method was first developed as a skin substitute for autografts when treating human patients with large area skin loss (Cuono et al., 1986; Langdon et al., 1988). The allogenic

dermal scaffold is relatively non-immunogenic and retains the durability and elastic properties of human dermis after manipulation in vitro, making it suitable for skin reconstructions (Compton et al., 1993; Hickerson et al., 1994; McKay et al., 1994). Importantly, the tissue can be lyophilized and stored at room temperature or preserved in glycerol for long term storage at 4 °C (Krejci et al., 1991; McKay et al., 1994). It was first shown that the human acellular dermis with human reconstructed epidermis could be successfully transplanted onto athymic mice to support a differentiated epithelium in the early 1990s (BenBassat et al., 1992; Krejci et al., 1991). A graft site can be established on the mouse dorsal back measuring approximately 1.5 cm2, and a full thickness surgical wound is prepared to exactly accept the xenograft - this is then stitched into place (Medalie et al., 1996). Eight weeks post surgery the graft is found to be mature, with a fully formed epidermal barrier, dermal components and blood vessels that reach the superficial papillary projections. This xenograft method has also been used to model skin disease (Choate et al., 1996). One major issue with this xenograft is the gradual replacement of human skin cells engrafted within the allogenic dermis by host mouse cells. However, the donor dermal matrix appears to remain intact when repopulated with host cells (Ben-Bassat et al., 1992). Replacement of human cells with host mouse cells can potentially lead to experimental variability if the human skin tissue is actually chimeric mouse and human tissue. This particular observation is not widely reported in the literature, however, we contend that other skin xenografting methods would also have this issue with chimeric grafts, albeit the cause remains unclear. b) Reconstructed 3D skin using type I collagen scaffold

Polymerized type I collagen gels are a widely used and relevant substitute dermal scaffolding material for 3D reconstructed skin material since collagens are abundant ECM proteins in the skin that can support fibroblast attachment and growth (Elsdale and Bard, 1972). Composite skin substitute models, as they were first described, are a reconstructed 3D skin with keratinocytes, fibroblasts and the collagen matrix as a dermal substitute (Bell et al., 1981a; Bell et al., 1981b; Gangatirkar et al., 2007). Initially these cultures contained collagen crosslinked with a glycosaminoglycan, together with cellular material that was stratified in vitro at an air-liquid interface prior to grafting onto athymic mice (Cooper and Hansbrough, 1991). Currently type I collagen derived from rat tail is preferred because of its availability, compatibility and relative cost benefits as compared to other collagens such as type IV that is abundant in human skin. Since its inception this composite skin substitute co-culture system has proven to be a successful xenograft model and was found to be superior to epidermal sheet grafts that were in use at the time, mainly due to its comparative durability and resilience to skin fragility (Banks-Schlegel and Green, 1980; Cooper et al., 1993; O'Connor et al., 1981). This technique involves grafting a collagen matrix encased with dermal fibroblasts and with human keratinocytes seeded on top that have been exposed to an air-liquid interface to initiate terminal differentiation and the formation of a stratified epidermis. This skin construct is then placed into a full-thickness wound bed created on the mouse dorsal back with graft size determined according to the in vitro reconstructed skin culture (Figure 2). The surgical graft site is then overlaid with a non-adhesive petroleum jelly gauze dressing to minimize cell damage and then a tie-over dressing is used to hold the graft in

place. Mice are observed daily after grafting, and experimentation can take place when the graft is fully mature at approximately 6 weeks (Figure 2)(Cooper et al., 1993). Histology with human or mouse specific antibodies can be used to identify the species origin of the tissue (Figure 2). This model is suitable for studying a number of human physiological skin processes,

including

cell

proliferation,

re-epithelialization,

epidermal

differentiation, dermal remodeling, basement membrane reorganization and wound healing (Geer et al., 2004; Greenberg et al., 2005). Of note, the dermal support consists overwhelmingly of type I collagen, even after the human fibroblasts have been resident in the pure type I collagen matrix for some time; this is different from the diversity of ECMs found in the human dermis (Ng and South, 2014). Additionally, the collagen used is typically type I collagen derived from the rat tail, which has subtle differences from human type I collagen. Unlike grafting using viable human skin or acellular human dermis, this method may not be as informative if the architecture of the tissue microenvironment is crucial to the experiment. c) Reconstructed 3D skin using fibrin scaffolds Skin tissue engineering using fibrin gel appeared as an experimental regenerative therapy driven by the serious need for stable coverage of extensive burn injuries in patients presenting with a lack of donor area for grafting. The fibrin gel is prepared by isolating fibrinogen from blood plasma and reconstituting in culture medium, thrombin and calcium chloride (Meana et al., 1998). The method was then used for an extensive variety of applications, such wound healing and disease modeling, including reconstructed 3D skin applications (Garcia et al., 2007). Acellular fibrin gels have also been used as

biological support for keratinocyte cultures, as have fibrin gels enriched with fibroblasts to support keratinocyte culture and transplantation (Ronfard et al., 2000). It has been reported that fibrin gels do not alter keratinocyte clonogenic capacity, growth level and long-term proliferative potential making fibrin a suitable scaffold material (Meana et al., 1998). This particular xenografting technique consists of creating a full thickness wound on the mouse dorsum. The bioengineered skin equivalent is placed in the wound bed and bandaged to protect the graft until the newly regenerated human skin is stably incorporated. Grafts adopting this approach have been reported to last more than 12 weeks and have been used to study several monogenic skin diseases (Carretero et al., 2011; Di et al., 2011; Garcia et al., 2011; Garcia et al., 2010; Hainzl et al., 2017; Larcher et al., 2007). This model has also be used to assess the clonogenic and immunogenicity of fetally derived human cells (Tan et al., 2014). Like all reconstruction methods, there is a limitation to the amount of complexity that can be regenerated with the short in vitro culture time and limited number of cell types and tissue structures that are introduced. One of the reported advantages for fibrin-based gels for xenografts is the reduction of graft size due to shrinkage as compared to type I collagen-based xenografts (Lorimier et al., 1998) d) Regenerated human skin using in vivo chamber housing In vivo human skin reconstruction inside silicon chambers is a method whereby a human cell mixture is allowed to regenerate and spontaneously self organize into a human stratified epidermis with an underlying human dermis enclosed within chamber housing that is surgically implanted on the back of the mouse.

This method was developed and adapted from earlier work for mouse reconstruction models (Fusenig et al., 1983; Lichti et al., 2008; Woo et al., 2013; Worst et al., 1974). Large scale in vitro cellular expansion is required for this method (reported at 6 × 106 keratinocytes and fibroblasts per mouse), however, in vitro 3D reconstructed skin equivalent generation is not required prior to grafting, potentially simplifying the cell culture procedures. A concentrated suspension of keratinocytes and fibroblasts in culture medium is directly transferred into the silicon chamber via syringe injection (Figure 2). Eventually the chamber is removed and the regenerated human skin outcome is very similar in structural architecture to normal in vivo human skin, with protein biomarkers expressed at the correct tissue compartments and cellular layers (Wang et al., 2000). This method of in vivo regeneration by spontaneous cell reorganization

appears

to

also

have

improved

subcellular

structural

characteristics such as keratin, hemidesmosome and anchoring fibril connections. These may have been stimulated by mouse paracrine factors or mechanical stress from the host organism during movements underpining the improved healing outcomes and graft stability that are reported compared to other reconstructed methods (Shimoyama et al., 1989; Wang et al., 2000). The silicon chamber in vivo reconstruction model has also been used successfully for hair regeneration with the hair follicle quality and density similar to normal human hair (Ehama et al., 2007). In an interesting mouse-based study, bone marrow cells were isolated and co-injected with the skin cells and showed that the bone marrow cells could transdifferentiate into various skin cell types (Kataoka et al., 2003).

A number of technical challenges need to be considered for successful outcomes with this xenografting technique. These include: leakage of cells from below the chamber, spreading to the surrounding mouse tissue, thereby impedeing successful graft outcomes; the silicon chambers are currently custommade with a relatively small internal diameter; the chamber is encompassed by a “skirt” that extends out under the peripheral mouse skin, which limits the graft size; and, contraction of the maturing graft can make the final human skin site smaller than the original internal chamber area (Lorimier et al., 1998). e) Next generation reconstructed 3D skin xenografts There has been a push to develop new 3D skin methods for better experimental outcomes and to incorporate chemically defined media and animal-origin free scaffolds as these are belived to be more suitable for eventual translation into humans. Clinical application demands an animal-free culture system since the exposure of human cells to animal material can increase the risk of infection and contamination, for example, by transmission of prion and viruses that infect human cells. The research environment therefore provides a vital experimental test bed for analysing cutting edge technologies and materials (MacNeil, 2007). For example, the development of dermal and epidermal tissue regeneration using hydrogel-based scaffolds has been positive as it offers appropriate 3D support for cellular proliferation and differentiation (Zhao et al., 2016). Cellular encapsulation within hydrogel scaffolds of cross-linked natural substances like agarose, fibrin, collagen or hyaluronic acid with elevated water concentration has provided encouraging results (Tibbitt and Anseth, 2009). Hydrogels can also be tailored to provide specific cellular growth and functional characteristics, by either encapsulating cells in synthetic scaffolds (Heywood et al., 2004;

Jongpaiboonkit et al., 2008) or populating the hydrogel with cells that migrate into the gel after seeding on the surface (Topman et al., 2013). Additive manufacturing technology, commonly referred to as 3D printing, could potentially provide a number of technical improvements to building complex tissue engineering scaffolds for skin (Lee et al., 2014). 3D bioprinting focuses on printing bioactive substances and biocompatible structures, with automated material deposition using computer-aided design and manufacturing technology (Derby, 2012). Numerous scaffold properties such as shape, size, texture, rigidity, porosity can be manufactured with controlled precision to provide new opportunities for experimental design (Trombetta et al., 2017). Laser-assisted bioprinting (LAB) is a 3D bioprinting technology using UV or near-UV wavelengths as energy sources to print with materials relevant to producing 3D skin suchas hydrogels, cells and ECM proteins. LAB has advantages over other 3D techniques such as that it is nozzle-free reducing the possibility of blockages, and it is a non-contact process with high resolution and precise delivery (Catros et al., 2011). The LAB technique has been used for the construction of bio-skin resulting in successful generation of skin substitutes that were transplanted into wound beds prepared on nude mice (Michael et al., 2013). Further to this, pathogenic cells have been incorporated into the biomaterials to study pathophysiology of common inflammatory skin diseases atopic dermatitis and psoriasis as well as models to study common skin cancers such as melanoma (Lee et al., 2014).

HUMAN SKIN CANCER XENOGRAFTS IN MICE

The accessibility of skin as the outer layer of the body has made it relatively easy to visibly track and follow the progression of epithelial cancers without the need for invasive surgeries. The traditional mouse model of skin carcinogenesis involves two stages of chemical application of 7,12-dimethylbenzo[a]anthracene (DMBA) and12-O-tetradecanoylphorbol-13-acetate (TPA) (reviewed in(Schwarz et al., 2013)).

This results in the appearance of numerous benign mouse

papillomas, of which a fraction develops into squamous cell carcinomas (SCCs). While this is a well established murine experimental model of carcinogenesis, it is worth noting that the development is different from human SCC which are predominantly UV induced and develop from precursor lesions like actinic keratosis instead of papillomas. Furthermore, while most human tumours tend to occur in the epithelia, most mouse tumours are usually sarcomas and lymphomas (Khavari, 2006). The reliability of using mouse models to predict toxicity is also called into question in the case of carcinogens, where certain rodent carcinogens were not found to be carcinogenic in human, and vice versa (Anisimov et al., 2005). To better model the human skin cancers, approaches have been developed to directly xenograft primary tumour tissues or cells into immunedeficient mice. There is often a high success rate owing to the highly proliferative nature of cancerous cells. In melanoma xenograft models, it is possible to either implant the human melanoma tumour fragments subcutaneously into the mouse flank, or intradermally inject cell suspensions of the isolated tumour cells to study metastasis (Rofstad and Lyng, 1996). Another approach is to genetically modify primary human cells to engineer a tumour phenotype and transplant them onto

mice. In a pioneering experiment by Khavari’s lab, primary human keratinocytes were retrovirally transduced to overexpress Sonic Hedgehog (SHH), seeded onto devitalised human dermis and transplanted onto the back of immuno-deficient mice (Fan et al., 1997). They demonstrated for the first time that a genetic alteration was sufficient to malignantly convert the tissue and recapitulate key features of human basal cell carcinoma (BCC) in vivo. In SCC xenografts, success rates of 75% have been reported with subcutaneous injections of primary human SCC cells into SCID mice (Purdie et al., 2011). The protocol involves the injection of 1 to 4 million primary human SCC tumour cells mixed with concentrated Matrigel into the flanks of female SCID Balb/c mice. Tumours with volumes of 100 mm2 were reported to form as early as 12 days. The histology of such xenograft tumours appears to be identical to that of the human SCC (Watt et al., 2011). A separate study found that a prior implantation of a stromal bed of 1 million normal human fibroblasts in Matrigel could result in higher successful engraftments of primary human SCC cells in immune-deficient mice (28 times out of 29) (Patel et al., 2012). In the case of genetic skin disease recessive dystrophic epidermolysis bullosa, xenograft models have been used in the study of tumourigenesis (Dayal et al., 2014), the tumour stromal microenvironment (Ng et al., 2012) and also in the testing of drug therapies (Woodley et al., 2004). Studies done using human xenografts are especially relevant, as human skin has evolved to be resistant to carcinogenesis. Studies have shown that the mutational burden is surprising high in normal skin, with observations of regular clones of p53-mutated keratinocytes in normal skin (Jonason et al., 1996) and an average of two to six mutations per megabase per cell in normal skin

(Martincorena et al., 2015). Furthermore, normal primary human keratinocytes have been shown to be able to suppress carcinogenesis in premalignant cells when grown in a 12:1 ratio in xenografted organotypic cultures in nude mice (Javaherian et al., 1998). Whether such heterogeneity can be captured in mouse skin remains unknown. With the xenografting of primary human skin tumours into mice being a relatively straightforward option, this method will probably eventually gain traction in the field for clinically relevant in vivo studies over the traditional chemically induced carcinogenesis mouse model.

HUMAN IMMUNE-RESPONSIVE, HUMANIZED SKIN XENOGRAFT MICE

To increase the scope of application of humanized mouse models, the combination of human immune system and other human tissue compartments can be generated. These humanized mouse models that contain several humanized compartments can be referred to as “multi-tissue” humanized mouse models (Legrand et al., 2009). The human skin is a major immune functioning organ with a large repository of myeloid and lymphoid immune cells, either resident or trafficking through the skin tissue compartments (Pasparakis et al., 2014). To more accurately replicate inflammatory human skin conditions, mouse models with a human immune system are required. Humanized mouse models with a functioning human immune system have been sought after to improve immunogenicity for vaccine development, studying disease and de-risking drug development. The confluence of the development of the humanized mouse with a

human immune system and that of human skin xenografts described earlier has led to the generation of multi-organ xenograft models with functioning human immune systems and human xenografted skin. Pioneering studies have investigated psoriasis, atopic dermatitis, and Staphylocuccus aureus infection to further describe the combined human immune and skin response (Carretero et al., 2016; Guerrero-Aspizua et al., 2010; Lewandowski et al.; Tseng et al., 2015). Generation of a humanized immune system requires an immune-deficient mouse for the best results when grafting human skin. Humanized mice or ‘huMice’ are constructed from immunodeficient mouse strains. Three widely used strains for this technique include (1) NOD.CgPrkdcscidIl2rgtm1Wjl (NSG), (2) NODShi.Cg-PrkdcscidIl2rgtm1Sug (NOG), and (3) C;129S4-Rag2tm1FlvIl2rgtm1Flv (BALB/c-Rag2nullIL2rgnull or BRG) mice. These immune-deficient mice have defective T, M and natural killer (NK) cells, as well as reduced macrophage and dendritic cell function (Ito et al., 2012; Shultz et al., 2012; Walsh et al., 2017). To reconstitute the human haematopoietic system, and therefore aspects of the human immune system in these immunocompromised mice strains, three main engraftment approaches are typically used: a)

PBMC humanized mice (hu-PBMC). Hu-PBMC mice are generated using adult peripheral blood mononuclear cells. These models are suitable for short-term studies with strong effector and memory T cell and NK cell function but do not allow for longer term observation due to graft versus host disease (GvHD) (Brehm et al., 2014; Shultz et al., 2012; Walsh et al., 2017).

b)

CD34+ humanized mice (hu-CD34).

Hu-CD34+ mice are generated with cord blood-derived or fetal liverderived HSCs. This method to reconstitute the immune system allows for a more complete reconstitution of several aspects of the immune system (B cells, T cells, myeloid cells and antigen-presenting cells) compared to PBMCs, but granulocytes, platelets and red blood cells are at low levels. Additionally, human T cells in this model are H2 and not HLA-restricted (Brehm et al., 2014; Shultz et al., 2012; Walsh et al., 2017). c) Bone marrow/liver/thymus (BLT) model The BLT model is generated by transplanting fragments of human fetal liver and thymus into immune-deficient mice, followed by IV injection of autologous fetal liver CD34+ HSCs. This model develops all lineages of the human immune system like the hu-CD34 model. In addition, their T cells mature in the human thymus and are HLA restricted. However, BLT mice develop GvHD, thus restricting the window of study (Brehm et al., 2014; Shultz et al., 2012; Walsh et al., 2017). In order to allow for differentiation and maturation of more human cell lineages, new strains of immune-deficient mice that carry human cytokine genes and human factors are used to generate improved humanized mice (Shultz et al., 2012). For example, the introduction of human CSF-1 improved differentiation and function of macrophages (Rathinam et al., 2011), while knocking in IL-3 and GM-CSF improved the development of human alveolar macrophages (Willinger et al., 2011). Chen et al. showed that introducing expression plasmids containing GM-CSF and IL-4 leads to increased dendritic cells in humanized mice (Chen et al., 2012).

In the context of skin disease, such “multi-tissue” humanized mouse models that comprise the human immune system and human skin could be invaluable models to study the pathogenesis of human skin diseases and tools for drug discovery and vaccine development. Based on the Pober laboratory’s humanized mouse model of human allograft rejection, de Oliveira et al. generated humanized mice with human skin using a huPML-SCID-huSkin allograft model (de Oliveira et al., 2012). Human reconstituted skin was first transplanted onto SCID mice and subsequently allogenic human PBMCs were introduced intra-peritoneally into these mice. Within 2 to 3 weeks, the human skin on the humanized mice displayed inflammation with acanthosis and aberrant epidermal marker expression. In addition, human T cells, chemokines and cytokines were detected within the inflamed skin. Marcela Del Rio’s laboratory developed a humanized mouse model for psoriasis (Guerrero-Aspizua et al., 2010).

They extracted peripheral blood,

keratinocytes and fibroblasts from psoriatic patients and healthy donors. Lymphocytes were isolated from peripheral blood and T cells were expanded and differentiated towards a T1 phenotype. The differentiated T cells and recombinant IL-22 were then injected into immune-deficient Rj: NMRI— Foxn1nu (NMRI nu) mice to reconstitute the immune system. To generate the psoriasis model, matched human keratinocytes and fibroblasts from healthy donors were used to generate human skin equivalents and were grafted on the humanized mice. They found that the combination of in vitro differentiated T1 cells, Th17 cytokines IL-17 and IL-22 and the disruption of the stratum corneum

with tape stripping resulted in a psoriatic phenotype. This model can potentially be used to assess drugs that target psoriasis. Following the initial psoriasis model, the Del Rio laboratory further developed an improved psoriasis model and, additionally, an atopic dermatitis skin-humanized mouse model (Carretero et al., 2016). The psoriasis model was constructed using naïve CD4+ T cells derived from healthy donors that have been activated in vitro and directed to differentiate into Th1 and/or Th17 cells, while the AD model was constructed using naïve CD4+ T cells derived from healthy donors that have been activated and directed to differentiate to Th2-type cells. In both models, human keratinocytes and fibroblasts derived from healthy donors were reconstructed to form skin equivalents and grafted onto immune-deficient mice. These skin grafts were tape stripped and the resultant skin showed indications of psoriasis or AD from studying relevant disease specific biomarkers, hence representing the respective diseases. A similar psoriasis model was constructed by Amy Paller’s laboratory where human keratinocytes and fibroblasts were used to construct a human 3D skin equivalent and grafted on immune-deficient NSG mice (Lewandowski et al., 2017). To induce a psoriasis-like phenotype, the mice were subjected to stress with tape-stripping and intradermal injections of human cytokines (IL-17A and IL-22). They observed alterations in psoriasis-related biomarkers, including epidermal differentiation, proliferation, cytokines and cytokine receptors. To demonstrate the model’s therapeutic potential these mice were treated with anti-TNF treatment and the gene expression associated with psoriatic induction was prevented.

Interestingly, skin xenografts may not always be necessary to recapitulate human skin diseases, as it has been shown that the addition of only a human immune system in mice is sufficient to result in a more physiological model for Staphylococcus aureus infection. Tseng et al. found that the inoculum of S. aureus required to induce an infection in such humanized mice became comparable to the amount required to induce infections in human. In addition, they used this humanized mouse model to confirm that a human-tropic bacterial factor, Panton-Valentine leukocidin (PVL) is important in S. aureus mediated dermonecrosis (Parker, 2017; Tseng et al., 2015). These investigations with S. aureus indicate that humanized immune models will also be relevant for studying scientific questions on the skin microbiome and skin infection.

FUTURE PERSPECTIVES

Significant progress has been made in the development and use of humanized skin xenograft mouse models, from the initial work with transplants of epithelial sheets and the simple models of injecting cancer cells into the flanks of animals (Figure 3). We are now at a stage where complex model reconstructions with multiple cell types can provide enhanced technical platforms to more fully understand human skin physiology. The technical advances in molecular and tissue engineering have provided us with the ability to genetically alter and then regenerate intact human skin on immune-deficient mice. These human tissue models therefore provide a complementary approach to the commonly used murine genetic models to understand complex mechanisms of skin biology. Additionally, these human tissue platforms may also provide vital information in

pathways and regulatory networks where murine models have failed previously. The studies described above highlight a wealth of human tissue-based studies investigating skin physiology and disease across the multiple methodologies. These studies provide a human relevance to laboratory models that will aid biological understanding and therapy development. A more recent important advance in the skin xenografting space has been the creation of ‘multi-tissue’ humanized mouse models that incorporate both the human immune system and human skin. This permits studies of complex multicellular pathways in vivo that would have been impossible in humans, and again, this will undoubtedly accelerate therapeutic development. Ideally, to recapitulate the human immune system the humanized mouse models should consist of the full human immune system. Currently, human hematopoietic lineages like granulocytes, platelets and red blood cells have yet to be established. In addition, it is not well substantiated that resident immune cells of the epidermis, such as Langerhans cells, are efficiently translocated and subsequently survey their territory. Hence, the ongoing efforts to develop new, improved mouse strains and the use of various engraftment methods to introduce the human immune system will lead to better humanized mouse models in the future. The use of ‘multi-tissue’ humanized mouse models will likely provide new insights in skin disease and yield potential therapies in the near future (Legrand et al., 2009; Shultz et al., 2012). All the same, the technology platforms described above are not trivial and a certain amount of technical skill and patience is required to master the ability to generate successful skin xenografts. In addition, the techniques need to be carefully selected to ensue that the chosen method will indeed provide the

endpoint that is required, such as celluar source, dermal scaffold. It is likely that technical advances will popularize skin xenograft models further. The possibility of using 3D printing of bio-compatible matrices and bio-skin reconstructions are already being investigated and utilized as xenografts. These technological advances will add additional experimental standardization and programmable simplification, yet at the same time facilitate increased biological complexity that will aid the improvement of graft generation.

ACKNOWLEDGEMENTS We would like to thanks Nick Barker, Zee Upton and Angeline Tay for providing critical reading of this review prior to submission. The authors are funded by the Biomedical Research Council (BMRC) of Singapore and A*STAR SPF grants for basic and translational skin research (IAF SPF 2013/004; IAF SPF 2013/005).

CONFLICTS OF INTEREST The authors declare no conflicts of interests

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Figure 1. Structural comparisons between mouse and human skin. Histology of the skin is show at 10x and inlay box 20x magnification for (A, C) mouse and (B, D) human skin tissue. The epidermis and dermis is thicker in the human skin as compared to the mouse, and the mouse skin has the presence of a high density of hair follicles. SC = stratum corneum.

Figure 2. Examples of grafting techniques. (A) Silicon chamber housing is grafted onto the back of the NSG mouse prior to injecting cell suspension in to the housing. (B) Alternatively, in vitro reconstructed skin using collagen type I is inserted into the wound bed and stitched in place, (C) the reconstructed skin is covered and bandged. (D) Approximately 6 weeks after grafting a mature human skin graft can be clearly observed. (E) Human keratinocytes used to generate the reconstructed skin was transduced with GFP for in situ tracking of the human patch

and

to

differentiation

between

mouse

and

human

skin.

(F)

Immunohistochemistry with anti-GFP antibody that recognizes only the transduced human cells expressing GFP confirms the localization of the human epidermis whereas the mouse epidermis remains unstained. Of note the human skin is thicker than the mouse skin mimicking the anatomical difference observed between human and mouse skin .

Figure 3. Xenografting timeline. Highlights and key event in human skin xenografting.

Table 1. Xenograft methodologies with tissue related properties.

Model

Human cell types

Genetic engineering

Matrix type

Human immunity

Full-thickness human skin

All human skin cells

Not possible

Living ex vivo human

Resident human immune cells

Reconstructed 3D skin using human acellular dermal scaffold

Keratinocytes and fibroblasts

Possible

Non-living ex vivo human

Currently no human immune cells tested

Reconstructed 3D skin using collagen type I scaffold

Keratinocytes and fibroblasts

Possible

Animal derived

Currently no human immune cells tested

Reconstructed 3D skin using plasma and fibrin scaffolds

Keratinocytes and fibroblasts

Possible

Animal or human derived

Currently no human immune cells tested

Regenerated human skin using in vivo chamber housing

Keratinocytes, fibroblasts and bone marrow

Possible

Regenerated human from fibroblasts in vivo

Currently no human immune cells tested

Reconstructed 3D skin using laserassisted bioprinting

Keratinocytes and fibroblasts

Possible

Animal or human derived

Currently no human immune cells tested

Direct injecting skin cell xenograft

Keratinocytes, fibroblasts and melanocytes

Possible

Host

Currently no human immune cells tested

Multi-tissue - human immune, human skin xenograft

Keratinocytes, fibroblasts, PBMCs, HSCs and T cells

Possible

Dependent on skin method choice

Partially human immunity

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