WO2016039687A1

WO2016039687A1 - Organotypic skin model

Info

Publication number: WO2016039687A1
Application number: PCT/SG2015/050302
Authority: WO
Inventors: Tong CAO; Mohammad Mehdi MOVAHEDNIA; Fahad KIDWAI
Original assignee: National University Of Singapore
Priority date: 2014-09-10
Filing date: 2015-09-04
Publication date: 2016-03-17
Also published as: GB201416006D0

Abstract

The disclosure relates to a method for the differentiation of pluripotent stem cells to fibroblasts and keratinocytes, their use in the production of a full depth organotypic skin model and the use of the model in the testing of pharmaceutical or cosmetic gents.

Description

Organotypic Skin Model

Field of the Invention The disclosure relates to a method for the differentiation of pluripotent stem cells to fibroblasts and keratinocytes; their use in the production of a full depth organotypic skin model and the use of the model in the testing of pharmaceutical or cosmetic agents. Background of the Invention

Human skin is the first line of defence for internal organs against invasion of pathogens and microorganisms. Accordingly, the skin serves as a vital protective layer for human body against water loss, and potential exogenous mechanical and chemical hazards (Bickers, Athar, 2006). The epithelial surface of skin and oral mucosa is a stratified squamous tissue consisting of cells tightly attached to each other and arranged in a number of distinct layers (basal, prickle cell, granular and keratinized layers). The outermost part of skin is composed of multi-layered differentiated keratinocytes to shape a self-keratinized structure, calling the epidermis. The epidermis is combined with supportive underlying layers of fibroblasts cells, called the dermis layer (Barker et al., 1991 ).

Due to disruption of skin barrier function by aging and diseases, there is great interest in developing skin treatments products. Further, in this regard and given the intrinsic barrier function of the skin, effective topical delivery of therapeutic compounds requires penetration across the superficial permeability barrier of the tissue. Successful translation of new therapeutics requires the ability to evaluate test agents in realistic model systems for cutaneous and mucosal delivery. The development of an in vitro model that can reproduce the appropriate mechanical and permeability characteristics of the normal tissue is critical to the formulation and delivery of therapeutic compounds and to study barrier properties of the protective surface of skin and oral mucosa, and represents an important tool for preclinical testing and for facilitating the translation of therapeutic compounds into clinical use. Various skin models exist including ex vivo human tissue biopsies or surgical specimens to study permeability and barrier properties of skin and oral mucosa, but there are numerous difficulties associated therewith including ethical issues, supply and experimental variability (Groeber et al., 201 1 ). Additionally, animal studies whilst proving to be useful have numerous drawbacks for studying barrier properties due to inherent cross-species variability. There is also a desire to move away from animal testing of medicinal agents (Hewitt et al., 2013). Current in-vitro organotypic models of keratinized stratified tissue may exhibit some of the structural characteristics observed in vivo but they are expensive, highly variable and do not reproduce the barrier properties of the parent tissue (Gibbs et al., 2007; Godin, Touitou. 2007j (Table 1 ).

For example, Oh et al [Journal of Investigative Dermatology 2013 vol 133], discloses organotypic skin culture comprising a scaffold populated with fibroblasts after which keratinocytes are seeded on top. Differentiation of keratinocytes occurs through lifting the scaffold above liquid-air interface. WO97/41208 discloses skin regeneration using mesenchymal stem cells which form a multilayer skin equivalent with essentially two layers comprising mesenchymal derived dermoblasts e.g. dermal fibroblasts (or a dermal fibroblast layer and a reticular dermal fibroblast layer) and keratinocytes employing also a scaffold, preferably collagen. WO2014132063 discloses a 3D skin model using fibroblasts and keratinocytes derived form a skin biopsy sample. WO2009/156398 discloses a method of culturing keratinocytes, derived from human pluripotent stem cells, on a cell culture surface coated with fibroblasts in the presence of a keratinocyte culture medium supplemented with BMP-4 and ascorbic acid. WO2007/125288 discloses a cell culture substrate combined with mesenchymal and pluripotent cells. Skin grafts with two layers comprising fibroblasts and keratinocytes, although not full depth skin equivalents, are also disclosed. Furthermore, Guo et al [Journal of Investigative Dermatology 2013 vol 133] discloses induced pluripotent stem cells that differentiate into keratinocytes to form a 3D skin equivalent when combined with iPSC fibroblasts on a collagen scaffold. A similar disclosure is provided in Guo et al [Stem Cell Research and Therapy 2013 vol 4] and Itoh et al [PLOS One 2013, vol 8(10). Alternatively, cell and tissue culture models can offer advantages in terms of availability of tissue, cost and safety. However, current cell culture monolayers do not show differentiation that accompanies skin tissue stratification in vivo and thus do not show the barrier properties of the normal tissue.

The growth of stratified, differentiated human epithelium to form organotypic 3D cultures potentially overcomes the disadvantages of cell monolayers. 3D culture systems are biochemically and physiologically more similar to in vivo tissue. However, in practice it has not proved easy to grow organ cultures that can effectively reproduce the barrier function of a normal skin explant. For example, measurements of permeability of organotypic skin cultures has shown permeability to a variety of compounds to be 3-100 fold greater than for normal skin (Robert et al, 1997; Garcia et al, 2002 ; Barai et al, 2008). Further, current techniques require unfavourable harvesting skin biopsies through surgical processes from individuals and expansion of obtained cells in laboratory conditions to provide sufficient number of cells for these models, which can result in loss morphology and functionality of these cells. Thus current models are both expensive and suffer from batch variability. These issues for full-thickness skin models worsen, since two different types of cells (i.e. dermal and epidermal) are desired in full thickness skin models.

There is therefore an unmet need for a representative and reproducible organotypic skin model that faithfully recapitulates the features of human skin which can facilitate identification of therapeutic agents and research into skin disease.

This disclosure relates to an organotypic skin model that is full-thickness and authentically differentiated using material of known genetic origin that is functionally stable and limits the introduction of adventitious infectious agents to provide superior stability and longevity compared to existing models, with application in the screening, development and evaluation the long-term effectiveness of cosmetics, pharmaceutical agents, and therapeutics. Statements of Invention

According to an aspect of the invention there is provided a method to induce the differentiation of a mammalian pluripotent stem cell comprising the steps:

i) forming a preparation of mammalian pluripotent stem cells and providing cell culture conditions to induce the formation of embryoid bodies comprising differentiated or differentiating mammalian fibroblasts derived from said mammalian pluripotent stem cells;

ii) forming an isolated fraction comprising mammalian fibroblast cells from said embryoid bodies to form an isolated, differentiated mammalian fibroblast cell preparation; or

iii) extracting and isolating from said embryoid bodies a mammalian fibroblast derived extracellular matrix fraction;

iv) forming a preparation comprising mammalian pluripotent stem cells and the isolated mammalian fibroblast derived extracellular matrix fraction and providing cell culture conditions to induce differentiation of said mammalian pluripotent stem cells to differentiated mammalian keratinocytes;

v) forming an isolated, differentiated mammalian keratinocyte cell preparation; and

vi) maintaining said differentiated keratinocyte preparation in a fibroblast feeder culture preferably comprising a fibroblast preparation according to step ii).

In a preferred method of the invention said mammalian fibroblast and keratinocyte is human.

In an alternative preferred method of the invention said mammalian fibroblast and keratinocyte is: non-human primate, mouse, rat or hamster. In a preferred method of the invention differentiation of pluripotent embryonic stem cells into keratinocytes and maintenance of differentiated keratinocytes comprises the use of cell culture media as set forth in Table 2. According to a further aspect of the invention there is provided an isolated differentiated mammalian fibroblast obtained or obtainable by the method according to the invention.

According to a further aspect of the invention there is provided an isolated differentiated mammalian keratinocyte obtained or obtainable by the method according to the invention.

According to a further aspect of the invention there is provided a method for the preparation of an organotypic skin model comprising the steps:

i) forming a preparation comprising differentiated mammalian fibroblasts according to the invention wherein said preparation is associated with a biocompatible polymeric cell culture substrate and cell culture conditions to form a dermal part of said organotypic skin model;

ii) contacting said dermal part with a preparation of differentiated mammalian keratinocytes according to the invention and providing cell culture conditions sufficient to form a differentiated, stratified organotypic skin model. In a preferred method of the invention, said mammalian keratinocytes are cultured at an Air-Liquid Interface.

Reference herein to the term Air-Liquid Interface (ALI) refers to the culture of cells such that their basal membrane is in contact with, or submerged in, liquid and their apical membrane is in contact with air. Advantageously, the keratinocytes consequently demonstrate apical-basal polarity in their differentiation resulting in the development of functional keratinised surfaces as seen in vivo. In a preferred method of the invention said mammalian keratinocytes and fibroblasts are primate, preferably human.

In a yet a further preferred method of the invention said mammalian keratinocytes and fibroblasts are derived from pluripotent cells such as, but not limited to, human embryonic stem cells (hESC), human embryonic germ cells, human induced pluripotent stem cells (hiPSC). Advantageously, this permits consistent epidermal and full-thickness skin models populated with dermal and epidermal cells with the requisite barrier properties to be generated by providing potentially an unlimited source of skin cells (fibroblast and keratinocytes). Further, by incorporation of hESC- derived cell lines into skin equivalent (SE) models, they offer a more true reflection of the cellular phenotypes observed in vivo.

More preferably still, said mammalian keratinocytes and fibroblasts are derived from human embryonic stem cells (hESC).

In a preferred method of the invention said mammalian fibroblasts cells and/or said keratinocytes are autologous. In an alternative preferred method of the invention said mammalian fibroblasts cells and/or said keratinocytes are allogeneic.

Reference herein to a cell support substrate refers to any material that is capable of supporting three-dimensional tissue cell culture by replicating an in vivo cellular environment including cell attachment, cellular signalling and diffusion and mechanical support. As will be appreciated by those skilled in the art, numerous different types of cell supports substrates exist and can be used in accordance with the invention such as cell culture scaffolds having, in particular, the requisite porosity to facilitate cell seeding and diffusion throughout the whole structure of both cells and nutrients.

An example of a cell culture support substrate is disclosed in US2010/04841 1 , the content of which is incorporated by reference. These substrates comprise microcellular polymeric materials which are described as "polyHIPE" polymers. These polymers form reticulate structures of pores that interconnect with one another to provide a substrate to which cells can attach and proliferate. The process for the formation of polyHIPEs allows pore volume to be accurately controlled with pore volume varying from 75% to 97%. Pore sizes can vary between 0.1 to 1000 micron and the diameter of the interconnecting members from a few microns to 100 microns. Furthermore the polyHIPEs can be combined with additional components that facilitate cell proliferation and/or differentiation. PolyHIPEs are therefore versatile substrates on which cells can attach and proliferate in a cell culture system. Processes for the preparation of polyHIPEs are well known in the art and also disclosed in WO2004/005355 and WO2004/004880. PolyHIPEs are commercially available and comprise for example oil phase monomers styrene, divinyl benzene and a surfactant, for example Span 80 sorbitan monooleate. In addition, the rigidity of the polymer formed during processing of the polyHIPE may be affected by the inclusion of a monomer such as 2-ethylhexyl acrylate. The process for the formation of polyHIPE from an emulsion is initiated by the addition of a catalyst such as ammonium persulphate.

In a preferred method of the invention, said cell support substrate comprises a biocompatible polymer based scaffold such as but not limited to a polyester including polystyrene, polylactic acid, polyglycolic acid, polycaprolactone, poly-dl- lactic-co-glycolic acid, or the like. Preferably, said cell support substrate is not degradable. Alternatively, the biocompatible support matrix comprises a natural polymer selected from the group: collagen, for example Matrigel®, hyaluronic acid, hyaluronic acid esters such as hyaluronic acid benzyl alcohol, fibrinogen scaffolds, thrombin scaffolds and combinations thereon.

Reference herein to a cell culture medium includes reference to a medium designed to support the growth of cells according to the invention, in particular stem cells or keratinocytes. Many different types of chemical medium can be used to support the growth of stem or progenitor cells in culture, such as but not limited to, feeder support system medium which is either supplemented with fetal bovine serum or serum replacer and feeder-free systems supplemented with defined culture mediums such as mTeSR™1 and TeSR™8. The disclosure also relates to serum- free medium composed of DMEM-F12 supplemented with serum replacer in feeder supported system.

Further, in yet a further preferred method of the invention said cell culture medium comprises at least one other compound, agent, or drug useful in supporting normal cellular survival, metabolism or differentiation, such as but not limited to retinoic acid, epidermal growth factor (EGF), hydrocortisone, insulin and bone morphogenetic proteins 4 (BMP4).

In a preferred method of the invention said cell culture comprises additional cell types such as but not limited to melanocytes, neural cells or endothelial cells. The co-culture of cells with melanocytes provides an epithelial skin model exhibiting pigmentation, permitting assessment of the effects of UV exposure and anti-UV materials on the skin. Similarly, use of neural and endothelial cells permits development of a vascularised and innervated tissue to recapitulate tissue in vivo; preferably said additional cell types are autologous. In a preferred method of the invention differentiation of pluripotent embryonic stem cells into keratinocytes and maintenance of differentiated keratinocytes comprises the use of cell culture media as set forth in Table 2.

In a preferred method of the invention the formation of a stratified epidermis comprises the use of cell culture media as set forth in Table 3.

Additionally, according to a preferred method of the invention, said additional cell types are derived from human embryonic stem cells (hESC). In a preferred method of the invention, said method comprises culturing said fibroblasts in step i) for at least 1 -20 days prior to step ii), or more ideally, 2-14 days, or more ideally still a number of days selected from the group comprising of: 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, and 14 days. According to a further aspect of the invention there is provided an organotypic skin model obtained or obtainable by the method according to the invention. According to a further aspect of the invention there is provided a cell culture vessel comprising an organotypic skin model according to the invention.

In a preferred embodiment of the invention said cell culture vessel comprises a cell culture insert, optionally removable, containing said organotypic skin model and in fluid contact with cell culture medium.

In a preferred embodiment of the invention said culture vessel comprises cell culture media as set forth in Table 2. In a preferred embodiment of the invention said culture vessel comprises cell culture media as set forth in Table 3.

According to an aspect of the invention there is provided an organotypic skin model according to the invention for use in the testing of test agents such as but not limited to therapeutics, cosmetics, compounds or biologically active xenobiotic agents, on skin cell function and permeability.

The term "xenobiotic agent" is herein given a broad definition and includes not only compounds but also gaseous agents. Typically, xenobiotic agent encompasses pharmaceutically active agents used in human and veterinary medicine and human cosmetics.

In yet a further preferred embodiment of the invention, said test agent can contact the cell culture by adding it to said cell culture medium. Alternatively, said test agent can contact the cell culture by adding it to the apical surface of said organotypic model. Advantageously, this permits delivery of test agents, including vapours, gases and dry air-borne powders, in addition to soluble test-agents, this is much more representative of events that occur in-vivo wherein the skin epithelium is one of the first lines of defence to a variety of different agents.

According to a further aspect of the invention there is provided a cell array wherein said array comprises a plurality of cell culture vessels according to the invention.

The screening of large numbers of agents requires preparing arrays of cells for the handling of cells and the administration of agents. Assay devices, for example, include standard multiwell microtitre plates with formats such as 6, 12, 20 48, 96 and 384 wells which are typically used for compatibility with automated loading and robotic handling systems. Typically, high throughput screens use homogeneous mixtures of agents with an indicator compound which is either converted or modified resulting in the production of a signal. The signal is measured by suitable means (for example detection of fluorescence emission, optical density, or radioactivity) followed by integration of the signals from each well containing the cells, agent and indicator compound.

In a preferred embodiment of this aspect of the invention, said mammalian keratinocytes are cultured at an Air-Liquid Interface.

According to a further aspect of the invention there is provided a method for the high throughput screening of test agents comprising the steps:

i) providing an array according to the invention;

ϋ) contacting the array with a plurality of agents to be tested;

iii) collating activity data obtained following (ii) above;

iv) converting the collated data into a data analysable form; and optionally

providing an output for the analysed data.

In a preferred method of the invention the organotypic model is contacted with at least one therapeutic, cosmetic, compound or xenobiotic agent. In a preferred method of the invention, said mammalian keratinocytes are cultured at an Air-Liquid Interface.

The culture method results in the advantageous formation of a stable dermal layer in the cell support substrate. Further, culture of keratinocytes upon said fibroblast/support substrate dermal layer at the Air-Liquid interface leads to keratinocytes demonstrating apical-basal polarity in their differentiation resulting in the development of functional keratinised surfaces with epidermal stratification as seen in vivo. Additionally, it has been found that without embedding fibroblasts within enclosed substrates cellular interactions between the skin layers can be explored. This therefore results in the formation of a reliable and realistic skin equivalent model with superior stability.

Additionally, the formation of dermal compartment in this system depending on the history and quality of the fibroblast cells can be investigated. Therefore, formation of aged dermal compartment with aged dermal cells versus young dermal cells can be investigated.

According to an aspect of the invention there is provided an organotypic skin model according to the invention for use in monitoring the ageing of skin.

According to a further aspect of the invention there is provided a method for preparation of aged skin comprising:

i) providing an isolated preparation of differentiated mammalian fibroblast cells obtained by the method according to the invention;

ii) serially passaging said fibroblasts at least once;

iii) forming a preparation comprising a fibroblast derived extracellular matrix fraction according to the method of the invention and mammalian pluripotent stem cells and providing cell culture conditions to induce differentiation of said mammalian pluripotent stem cells to differentiated mammalian keratinocytes;

iv) forming a preparation comprising differentiated mammalian fibroblasts according to step (ii) above wherein said preparation is associated with a biocompatible polymeric cell culture substrate and cell culture conditions to form a dermal part of said organotypic skin model;

v) contacting said dermal part with a preparation of differentiated mammalian keratinocytes according to step (iv) above and providing cell culture conditions sufficient to form a differentiated, stratified organotypic skin model;

vi) monitoring the structural and functional properties of said stratified organotypic skin model as a measure of skin age.

In a preferred method of the invention said fibroblasts are passaged at least 8 times.

In a further preferred method of the invention said fibroblasts are passaged at least 16 times.

In a yet further preferred method of the invention said fibroblasts are passaged at least 24 times.

Preferably, said fibroblasts are serially passaged 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24 or more times.

According to a further aspect of the invention there are provided serially passaged fibroblasts obtained or obtainable by the method according to the invention.

Any further aspect of the invention may, in preferred embodiments, include or be characterised by any of the aforementioned features. Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", means "including but not limited to", and is not intended to (and does not) exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise. Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

Moreover, unless stated otherwise, any feature disclosed herein may be replaced by an alternative feature serving the same or a similar purpose.

No admission is made that any reference referred to herein constitutes prior art. Further, no admission is made that any of the prior art constitutes part of the common general knowledge in the art.

An embodiment of the invention will now be described by example only and with reference to the following figures: Figure 1 : Differentiation of Human embryonic stem cell (hESC) into fibroblast and keratinocyte. Single hESC source was differentiated to fibroblast and keratinocyte to provide an autologous cell lines for skin models. Both fibroblasts and keratinocytes were characterized for their respective markers at protein level by immunofluorescence staining;

Figure 2: Characterization of MLE at histological level; stained with Hematoxylin and eosin (H&E) staining and reproduce at the same magnification (100x). (a) H&E staining of HaCaT derived MLE at day 14. (b) H&E staining of hESCs-Kert derived MLE at day 14. Abbreviations: hESC, human embryonic stem cell; MLE, multilayer epithelium; HaCaT, Immortalized control cell line; HaCaT-MLE, HaCaT-derived multilayer epithelium; hESCs-MLE, hESCs-derived multilayer epithelium.

Figure 3: Characterization of MLE at mRNA level. Expression of terminally differentiated keratinocyte markers. β-Actin was taken as loading control. Abbreviation: β Act, β Actin; HaCaT, Keratinocyte immortalized cell line; hESC, human embryonic stem cell; MLE, multilayer epithelium; Figure 4: Confocal microscopy for Involucrin and Filaggrin expression by hESCs- MLE (a-b) and (c) HaCaT derived MLE. Expression assessed at day 14 of terminal differentiation in air culture. Magnification = 100x. Abbreviations: MLE: multilayer epithelium; hESC, human embryonic stem cell; hESCs-MLE, hESCs-derived multilayer epithelium;

Figure 5: Confocal microscopy for K14 and Collagen IV expression by hESCs-MLE (a-b) and HaCaT-MLE (c). Images were taken on day Day 14 of terminal differentiation at 100x magnification. Abbreviations: MLE: multilayer epithelium; hESC, human embryonic stem cell; hESCs-MLE, hESCs-derived multilayer epithelium;

Figure 6: Protein expression of hESCs-MLE. Western Blot results showing the expression of Involucrin and Filaggrin protein by hESCs-MLE at day 14. HaCaT- MLE was taken as reference control. β-Actin was taken as loading control. Abbreviations: MLE: multilayer epithelium; hESC, human embryonic stem cell; hESCs-MLE, hESCs-derived multilayer epithelium; Control, HaCaT-derived MLE;

Figure 7: Scan Electron Microscopy of MLE (a, b). SEM images of MLE derived from hESCs (a) and HaCaT (b). Abbreviations: C, keratinized layer; SK, stratified keratinocytes. HaCaT derived keratinocyte was taken as control. All images in (a) are the same sample but different magnifications 200x (Ai), 800x (Aii) and 2000x (Aiii). Image (b) was taken at 500x magnification; Figure 8: Hematoxylin and eosin (H&E) staining of paraffin embedded full-thickness skin construct after 9 weeks. Morphological analysis indicates the formation distinct dermal and epidermal compartments and shaping the well-defined stratum corneum at the top of skin construct. Scale bar indicate 100 μηι. Figure 9: Permeability testing. (A) DPM values of titrated water across different samples. (B) Kp index values of titrated water across different samples. Error bars = mean +/- SEM (n=3). Abbreviation: Time, in hours; hESC, human embryonic stem cell; hESCs-Kert, hESCs-derived MLE comprising of hESCs-Kert only; hESCs- Kert/hESCs-ebF, hESC-derived MLE comprising of hESCs-Kert (epidermal compartment) and hESCs-derived fibroblast (dermal compartment); Control Keratinocyte, MLE comprising of HaCaT only (control keratinocyte cell line); Control Kert/hESCs-ebF, MLE comprising of control cell line (epidermal compartment) and hESC-derived fibroblast (dermal compartment); hESC-ebF, inserts with hESC- derived fibroblast only; Insert without cells was taken as negative control;

Figure 10: Illustration of the organotypic culture system. hESCs-derived fibroblasts are seeded on highly porous polystyrene scaffold for 7 days in humidified chamber at 37 °C to shape dermal compartment. At day 8 the hESC-derived keratinocytes are seeded on constructed dermal compartment in contact with air to trigger the stratification process. This organotypic model allows direct contact of dermal and skin basal layer at all time. The skin models stable for 9 weeks inside humidified chamber;

Figure 1 1 : Immunofluorescent analysis of dermal layer after long-term cultivation: (A) Collagen I, the main dermal layer extra cellular protein, was extensively deposited in dermal layer. (B) Collagen IV, the basement membrane protein, was deposited at the dermal-epidermal junction. Scale bar represents 100 μηι;

Figure 12: Immunofluorescent analysis of epidermal layer after long-term cultivation of SE model indicates the normal hierarchy of differentiation from basal layer to superficial layers. (A) K14 expression at the basement layer indicating the proliferative potential of residing keratinocytes cells at basal layer. (B) By migration of basal layer keratinocytes K10 the early marker of keratinocytes differentiation was expressed at upper layers of basal layer. By progression of differentiation process, late terminal differentiation markers (C) Filaggrin, (D) Involucrin, and (E) Loricrin were evident. The expression of these proteins is essential for formation of barrier function in SE model and presence of these markers indicates the formation of highly cross-linked epidermis barrier at superficial layer of SE model.

Figure 13: Hematoxylin and eosin (H&E) staining demonstrated the formation of stratified multi-layered epithelium after 15 days culture at air-liquid interface. Immunohistochemistry confirmed expression laminin V at the basement membrane and high expression K10 at superficial layers. Filaggrin and involucrin were expressed at superficial layers. Scale bars represent 50 μηι; Figure 14: Immunohistochemical analysis of organotypic skin cultures generated with serially passaged hESC-derived fibroblasts and subsequent effects on epidermis formation, (a) Morphological analysis with Hematoxylin and eosin (H&E) staining showed diminished dermal compartment in organotypic skin cultures generated with hESC-derived fibroblasts cells at passage 16 compared with hESC- derived fibroblast cells at passage 8 (young). Furthermore, significant thinner epidermal compartment and stratum corneum formed in organotypic skin cultures generated with hESC-derived fibroblast cells at passage 16 (semi aged). There was no distinct formation of dermal and epidermal compartment in organotypic skin cultures generated with hESC-derived fibroblast cells at passage 24 (aged). The dermal and epidermal compartments thicknesses were measured using image processing software at 10 different levels of each organotypic skin culture. The results are presented as mean ± SD. Scale bars 100 μηι. (b) Immunohistochemical analysis of showed diminished expression of terminal differentiation markers in organotypic skin models generated with hESC-derived fibroblasts at passage 16, compared to those generated with hESC-derived fibroblast at passage 8. K14 and collagen type IV was abundantly expressed in BMs of generated organotypic skin cultures without notable differences. The dashed line indicates the formed BM. Scale bars 100 μηι. Abbreviations: BM, basement membrane, hESC-ebF, hESC- derived fibroblast; p, passage number;

Figure 15: mRNA expression levels of UVA-induced cytokines after acute exposure of monolayer culture to 10 J/cm² UVA intensity. There was no significant difference in mRNA expression levels between two differentiated batches of hESC- derived keratinocytes. In contrary, primary keratinocytes exhibited donor-dependent expression levels of IL-1a and IL-6. ^*P <0.05, ^**P <0.01 versus primary keratinocyte obtained from donor 2 at the same time point. Error bars indicate mean ± SD. Abbreviations: hESC-Kert, hESC-derived keratinocytes; TNF-a, tumor necrosis factor a; IL-1a, interleukin 1a; IL-6, interleukin 6; IL-8, interleukin 8; Figure 16: mRNA expression levels of UVA-induced cytokines after acute exposure of monolayer culture to 20 J/cm² UVA intensity. There was a consistent mRNA expression level in hESC- derived keratinocytes at different differentiation batches. In contrary, primary keratinocytes exhibited donor-dependent expression levels of TNF-a, IL-1a, IL-6 and IL-8. ^*P <0.05, ^**P <0.01 versus primary keratinocyte obtained from donor 2 at the same time point. Error bars indicate mean ± SD. Abbreviations: hESC-Kert, hESC-derived keratinocytes; TNF-a, tumor necrosis factor a; IL-1a, interleukin 1a; IL-6, interleukin 6; IL-8, interleukin 8.

Figure 17: Evaluation of the photoprotective effect of a-tocopherol against UVA- induced cytokines expression levels after acute exposure of hESC-derived keratinocyte to 10 J/cm² and 20 J/cm² UVA. a-T inhibited cytokine expressions under 10 J/cm² UVA intensity. By supplementation of a-T, pronounced down- regulation of cytokine expression levels could be observed under 20 J/cm2 in hESC- derived keratinocytes. ^*P <0.05, ^**P <0.01 versus a-T supplemented groups at the same time point, # P <0.05 versus "non-irradiated" group. Error bars indicate mean ± SD. Abbreviations: hESC-Kert, hESC-derived keratinocytes; +a-T, α-tocopherol supplemented group; TNF-a, tumor necrosis factor a; IL-1a, interleukin 1 a; IL-6, interleukin 6; IL-8, interleukin 8.

Table 1 Examples of Existing Skin Model Systems

variations

• General genetic health problems

• Expensive to obtain

• Ethical issues.

Epidermal • Represent the three- • Lack of dermal layer

organotypic dimensional structure of

• Not representative of human skin biology models epidermal layer

(composed of • Lack of dermal and epidermal interactions

• Live model with metabolic

only epidermal

activity • Donor dependency, batch to batch compartment) variations

• Provide some degree of

barrier properties • Variations in each model barrier function

• Complex process of quality assurance and donors' background checks

• Expensive to obtain

• Ethical issues.

Full-thickness • Full-thickness model • Batch to batch variations

organotypic

• Provide realistic skin three- • Variations in each model barrier function skin models

dimensional structure

(composed of • Complex process of quality assurance and dermal and • Represent the dermal- donors' background checks

epidermal epidermal interactions

• Expensive to obtain

compartment) • Metabolically active • Ethical issues.

• Enhanced barrier properties

in comparison to epidermal

oragnotypic model

Table 2 is a summary of the media used for differentiation of hESC to keratinocytes and subsequent keratincoytes growth.

Table 3 is a summary of the media used for epidermal stratification in epidermal and full-thickness skin models.

MATERIALS AND METHODS

Expansion of hESCs

In this invention we used H1 hESCs obtained from WiCell Research Institute (Madison, Wl) as the source of keratinocyte and fibroblast cell lines. hESCs were cultured on mouse embryonic fibroblasts in hESC medium which composed of DMEM-F12 (Biowest) supplemented with 20% Knockout serum replacer, 1 mM L- glutamine, 4 ng/mL FGF-2 (all from Life Technologies), 1 % nonessential amino acids and 0.1 mM β-mercaptoethanol (all from Sigma).

Differentiation of hESC to fibroblasts

To differentiate hESCs to fibroblasts, hESCs aggregates were transferred on low- attachment plates (Corning) to form embryoid bodies (EB) in hESCs medium without supplementation of FGF-2. After 5 days formed EB aggregates were transferred onto 0.1 % gelatin-coated culture flasks in fibroblast growth medium composing of DMEM high glucose and 10% fetal bovine serum (FBS; Biowest) for 3 weeks, before passaging on new gelatin-coated culture flasks. The cells were cultured at least for 8 passages before incorporation into skin constructs.

Differentiation of hESC to keratinocytes

H1 hESCs were induced to epidermal lineage in defined culture milieu by utilizing the extracellular matrix (ECM) extracted from hESC-derived fibroblasts as a novel autogenic microenvironment. To deposit and extract the ECM components from the cells, 2.5x 10⁵ hESC-derived fibroblasts were seeded onto gelatin-coated plates in fibroblast growth medium. On the next day, the fibroblast growth medium was changed to crowding medium composing of Ficoll cocktail (37.5 mg/mL Ficoll 70 KDa and 25 mg/mL Ficoll 400 KDa; GE Healthcare Life Science) in DMEM medium supplemented with 0.5% FBS, 50 μg/mL ascorbic acid (Sigma). hESC-derived fibroblasts were cultured in crowding medium for 7 days before performing cell lysis and extraction of deposited ECM. Cell lysis was performed by 3-4 repeats of incubation with 0.5% sodium deoxycholate (Sigma) in 0.5X complete protease inhibitor solution (Roche Diagnostics GmbH) and 2 repeats of incubation with 0.5% sodium deoxycholate in PBS. The DNA remnants were removed by incubation of monolayer culture with DNAse solution composing of 10 Mm Tris, 2.5 mM MgCL2, 0.5 mM CaCI2 and 1 U/μΙ DNAse (all from Sigma) at 37 °C for 60 min. The hESCs were differentiated toward epidermal progenitors on the obtained ECM for 10 days in defined keratinocytes serum free medium (DKSFM; Life Technologies) supplemented with 1 μΜ retinoic acid (RA; Sigma) 50 μg/mL ascorbic acid and 0.4 μg/mL hydrocortisone (Sigma). Additionally, the culture medium supplemented with 25 ng/mL bone morphogenetic protein 4 (BMP4: R&D systems) for the first 3 days. Subsequently, the cells were transferred onto collagen IV-coated culture flasks and expanded in DKSFM for at least 30 days before incorporation in SE.

Multilayer epithelium and full-thickness skin model construction

In order to construct multilayer epithelium (MLE), 5x10⁴ cells/cm² hESC-derived keratinocytes or HaCaT cell lines were seeded on polycarbonate culture inserts with 0.4 μηι diameter pore size (Griener Bio-One) in DKSFM for 7 days. The epidermal stratification process was started by supplementing DKSFM with 1 .5 mM of calcium chloride (Sigma), 50 μg/mL ascorbic acid, 0.4 μg/mL hydrocortisone, 1 n ng/mL epidermal growth factor (Sigma) and exposing the cells monolayer surface to Air- Liquid interface. The stratification process was continued for 14 days and medium was fed from the bottom of polycarbonate inserts every two days intervals.

To establish full-thickness skin construct with long-term culture ability and stable dermal compartment, Alvatex® polystyrene scaffold (Reinnervate) was utilized for dermal compartment construction. Briefly, 5χ 10⁵ hESC-derived fibroblasts were seeded in each scaffold and cultured in fibroblast growth medium for 7 days. After formation of dermal compartment in day 8, 5χ 10⁵ hESC-derived keratinocytes were seeded on top of dermal compartment in presence of DKSFM and scaffold submerged in in DKSFM for 7 days before starting the stratification process. In order to trigger epidermal stratification the scaffold surface exposed to Air-Liquid interface and DSKFM was changed to stratification medium designed to maintain dermal and epidermal cells viability over long-term culture which is composed of 3:1 DMEM:DMEM-F12, 5% FBS, 5 ng/mL human insulin, 0.4 μg/mL/mL hydrocortisone, 5 ng/mL bovine transferrin, 10 ng/mL human recombinant epidermal growth factor and 1 .5 mM calcium chloride (all from Sigma otherwise stated). The stratification continued for 9 weeks and full-thickness constructs were fed from the bottom in every 2 days intervals.

Reverse transcriptase - Polymerase Chain Reaction (RT-PCR) analysis

Total RNA was extracted by RNeasy Mini Kit (Qiagen) following manufacturer's instructions. The RNA samples were converted to cDNA using iSCRIPT™ cCDNA synthesis kit (Bio-Rad) according to manufacturers' protocol. The cCDNA samples were amplified by PCR machine (Bio-Rad) and 20 μΐ of amplified PCR products were examined by electrophoresis on 2% agarose gel supplemented with O^g/mL ethidium bromide. After 35 min electrophoresis, the gels were visualized under UV light. In this invention β-actin was served as the internal control.

Immunocytochemistry and frozen section staining

For immunocytochemistry staining of monolayer culture, the cells were fixed for 15 min and then permeabilized for 15 min in 4% paraformaldehyde and 0.4% Triton X- 100 respectively. Blocking was performed by 2% bovine serum albumin for 60 min before overnight incubation of samples with primary antibodies in blocking solution. The samples were washed with 0.05% Tween-20 twice and incubated with fluorochrome-conjugated secondary antibodies for 45 min. For immunohistochemistry, the constructed skin models were frozen in OCT compound (Sakura) and sectioned at 20 μηι thickness before mounting on Poly-L-Lysine slides. The mounted samples subjected to the same process of staining as described above. Fluorescent-stained samples were counterstained with 4',6-diamidino-2- phenylindole (DAPI) before examination by 1X70 inverted fluorescence microscope (Olympus). For morphological analysis of constructed skin models, hematoxylin and eosin (H&E) staining was performed after 5 μηι sectioning of paraffin-embedded samples.

Permeability assay on multilayer epitheliums

Disks containing constructed multilayer epitheliums were mounted in the perfusion chambers. The perfusion chambers were maintained in 37°C in thermo-stated holders. A solution containing permeates was introduced onto the donor chamber from the top while PBS was pumped through the receptor chamber at the bottom at the rate of 1 mL/hour. The receptor samples were collected from each donor chamber at every 30 min intervals. The collected effluent samples were mixed with scintillation fluid and the radioactivity was determined using a liquid scintillation counter. By determining the concentration of penetrant in the donor and receptor, the flux was determined as the amount of penetrant moving across the tissue per unit time and area. The flux was expressed as DPM/cm² /min.

Flux values at each sampling interval were plotted against time until there was no increase in value. Kp as a permeability constant were calculated as: Kp = J/AC which J is the flux at steady state, and AC is the average concentration gradient cpm/cm³.

Example 1

Differentiation of hESC into skin cells

Cell cultures of hESCs were differentiated into pure populations of both fibroblasts and keratinocytes to provide autologous functional cell lines for use in establishment of the skin cell model. Cell phenotype was confirmed through marker expression (figure 1 ). Use of hESC-derived cell lines to form skin equivalent (SE) models permits generation of a perfect replica of human skin and delivers a robust model with minimal variability. Example 2

Generation of a stable stratified Multiple Layered Epithelium (MLE) Model

Existing Skin Equivalent (SE) models suffer from the inherent drawback of poor stability of the dermal component which restricts their usability to, at best, less than a month from generation. Typical models must be used within 1 week. In order to provide full-thickness skin models populated with the hESC-derived cell line, we utilized a polystyrene scaffold for the first time for hESC-derived skin cells (figure 2). The long-term support of scaffold enables us to provide a SE model with significant enhanced stability. Additionally, despite the common scaffolds which avoid the direct epidermal and dermal contacts, the application of this particular scaffold permits direct contact of dermal and epidermal compartments and mimics the in vivo conditions. The direct contact of epidermal and dermal cells can provide another unique advantage in providing the aged-like skin models characterized with dermal atrophy and reduced fibroblast proliferation for wide applications in aging studies. Furthermore, the long-term stability of scaffold permits to investigate over the long- term effects of supplementing the desired test substances. Due to long-term support of scaffold from dermal layer, skin constructs were found to be stable up to 9 weeks. hESCs were able to form MLE in 14 days using an Air-Liquid interface culture method. In previous models, stratification of keratinocytes in an in vitro constructed were not found to be similar as the in vivo constructed models in which stratified squamous tissue cells closely attach to each other in a number of distinct layers. We found that hESCs-MLE consists of 4-5 layers terminally differentiated keratinocytes with the keratinized layer on the top replicating that found in vivo.

Example 3

Characterization of in vitro hESCs-MLE construct at mRNA and protein level

The MLE was characterized at mRNA and protein level. Cytokeratin proteins K5, K6, K14, K16, and K17 are constitutively produced by cells of all terminally differentiated keratinocytes of squamous epithelia in culture. Moreover, K1 , K10, K13, K19, Involucrin and Filaggrin production is useful as specific markers for assessing differentiation of epithelial cells. After 14 days culture of hESCs-derived MLE in contact with Air-Liquid interface, characterization of hESCs-derived MLE at mRNA level confirmed hESCs-derived keratinocytes were able to undergo process of terminal differentiation. Furthermore, the gene expression of the stratified hESCs-derived MLE was found to be similar to the human keratinocytes as our study control (HaCaT-MLE) indicates (Figure 3).

We analyzed the expression of terminal differentiation cytokeratin markers Filaggrin and Involucrin at protein level by immunostaining and western blot in hESCs-derived MLE. Furthermore, in superficial layers, Filaggrin and Involucrin are found to be associated with keratohyalin granules which are thought to facilitate the aggregation and formation of cross-links between the cytokeratin filaments which results in the formation of skin barrier at superficial layers. Therefore, it was of our interest to analyze the expression of Filaggrin and Involucrin in hESCs-derived MLE at protein level to examine the formation of barrier at superficial layers. hESCs-derived -MLE was able to express Filaggrin and Involucrin at MLE surface (Fig. 4B). Our results indicate the hESCs-derived MLE show similar expression of Filaggrin and Involucrin as human keratinocytes (Fig. 4C). Furthermore, western blot analysis demonstrates there is no difference in level of expression Filaggrin and Involucrin between hESCs- derived MLE and HaCaT-MLE (Fig. 6).

At the next step we analyzed the expression of K14 and Collagen IV at MLE. Our finding indicated hESCs-derived MLE exhibits same expression of Collagen IV and K14 as HaCaT-MLE (Fig 5).

Morphological analysis by SEM revealed a stratified cellular arrangement (figure 7). Example 4 Application of hESCs-derived MLE for Permeability Assay

Formation of skin barrier properties leads to organization of permeable lipid structure at the outermost layer of skin which not only serves as a functional barrier toward penetration of exogenous hazards, but also provides a transportation avenue for drugs, reagents and active ingredients. Therefore, it was our interest to test the permeability of our epidermal SE model to evaluate both barrier properties and the functionality of SE model. For permeability testing; we used tritiated water as it is chemically inert and a sensitive indicator of changes in barrier functions because of its relatively high tissue permeability. We found that hESCs-derived MLE showed similar trend of permeability and Kp index compared to control (Fig. 9). Interestingly, we found that MLE with a layer of dermal compartment showed slightly more resistance than MLE without dermal compartment. The possible reason for this result could be due to the construction of more homogenous and more organized MLE in the presence of fibroblast compared to MLE without dermal compartment. Furthermore, our results demonstrated that hESCs-ebF were not able to show any permeability, however it seems that they play an important indirect role in keratinization of MLE which resulted in increase in permeability of MLE. Figure 8 illustrates hematoxylin and eosin staining of paraffin embedded full-thickness skin organotypic skin construct after 9 weeks. Morphological analysis indicates the formation distinct dermal and epidermal compartments and shaping the well-defined stratum corneum at the top of skin construct.

Example 6 hESC dual cell MLE model is stable after long term culture

Human skin is composed of two distinct physiological compartments, dermis and epidermis, which work in concert to establish skin structure. The communication between these two different layers beside the dermal-epidermal interactions is vital for human skin homeostasis, maintenance and structural integrity. Additionally, without embedding the fibroblasts in enclosed matrix like collagen gel it will be possible to investigate directly over skin dermal layer and the effect of uninterrupted interactions between fibroblasts and keratinocytes in realistic in vivo like conditions. hESC-derived fibroblasts were embedded in a polystyrene scaffold before seeding the keratinocytes. After one week fibroblasts proliferated and migrated inside the porous scaffold to form thick and distinguished dermal layer. In order to provide epidermal compartment hESC-derived keratinocytes were seeded on top of the formed dermal layer and allow them to be in contact to Air-Liquid interface in incubator at 37°C to trigger the epidermal stratification process (fig. 10).

The dermal layer consists of fibroblasts with mainly deposited collagen I. Immunofluorescent analysis showed high deposition of cross-linked collagen I in dermal layer in our SE model (Fig 1 1 .A). The dermal and epidermal compartment interconnected through the basement membrane which is full of Collagen IV and Laminin 5 (Laminin 332) deposited by proliferative basal keratinocytes. Our results indicated that distinct Collagen IV deposited in dermal-epidermal junction (Fig 1 1 .B). The proliferative keratinocytes adjacent to basement membrane are responsible for epidermis homeostasis, differentiation and regeneration. As basal keratinocytes undergo complex process of differentiation from basal layer to the superficial layers, they express distinct proteins with progression of differentiation depending on cells' position in epidermis layers. The proliferative population residing at basal layer expresses k14 (Fig 12.A) by progression of differentiation process the basal layer residing cells traverse upward to shape different epidermis layers and finally shaping impermeable highly cross-linked lipid barrier, stratum corneum. As keratinocytes leave the basal layer and start to migrate to the superficial layers they immediately downregulate K14 expression and express early terminal differentiation markers such as K10 (Fig 12.B). As differentiation progresses, late terminal differentiation markers such as Involucrin and Filaggrin are expressed at the superficial layers followed by expression of Loricrin (Fig 12.C,D,E), which indicates shaping of extensively cross-linked cornified envelops structure to form skin barrier. Cornified envelopes are cross-linked protein-lipid responsible for skin barrier function. Furthermore, Involucrin, Filaggrin and Loricrin are essential proteins for epidermis barrier formation and reinforcement of cornified envelope barrier function. Immunofluorescent analysis of frozen sectioned SE model after long term cultivation showed that the SE populated with hESC-derived cells exhibits normal hierarchy of differentiation markers from basal layer to surface and able to form right level of barrier at the superficial layers in comparison to hESCs-derived MLE. We have developed a full-thickness skin equivalent (SE) model, composing of both dermal and epidermal compartments, with superior long-term stability. Further, the formation of a stratified keratinized epithelium provides requisite permeability reflective of that observed in vivo. Use of skin cell populations from hESC to populate the model also provides unlimited autologous cell lines for development of same, obviating the need to obtain skin cells from human donors and giving rise to a reproducible, standardized, realistic and stable human skin model. This therefore represents and in vitro clinical platform for examination of drug delivery and early pathological events in cutaneous and mucosal tissues, including microbial invasion and colonization, and will also increase our understanding of skin repair and regeneration, maintenance process and wound healing therapies in a defined environment. This will also reduce our reliance on animal and human for scientific experimentation.

Example 7

Autologous organotypic skin models on collagen lattice

In order to examine the functionality of hESC-Kert, the formation stratified epithelium in organotypic culture was evaluated as described previously for primary cell lines (Stark, Baur et al. 1 999, Selekman, Grundl et al. 201 3) . Initially, collagen dermal compartment populated with hESC-derived fibroblast cells at cell density of 1 x 1 0⁵ cells/ml was constructed. The formed dermal compartment was supplemented with hESC-derived keratinocytes at cell density of 3 x 1 0⁵/cm² and kept in contact with air- liquid interface over 1 5 days to allow formation of stratified epithelium. For construction of dermal equivalents, 8 volumes of acidic collagen type I solution from rat tail (Life Technologies) were supplemented with 1 volume of 1 0X DMEM (Sigma) and neutralized with 1 N sodium hydroxide (NaOH), before addition of 1 volume FBS containing 1 x1 0⁵ hESC-derived fibroblast cells. The dermal equivalents were cast into tissue culture insert with the pore size of 1 μηι and incubated for 6 days to allow the gel contraction. The formed dermal equivalents were supplemented with hESC-Kert. The tissue construct submerged with culture medium composing of 1 :3 Ham's F12: DMEM (FAD medium) and 10% FBS supplemented with 5 μg/ml insulin, 0.4 μςΛηΙ hydrocortisone, and 50 μg/ml AA and 1 ng/ml epidermal growth factor (all from Sigma) for 3 days before the medium was lowered to the bottom of insert to provide Air-Liquid interface. The medium for during Air-Liquid exposure was changed to full-thickness skin stratification medium (Table 3) and supplemented for 15 days (Figure 13).

Example 8

Application of serially passaged human embryonic stem cell-derived fibroblasts in modeling intrinsic skin aging phenotype

To establish novel organotypic skin models for research and clinical application the effect of chronic aging human dermal cells and subsequent effect of epidermal regeneration ability was investigated in 3D microenvironment. Therefore, hESC- derived fibroblast were serially passaged and passage numbers 8, 16 and 24, where the hESC-derived fibroblast cells entered senescence phase, were selected. Serially passaged hESC-derived fibroblast at three different passages are representing young, semi-aged and aged fibroblast cells respectively. Dermal compartments populated with hESC-derived fibroblast cells at different passages were supplemented with hESC-derived keratinocytes and co-cultured over 2 weeks in contact with Air-Liquid interface at the presence of full-thickness skin stratification medium (Table 3). After 2 weeks, pronounced differences were observed between each constructed organotypic model. Notably, diminished thickness of dermal compartment from 350 ± 103 μηι to 120 ± 44 μηι [P < 0.01 ] and epidermal compartment from 195 ± 49 μηι to 1 10 ± 23 μηι [P < 0.05] was evident between organotypic cultures generated by early and mid-passage of hESC-derived fibroblasts. However, organotypic skin cultures generated with hESC-derived fibroblast in late passage (p24) failed to construct distinct dermal and epidermal compartment and only 3-4 cell layers were evident. Furthermore, expression analyses of the specific epidermal compartment revealed, notably decreased expression of terminal differentiation markers such as filaggrin, involucrin and cytokeratin 10. We could not observe any significant changes in expression of basal marker K14. Moreover, expression of basement membrane ECM protein, collagen IV, did not show significant change between organotypic skin models generated with early and mid-passaged population of dermal cells. (Figure 14)

Example 9

Assessment of UVA-mediated oxidative damage in hESC-derived epidermal cells and the photoprotective effects of a-tocopherol

UVA is the major part of the UV spectrum (-95%) reaching the earth surface (Svobodova, Zdarilova et al. 2007). UVA-induced damage to the cells mainly results from oxidative stress and accumulation of ROS in the cells (Rittie and Fisher 2002, Pillai, Oresajo et al. 2005). Because the main pathway of UVA-mediated damage to skin cells is through the generation of free radicals, the exogenous application of antioxidant may eliminate or mitigate UVA-mediated damage to the skin cells (Masaki 2010). This example assessed the ability and novelty of hESC-derived epidermal cells in photoaging studies and evaluated the reliability and consistency of these epidermal models in studying effect of UVA on both hESC-derived keratinocytes and primary human keratinocyte.

The effect of UVA radiation on cytokine expressions and the possible similarities and differences between hESC- derived keratinocytes and primary keratinocyte was evaluated. Therefore the cells in monolayer culture were irradiated with UVA and analyzed for mRNA expression at 3, 6, 12 and 24 h post-radiation. In order to evaluate the consistent functionality of hESC-derived keratinocytes cell lines, two different batches of differentiated hESC-derived keratinocytes from same hESC cell source were exposed to UVA at 10 J/cm² and 20 J/cm². Similarly, two primary keratinocyte cell populations, obtained from two different donors, were irradiated with UVA at two intensities. The quantitative RT-PCR results demonstrated a rapid increase in expressions of TNF-a, IL-1a and IL-8 expression which were heightened after 3 hours exposure time in both hESC-Kert and primary keratinocytes under 10 J/cm² and 20 J/cm² UVA exposure. The cytokine expression levels returned to the control levels after 12 h post-irradiation. Notably, the expression levels of TNF-a, IL- 1 α, IL-6 and IL-8 in hESC- derived keratinocytes cell lines at different differentiation batches were relatively consistent as no significant difference could be detected at different time points [P > 0.1 ]. In contrary, primary keratinocyte from two different donors exhibited differential expression levels of IL-1a and IL-6 [P <0.05]. There was no significant difference in expression level of TNF-a and IL-8 at 10 J/cm² [P > 0.2] (Figure 15).

In order to evaluate the cytokine expressions in hESC-derived keratinocytes and primary keratinocytes under high UVA intensities, the monolayer cultures were irritated with 20 J/cm² UVA. As illustrated in the Figure 16, the similar expression trends of TNF-a, IL-1 a, IL-6 and IL-8 under 20 J/cm² UVA intensity were observed. Higher expression levels of all cytokines under 20 J/cm² intensities suggested the dose-dependent expression manner of cytokines in both hESC- derived keratinocytes and primary keratinocytes. However, primary keratinocytes showed pronounced donor-dependent expression levels of TNF-a, IL-1 a, IL-6 and IL-8, whilst hESC- derived keratinocytes at different production batches did not show significant differences in expression levels [P >0.1 ].

There were higher expression levels of TNF-a, IL-1 a and IL-8 in hESC- derived keratinocytes compared to primary keratinocytes. However, the expression level of IL-6 did not display pronounced up-regulation at both 10 J/cm² and 20 J/cm² UVA intensities as compared with non-irradiated group. The same low expression level of IL-6 was detected in primary keratinocyte from donor 2. On the other hand, the general higher cytokine expressions, including IL-6, could be observed in primary keratinocyte from donor 1 . These data suggest the donor-dependent production of some cytokines in keratinocytes exposed to UVA irradiation.

Example 10

Evaluation the effects of a-T as a photoprotective reagent on hESC- derived keratinocytes

Vitamin E is the main chain-breaking endogenous antioxidant, preventing oxidative stress in skin. Natural vitamin E is present in eight isoforms which depends on the position and number of methyl groups positioned in a chromanol ring (α- β-, γ- and δ- isoforms of tocopherols and tocotrienol) (Mijiler, Theile et al. 2010). Amongst these homologues, the a-homologous possess the greatest ROS scavenging ability (Wu, Gao et al. 2008). There is a wealth of published data, suggesting the application of antioxidants/ free radical scavengers can synergize the antioxidative defence ability of human skin and exert protection against photo-oxidative stress (Kaur, Kapila et al. 2007, Wu, Gao et al. 2008, Masaki 2010). In this example, the antioxidant ability of a-T was evaluated on hESC-Kert, since a-T is among the most studied photoprotective reagents against photooxidative stresses in human skin (Zigman, McDaniel et al. 1995, Thevanayagam, Mohamed et al. 2014).

In order to evaluate the photoprotective effect of a-T against low and high intensities of UVA, the hESC-derived keratinocytes monolayer cultures were supplemented with 100 g/ml a-T and incubated for 48 h before exposure to UVA. The mRNA expression analysis revealed the down-regulation of cytokine expressions such as TNF-a, IL-1a and IL-8 to almost control levels in hESC-derived keratinocytes at 10 J/cm² intensity. At 20 J/cm² UVA intensity, the significant reduction in expression of TNF-a, IL-1a and IL-8 upon supplementation of a-T could be detected. However, still significant up-regulation of cytokines compared to control levels could be detected [P <0.05]. These results are in harmony of ROS production level measurement and confirmed the abolishment of ROS production under 10 J/cm² resulted in photoprotective effect and suppression of cytokine expressions (Figure 17). The expression levels of IL-6 under both high and low intensities did not show any significant changes [P >0.1] indicating there was no effect of a-T on modulation of IL-6 expression in hESC-derived keratinocytes.

References

Barai ND, Boyce ST, Hoath SB, et al. (2008) Improved barrier function observed in cultured skin substitutes developed under anchored conditions. Skin Research and Technology 14:418-24.

Barker JN, Mitra RS, Griffiths CE, et al. (1991 ) Keratinocytes as initiators of inflammation. Lancet 337:21 1 -4. Bickers DR, Athar M (2006) Oxidative stress in the pathogenesis of skin disease. The Journal of investigative dermatology 126:2565-75. Garcia N, Doucet O, Bayer M, et al. (2002) Characterization of the barrier function in a reconstituted human epidermis cultivated in chemically defined medium. Int J Cosmet Sci 24:25-34.

Gibbs S, van de Sandt JJM, Merk HF, et al. (2007) Xenobiotic Metabolism in Human Skin and 3D Human Skin Reconstructs: A Review. Current Drug Metabolism 8:758- 72.

Godin B, Touitou E (2007) Transdermal skin delivery: predictions for humans from in vivo, ex vivo and animal models. Adv Drug Deliv Rev 59:1 152-61 .

Groeber F, Holeiter M, Hampel M, et al. (201 1 ) Skin tissue engineering -In vivo and in vitro applications. 128:352-66.

Guo et al., Generation of 3D full-thickness skin equivalents exclusively from human induced pluripotent stem cell (iPSC)-derived keratinocytes and fibroblasts Journal of Investigative Dermatology, 2013; Vol 133, Suppl. 1 , page S258, Abstract #1516.

Hewitt NJ, Edwards RJ, Fritsche E, et al. (2013) Use of human in vitro skin models for accurate and ethical risk assessment: metabolic considerations. Toxicological sciences : an official journal of the Society of Toxicology 133:209-17.

Ji Won Oh et al., Organotypic Skin Culture , J Investigative Dermatology, 2013 Nov; 133(1 1 ): e14. Kaur IP, Kapila M, Agrawal R (2007) Role of novel delivery systems in developing topical antioxidants as therapeutics to combat photoageing. Ageing research reviews 6:271 -88. Masaki H (2010) Role of antioxidants in the skin: anti-aging effects. Journal of dermatological science 58:85-90.

MGIIer L, Theile K, Bohm V (2010) In vitro antioxidant activity of tocopherols and tocotrienols and comparison of vitamin E concentration and lipophilic antioxidant capacity in human plasma. Molecular Nutrition & Food Research 54:731 -42.

Munenari Itoh et al., Generation of 3D Skin Equivalents Fully Reconstituted from Human Induced Pluripotent Stem Cells (iPSCs), Plos One, Vol 8, no 10, 2013.

Pillai S, Oresajo C, Hayward J (2005) Ultraviolet radiation and skin aging: roles of reactive oxygen species, inflammation and protease activation, and strategies for prevention of inflammation-induced matrix degradation - a review. Int J Cosmet Sci 27:17-34.

Rittie L, Fisher GJ (2002) UV-light-induced signal cascades and skin aging. Ageing research reviews 1 :705-20.

Robert M, Dusser I, Muriel MP, et al. (1997) Barrier Function of Reconstructed Epidermis at the Air-Liquid Interface: Influence of Dermal Cells and Extracellular Components. Skin Pharmacology and Physiology 10:247-60.

Svobodova A, Zdarilova A, Maliskova J, et al. (2007) Attenuation of UVA-induced damage to human keratinocytes by silymarin. Journal of dermatological science 46:21 -30.

Thevanayagam H, Mohamed S, Chu W-L (2014) Assessment of UVB- photoprotective and antioxidative activities of carrageenan in keratinocytes. J Appl Phycol 26:1813-21 .

Wu S, Gao J, Dinh QT, et al. (2008) IL-8 production and AP-1 transactivation induced by UVA in human keratinocytes: roles of D-alpha-tocopherol. Molecular immunology 45:2288-96. Zigman S, McDaniel T, Schultz JB, et al. (1995) Damage to cultured lens epithelial cells of squirrels and rabbits by UV-A (99.9%) plus UV-B (0.1 %) radiation and alpha tocopherol protection. Mol Cell Biochem 143:35-46.

Zongyou Guo et al., Building a microphysiological skin model from induced pluripotent stem cells, Stem Cell Research & Therapy, 2013, Vol 4, Suppl 1 .

Claims

A method for the preparation of an organotypic skin model comprising the i) forming a preparation of mammalian pluripotent stem cells and providing cell culture conditions to induce the formation of embryoid bodies comprising differentiated or differentiating mammalian fibroblasts derived from said mammalian pluripotent stem cells;

ii) forming an isolated fraction comprising mammalian fibroblast cells from said embryoid bodies to form an isolated, differentiated mammalian fibroblast cell preparation; or iii) extracting and isolating from said embryoid bodies a mammalian fibroblast derived extracellular matrix fraction; iv) forming a preparation comprising mammalian pluripotent stem cells and the isolated mammalian fibroblast derived extracellular matrix fraction and providing cell culture conditions to induce differentiation of said mammalian pluripotent stem cells to differentiated mammalian keratinocytes;

v) forming a preparation comprising differentiated mammalian fibroblasts according to step (ii) above wherein said preparation is associated with a biocompatible polymeric cell culture substrate and cell culture conditions to form a dermal part of said organotypic skin model; and vi) contacting said dermal part with a preparation of differentiated mammalian keratinocytes according to step (iv) above and providing cell culture conditions sufficient to form a differentiated, stratified organotypic skin model.

2. The method according to claim 1 wherein said mammalian keratinocytes are cultured at an Air-Liquid Interface.

3. The method according to any one of claims 1 or 2 wherein said mammalian keratinocytes and fibroblasts are primate.

4. The method according to claim 3 wherein said primate is human.

5. The method according to any one of claims 1 to 3 wherein said mammalian keratinocytes and fibroblasts are derived from: human embryonic stem cells (hESC), human embryonic germ cells, human induced pluripotent stem cells (hiPSC).

6. The method according to claim 5 wherein said mammalian keratinocytes and fibroblasts are derived from human embryonic stem cells (hESC).

7. The method according to any one of claims 1 to 6 wherein said mammalian fibroblasts cells and/or said keratinocytes are autologous.

8. The method according to any one of claims 1 to 6 wherein said mammalian fibroblasts cells and/or said keratinocytes are allogeneic.

9. The method according to any one of claims 1 to 8 wherein said cell support substrate comprises a biocompatible polymer based scaffold such as but not limited to a polyester including polystyrene, polylactic acid, polyglycolic acid, polycaprolactone, poly-dl-lactic-co-glycolic acid, or the like.

10. The method according to any one of claims 1 to 8 wherein biocompatible support matrix comprises a natural polymer selected from the group: collagen, for example Matrigel®, hyaluronic acid, hyaluronic acid esters such as hyaluronic acid benzyl alcohol, fibrinogen scaffolds, thrombin scaffolds and combinations thereon.

1 1 . The method according to any one of claims 1 to 10 wherein said cell culture comprises additional cell types such as but not limited to melanocytes, neural cells or endothelial cells.

12. The method according to claim 1 1 wherein said additional cell types are derived from human embryonic stem cells (hESC), preferably said additional cell types are autologous.

13. The method according to any one of claims 1 to 12 wherein said method comprises culturing said fibroblasts in step i) for at least 1 -20 days prior to step ii), or more ideally, 2-14 days.

14. The method according to any one of claims 1 to 13 wherein differentiation of pluripotent embryonic stem cells into keratinocytes comprises cell culture media as set forth in Table 2.

15. The method according to any one of claims 1 to 1 wherein the formation of a stratified epidermis comprises cell culture media as set forth in Table 3.

16. An organotypic skin model obtained or obtainable by the method according to any one of claims 1 to 15.

17. A cell culture vessel comprising an organotypic skin model according to claim 16.

18. The cell culture vessel according to claim 17 wherein said cell culture vessel comprises a cell culture insert, optionally removable, containing said organotypic skin model and in fluid contact with cell culture medium.

19. The cell culture vessel according to claim 17 or 18 wherein said vessel comprises cell culture media as set forth in Table 3

20. An organotypic skin model according to claim 16 for use in the testing of test agents such as but not limited to therapeutics, cosmetics, compounds or biologically active xenobiotic agents, on skin cell function and permeability.

21 . A cell array wherein said array comprises a plurality of cell culture vessels according to any one of claims 17 to 19.

22. A method for the high throughput screening of test agents comprising the steps:

i) providing an array according to claim 21 ;

ii) contacting the array with a plurality of agents to be tested;

iii) collating activity data obtained following (ii) above; iv) converting the collated data into a data analysable form; and optionally

v) providing an output for the analysed data.

23. An organotypic skin model according to claim 16 for use in monitoring the ageing of skin.

24. A method for monitoring the ageing of skin comprising:

i) providing an isolated preparation of differentiated mammalian fibroblast cells obtained by the method according to claim 1 (ii);

ii) serially passaging said fibroblasts at least once;

iii) forming a preparation comprising a fibroblast derived extracellular matrix fraction according to claim 1 (iii) and mammalian pluripotent stem cells and providing cell culture conditions to induce differentiation of said mammalian pluripotent stem cells to differentiated mammalian keratinocytes;

iv) forming a preparation comprising differentiated mammalian fibroblasts according to step (ii) above wherein said preparation is associated with a biocompatible polymeric cell culture substrate and cell culture conditions to form a dermal part of said organotypic skin model; v) contacting said dermal part with a preparation of differentiated mammalian keratinocytes according to step (iv) above and providing cell culture conditions sufficient to form a differentiated, stratified organotypic skin rrYodel; and optionally vi) monitoring the structural and functional properties of said stratified organotypic skin model as a measure of skin age.

25. The method according to claim 24 wherein said fibroblasts are passaged at least 8 times.

26. The method according to claim 24 wherein said fibroblasts are passaged at least 16 times.

27. The method according to claim 24 wherein said fibroblasts are passaged at least 24 times.

28. The method according to claim 24 wherein said fibroblasts are serially passaged 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24 or more times.

29. A preparation of fibroblasts obtained by the method according to claim 24 or 25.

30. A preparation of fibroblasts obtained by the method according to claim 24 or 26.

31. A preparation of fibroblasts obtained by the method according to claim 24 or 27.

32. A preparation of fibroblasts obtained by the method according to claim 24 or 28.