Engineering epithelial-stromal interactions in vitro for toxicology assessment

Abstract

Crosstalk between epithelial and stromal cells drives the morphogenesis of ectodermal organs during development and promotes normal mature adult epithelial tissue homeostasis. Epithelial-stromal interactions (ESIs) have historically been examined using mammalian models and ex vivo tissue recombination. Although these approaches have elucidated signaling mechanisms underlying embryonic morphogenesis processes and adult mammalian epithelial tissue function, they are limited by the availability of tissue, low throughput, and human developmental or physiological relevance. In this review, we describe how bioengineered ESIs, using either human stem cells or co-cultures of human primary epithelial and stromal cells, have enabled the development of human in vitro epithelial tissue models that recapitulate the architecture, phenotype, and function of adult human epithelial tissues. We discuss how the strategies used to engineer mature epithelial tissue models in vitro could be extrapolated to instruct the design of organotypic culture models that can recapitulate the structure of embryonic ectodermal tissues and enable the in vitro assessment of events critical to organ/tissue morphogenesis. Given the importance of ESIs towards normal epithelial tissue development and function, such models present a unique opportunity for toxicological screening assays to incorporate ESIs to assess the impact of chemicals on mature and developing epidermal tissues.

Keywords: Bioengineering; Development; Epithelial cells; Epithelial-stromal interactions; Morphogenesis; Multipotent stromal cells; Organotypic; Palate fusion; Stromal cells.

Fig. 1. Methods to Generate 3D Organotypic Culture Models (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Schematic demonstration of organotypic culture of epithelial (green) and stromal (blue) cells derived from primary sources or from immortalized human cell lines. A: Recombination of epithelial and stromal cells using in a layer-by-layer assembly approach has been used to generate engineered skin, cornea, mucosa, or vesicals either in submerged or air-liquid interface culture. B: Schematic of primitive skin tissue comprising keratinocytes (white), epithelium (light green), melanocytes (pink), basement membrane (BM; purple), blood vessels (BV; dark blue, red), adipocytes (yellow), ECM, and fibroblasts (blue). C: Recombination and self-assembly of epithelial and stromal cells to hair follicle-like structure after seeding on poly (ethylene-co-vinyl alcohol) (pEVAL) substrates. D: Schematic of primitive hair follicle, comprising hair shaft, inner root sheath (IRS; light green), outer root sheath (white), BM (purple), dermal papilla (DP; blue), BV (dark blue/red), sebaceous glands, and bulge region (stem cell niche). E: 3D encapsulation of epithelial and stromal cells has previously been used to generate mammary gland or pulmonary tissue through culture either in survival factors or morphogens (e.g. HGF, FGF). F: Schematic of primitive mammary gland comprising adipocytes (yellow), epithelial alveolus/acinus (light green), BM (purple), fibroblasts (blue), and BV (dark blue/red). G: The organ germ method encompasses 1. dissociation of embryonic tissues and segregation of epithelial and mesenchymal tissues, 2. encapsulation of epithelial and mesenchymal cells, 3. implantation of ‘organ germs’ to mouse sub-renal capsule, and 4. transplantation of engineered organ germs in situ to assess their function. The organ germ method has been used to generate salivary and lacrimal gland, tooth, hair follicle, and renal tissue. H: Schematic of primitive tooth crown at the late bell stage of development, showing the outer epithelial sheath (dark green), stellate reticulum (purple shading), ameloblasts (light green), enamel (white), dentin (pink shading), and a dental papilla (light blue shading) consisting of odontoblasts (dark blue), neural crest (NC) mesenchyme (light blue), and BV (blue/red).

Fig. 2. Leveraging Mesenchymal-Epithelial Transition in Bioengineered Tissues

Schematic representation of the generation of stratified epidermal tissue (A) and kidney organoids (B) using pluripotent stem cells. A: Organotypic layer-by-layer assembly of encapsulated hESC-derived stromal cells (CD73+/CD105+/CD106+/CD44+/CD10+/CD13+) and monolayer epithelial-like (CD73−/CD105+/CD106−/CD44+/CD10−/CD13+) cells gives rise to a stratified epidermal-like tissue with CK12+ keratinized epithelial layer and CK18+ stromal layer separated by a collagen IV basement membrane B: Pellet culture of metanephric mesenchyme cells generates kidney organoids with renal tubular structures (ECAD−/LTL+ or ECAD+/LTL−), collecting duct structures (ECAD+/GATA3+), glomerulus structures (WT1+), and stromal tissue comprising CD31+/KDR+/Sox17+ primitive vascular networks associated with PDGFRA+ stromal pericyte-like cells.