Elsevier

Acta Biomaterialia

Volume 82, December 2018, Pages 93-101
Acta Biomaterialia

Full length article
Development of an innervated tissue-engineered skin with human sensory neurons and Schwann cells differentiated from iPS cells

https://doi.org/10.1016/j.actbio.2018.10.011Get rights and content

Abstract

Cutaneous innervation is increasingly recognized as a major element of skin physiopathology through the neurogenic inflammation driven by neuropeptides that are sensed by endothelial cells and the immune system. To investigate this process in vitro, models of innervated tissue-engineered skin (TES) were developed, yet exclusively with murine sensory neurons extracted from dorsal root ganglions. In order to build a fully human model of innervated TES, we used induced pluripotent stem cells (iPSC) generated from human skin fibroblasts. Nearly 100% of the iPSC differentiated into sensory neurons were shown to express the neuronal markers BRN3A and β3-tubulin after 19 days of maturation. In addition, these cells were also positive to TRPV1 and neurofilament M, and some of them expressed Substance P, TrkA and TRPA1. When stimulated with molecules inducing neuropeptide release, iPSC-derived neurons released Substance P and CGRP, both in conventional monolayer culture and after seeding in a 3D fibroblast-populated collagen sponge model. Schwann cells, the essential partners of neurons for function and axonal migration, were also successfully differentiated from human iPSC as shown by their expression of the markers S100, GFAP, p75 and SOX10. When cultured for one additional month in the TES model, iPSC-derived neurons seeded at the bottom of the sponge formed a network of neurites spanning the whole TES up to the epidermis, but only when combined with mouse or iPSC-derived Schwann cells. This unique model of human innervated TES should be highly useful for the study of cutaneous neuroinflammation.

Statement of Significance

The purpose of this work was to develop in vitro an innovative fully human tissue-engineered skin enabling the investigation of the influence of cutaneous innervation on skin pathophysiology. To reach that aim, neurons were differentiated from human induced pluripotent stem cells (iPSCs) generated from normal human skin fibroblasts. This innervated tissue-engineered skin model will be the first one to show iPSC-derived neurons can be successfully used to build a 3D nerve network in vitro. Since innervation has been recently recognized to play a central role in many human skin diseases, such as psoriasis and atopic dermatitis, this construct promises to be at the forefront to model these diseases while using patient-derived cells.

Introduction

Although the primary purpose of cutaneous sensory innervation is the perception of sense of touch, pain and temperature, it also plays a major role in skin physiology and pathology. Indeed, sensory neurons are able to trigger a neurogenic inflammation upon stimulation through the release of neuropeptides. This process induces microvascular vasodilation and an increase in vascular permeability, along with immune cells chemotaxis and activation [1]. Consequently, neuronal triggering is intimately combined with the local innate immune response early after an injury to promote tissue inflammation. Moreover, it has more recently been shown that sensory neurons are also involved in the direct recognition of pathogens through the same molecular pathways as immune cells, such as Toll Like Receptors (TLRs) [2]. The density of cutaneous innervation as well as the high speed of neural action potentials constitute major advantages for the detection of danger signals. Thus, the in vitro development of a human skin integrating a nerve network represents an essential first step towards detailed studies on the interaction of nociceptor neurons with endothelial and immune cells and the regulation of neurogenic inflammation. We previously created an innervated TES including sensory neurons obtained from mouse embryos. These neurons were cultured underneath the tissue and were shown to build a dense nerve network that reached the epidermis through the whole thickness of the dermal compartment [3], [4]. Neurons released neuropeptides such as SP that was shown to enhance reepithelialization in an in vitro model of wound healing [5]. Whereas murine neuropeptides successfully activated human cells, their animal origin in a human tissue-engineered skin model could be a limitation for further studies, all the more that dorsal root ganglion neurons are extracted at an early embryonic developmental stage (E12 to E14). To develop a more reliable innervated skin model, the use of human sensory neurons is highly desirable, especially for cosmetic research and development in Europe where the use of animal cells is banned.

Fortunately, the technology allowing the differentiation of neuronal cells from human skin fibroblasts induced to become pluripotent stem cells (iPSC) is now widely available [6].

Some research groups succeeded in differentiating iPSC into sensory neurons, albeit with low degrees of purity and cell yields, and these studies did not attempt to create tridimensional networks [7], [8], [9], [10], [11], [12], [13], [14]. To study neurogenic inflammation, it is crucial that the sensory neurons differentiated from human iPS cells express the transient receptor potential cation channel subfamily (TRPV1, TRPV2, TRPV3, TRPA1) that regulate neuropeptides release [15]. Indeed, a challenge with hot temperature or irritant compounds like capsaicin or eugenol should drive activation of TRPVs and subsequent release of neuropeptides, mainly Substance P (SP) and Calcitonin gene-related peptide (CGRP), from peptidergic sensory neurons. Moreover, sensory neurons should express the tropomyosin receptor kinase A (TRKA) responsible for the binding to nerve growth factor (NGF), necessary for their survival [12].

Here, we propose a complete method to differentiate pure, functional sensory neurons and Schwann cells from human iPSCs and their combination in a 3D culture system to generate a fully human tissue-engineered skin model. It will be a highly valuable tool to study in vitro the relationship between human sensory nerves and skin physiopathology.

Section snippets

Cell culture

All human cells used in this work were obtained with informed consent from the donors; The study was approved by the CHU de Quebec-Université Laval research ethics review board.

Fibroblasts and keratinocytes were isolated from human skin biopsies after breast reductive surgeries as previously described [16].

Human keratinocytes were cultured in flasks on a feeder layer of irradiated 3T3 mouse fibroblasts in DHc medium consisting of a 3:1 ratio of DMEM/Ham's F12 (Gibco, Gaithersburg, MD) with

Sensory neurons were differentiated from human iPS cells with high purity, high yield and expressed specific markers

Human fibroblasts-derived iPSCs cultured on Geltrex-coated 6-well plates (2D culture) were allowed to differentiate for 22 days and were stained with specific markers for sensory neurons to confirm their identity. Based on the expression of BRN3A (a transcription factor expressed in the developing sensory neurons [21], [22], neurofilament M (NFM, 150 kDa [23]) and TRPV1, the optimal maturation time was observed at day 19, and was followed by a decrease in the number of cells at day 22 (

Discussion

The aim of this work was to generate human iPSC-derived sensory neurons to be used in place of rodent neurons to prepare innervated tissue-engineered skin models. The animal origin of neurons in these models does not seem to critically compromise their ability to interact with most human skin cells, notably because neuropeptides are highly conserved among species. However, interspecies differences cannot be ruled out. Finally, the use of animal cells has been banned in the European Union for

Declaration of interest

None.

Author contribution

QM and M-JB contributed to the conception, design and achievement of experiments.

TDSB and SB participated to the acquisition of data.

VF participated in interpretation of data and critically revised the article.

FB contributed to the study conception and design, analysis of data, drafting of the article and gave final approval of the submitted version.

Acknowledgments

The authors acknowledge Dr. Jack Puymirat and the iPS-Quebec platform of the CHU de Québec-Université Laval research center for the production of the human iPS cell lines used in this work.

Funding: This work was supported by the CQDM (Centre Québécois pour la Découverte du Médicament), Pierre Fabre Dermocosmétique (Toulouse, France) and the Quebec Network for Cell and Tissue Therapies – ThéCell (a thematic network supported by the Fonds de recherche du Québec–Santé). QM and VF were supported by

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