Generation of 3D Skin Equivalents Fully Reconstituted from Human Induced Pluripotent Stem Cells (iPSCs)

et al. (2013) Generation of 3D Skin Equivalents Fully Reconstituted from Human Induced Pluripotent Stem Cells (iPSCs). PLoS ONE 8(10): e77673. doi:10.1371/journal.pone.0077673 Generation of 3D Skin Equivalents Fully Reconstituted from Human Induced Pluripotent Stem Cells (iPSCs) Munenari Itoh 0 1 Noriko Umegaki-Arao 0 1 Zongyou Guo 0 1 Liang Liu 0 1 Claire A. Higgins 0 1 Angela M. 0 1 Richard L. Eckert, University of Maryland School of Medicine, United States of America 0 Current address: Department of Dermatology, the Jikei University School of Medicine , Tokyo , Japan 1 1 Department of Dermatology, Columbia University, College of Physicians & Surgeons , New York , New York, United States of America, 2 Department of Genetics & Development, Columbia University, College of Physicians & Surgeons , New York, New York , United States of America Recent generation of patient-specific induced pluripotent stem cells (PS-iPSCs) provides significant advantages for cell- and gene-based therapy. Establishment of iPSC-based therapy for skin diseases requires efficient methodology for differentiating iPSCs into both keratinocytes and fibroblasts, the major cellular components of the skin, as well as the reconstruction of skin structures using these iPSC-derived skin components. We previously reported generation of keratinocytes from human iPSCs for use in the treatment of recessive dystrophic epidermolysis bullosa (RDEB) caused by mutations in the COL7A1 gene. Here, we developed a protocol for differentiating iPSCs into dermal fibroblasts, which also produce type VII collagen and therefore also have the potential to treat RDEB. Moreover, we generated in vitro 3D skin equivalents composed exclusively human iPSC-derived keratinocytes and fibroblasts for disease models and regenerative therapies for skin diseases, first demonstrating that iPSCs can provide the basis for modeling a human organ derived entirely from two different types of iPSC-derived cells. - Funding: This work was supported in part by the Dr. Ines Mandl Research Fellowship (Columbia University), and grants from NYSTAR (The New York State Foundation for Science, Technology and Innovation), SDH C024321 (New York State Stem Cell Science), U18TR000561-01 (As part of Tissue Chip Awards from National Institutes of Health/National Center for Advancing Translational Sciences) and Helmsley Stem Cell Starter Grants (Columbia University). This work was supported by the Core facilities of the Skin Disease Research Center at Columbia University (P30AR044535 from NIH/NIAMS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. These authors contributed equally to this work. Induced pluripotent stem cells (iPSCs) are stem cells generated from individual somatic cells by exogenous expression of several transcription factors to initiate the reprogramming process [1]. In recent years, patient-specific iPSCs (PS-iPSCs) have been derived from patients with several human diseases to investigate unknown disease mechanisms and perform pre-clinical testing in various models [2]. Autologous PS-iPSCs have the potential to provide an unlimited source of cells for gene and cell therapies for specific human diseases, since they are believed to have unlimited proliferative capacity and extensive differentiation capability into a wide range of cell types. Recessive dystrophic epidermolysis bullosa (RDEB) is an inherited blistering disorder caused by mutations in the COL7A1 gene encoding type VII collagen, the major component of anchoring fibrils at the basement membrane zone (BMZ) at the epidermal-dermal junction of the skin. Since anchoring fibrils provide functional integrity to the skin, defective anchoring fibrils caused by lack or deficiency of type VII collagen leads to skin fragility. Therefore, patients with RDEB develop severe skin phenotypes, including repeated skin blistering from minor trauma, as well as mutilating scarring, alopecia, corneal erosions, tooth and nail dystrophy, esophageal strictures, joint contractures, and fusion of fingers and toes [3]. RDEB patients also present an increased risk of developing squamous cell carcinomas (SCCs) in early adulthood as a result of repeated and aberrant wound repair and chronic inflammation. Furthermore, since SCCs in patients with RDEB are highly aggressive and metastatic, SCCs are one of the most life-threatening complications for RDEB patients [4]. There is currently no cure for RDEB. One promising strategy for RDEB treatment is to increase the amount of type VII collagen at the epidermal-dermal junction in patients with RDEB to restore skin integrity [5]. Various trials are being pursued using gene-, protein-, drug- and cell-based approaches to address this challenge. Although keratinocytes produce the majority of collagen VII in the skin in vivo, several studies using mouse models and mouse/human fibroblasts have shown that dermal fibroblasts also synthesize type VII collagen, suggesting that both cell types are potentially useful as a source of cells for treating RDEB [610]. Previous clinical studies have shown that a single intradermal injection of allogeneic fibroblasts led to increased expression of type VII collagen at the epidermal-dermal junction in patients with RDEB [10]. Although donor fibroblasts were undetectable in recipient skin 2 weeks after injection, the newly produced type VII collagen persisted for 3 months, suggesting that cell-based approaches that introduce exogenous type VII collagenproducing cells, such as fibroblasts, might have a therapeutic benefit. We previously reported the generation of PS-iPSCs from patients with RDEB and efficient derivation of functional keratinocytes from normal and RDEB PS-iPSCs, thereby demonstrating the utility of PS-iPSCs for establishing cell- and gene-based therapy for RDEB [11]. In contrast, the methodology for generating dermal fibroblasts from ESCs/ iPSCs has not yet been fully defined. Therefore, the goal of this study was to establish proof-of-principle for differentiating iPSCs into dermal fibroblasts with high efficiency to provide a new source of cells for RDEB treatment, and further to develop 3D skin equivalents derived exclusively from human iPSCs. Derivation of Fibroblasts from Human iPSCs We first aimed to develop a differentiation protocol to generate dermal fibroblasts from iPSCs in vitro. In previous studies, ESCs have been differentiated into mesodermal lineages using several methods and reagents. Ascorbic acid is well known to induce mesodermal differentiation of ESCs [12,13] and increase collagen synthesis [14], and is widely used to generate mesodermal components from ESCs [15,16]. Moreover, it has been reported that members of the TGF family can also enhance not only mesodermal differentiation from stem cells [1719], but also collagen production (...truncated)