Sonya Ruiz-Torres1, Paul F Lambert2, Kathryn A Wikenheiser-Brokamp3,4, Susanne I Wells5,6. 1. Division of Oncology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA. 2. McArdle Laboratory for Cancer Research, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA. 3. Division of Pathology and Laboratory Medicine and The Perinatal Institute Division of Pulmonary Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA. 4. Department of Pathology and Laboratory Medicine, University of Cincinnati College of Medicine, Cincinnati, OH, USA. 5. Division of Oncology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA. Susanne.Wells@cchmc.org. 6. Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, USA. Susanne.Wells@cchmc.org.
Abstract
BACKGROUND: Pluripotent stem cells (PSCs) produced by somatic cell reprogramming self-renew in culture and can differentiate into any cell type, representing a powerful tool for disease modeling, drug screening, regenerative medicine, and the discovery of personalized therapies to treat tissue-specific pathologies. We previously reported the directed differentiation of human PSCs into epidermal stem and progenitor cells (ESPCs) and 3D epidermis to model the inherited syndrome Fanconi anemia (FA), wherein epidermal cell-junctional defects discovered using this system were validated in patient populations. Here, we describe in detail the corresponding protocol for generating PSC-derived keratinocytes using a distinct, normal PSC line (209.2 PSC). METHODS AND RESULTS: Our approach modifies previous protocols to minimize spontaneous cell death and terminal differentiation, eliminate cell stress-inducing keratinocyte selection steps, and reduce total protocol duration and cost. Independent donor-derived PSC lines were converted into ESPCs through the addition of relevant morphogens and a ROCK inhibitor. Results for the 209.2 PSC line highlight consistencies in 2D and also variable features in 3D epidermis compared to the previously published FA-PSC lines. 209.2 PSC-derived ESPCs exhibited a basal cell phenotype while maintaining the capacity to form epidermal organotypic rafts with morphology consistent with fetal epidermis. Transcriptional analyses demonstrated 209.2 ESPCs express epidermis-selective markers and not early endoderm markers, thus supporting an immature stage of p63+ epidermal development. CONCLUSIONS: This protocol provides an accelerated path for the generation of human ESPCs and 3D epidermal models to study normal epidermal development and homeostasis, elucidate mechanisms of epidermal disease pathogenesis, and provides a platform for developing personalized therapies.
BACKGROUND: Pluripotent stem cells (PSCs) produced by somatic cell reprogramming self-renew in culture and can differentiate into any cell type, representing a powerful tool for disease modeling, drug screening, regenerative medicine, and the discovery of personalized therapies to treat tissue-specific pathologies. We previously reported the directed differentiation of human PSCs into epidermal stem and progenitor cells (ESPCs) and 3D epidermis to model the inherited syndrome Fanconi anemia (FA), wherein epidermal cell-junctional defects discovered using this system were validated in patient populations. Here, we describe in detail the corresponding protocol for generating PSC-derived keratinocytes using a distinct, normal PSC line (209.2 PSC). METHODS AND RESULTS: Our approach modifies previous protocols to minimize spontaneous cell death and terminal differentiation, eliminate cell stress-inducing keratinocyte selection steps, and reduce total protocol duration and cost. Independent donor-derived PSC lines were converted into ESPCs through the addition of relevant morphogens and a ROCK inhibitor. Results for the 209.2 PSC line highlight consistencies in 2D and also variable features in 3D epidermis compared to the previously published FA-PSC lines. 209.2 PSC-derived ESPCs exhibited a basal cell phenotype while maintaining the capacity to form epidermal organotypic rafts with morphology consistent with fetal epidermis. Transcriptional analyses demonstrated 209.2 ESPCs express epidermis-selective markers and not early endoderm markers, thus supporting an immature stage of p63+ epidermal development. CONCLUSIONS: This protocol provides an accelerated path for the generation of human ESPCs and 3D epidermal models to study normal epidermal development and homeostasis, elucidate mechanisms of epidermal disease pathogenesis, and provides a platform for developing personalized therapies.
Authors: Sonya Ruiz-Torres; Marion G Brusadelli; David P Witte; Kathryn A Wikenheiser-Brokamp; Sharon Sauter; Adam S Nelson; Mathieu Sertorio; Timothy M Chlon; Adam Lane; Parinda A Mehta; Kasiani C Myers; Mary C Bedard; Bidisha Pal; Dorothy M Supp; Paul F Lambert; Kakajan Komurov; Melinda Butsch Kovacic; Stella M Davies; Susanne I Wells Journal: Cell Stem Cell Date: 2020-11-23 Impact factor: 24.633
Authors: Tobias Hirsch; Tobias Rothoeft; Norbert Teig; Johann W Bauer; Graziella Pellegrini; Laura De Rosa; Davide Scaglione; Julia Reichelt; Alfred Klausegger; Daniela Kneisz; Oriana Romano; Alessia Secone Seconetti; Roberta Contin; Elena Enzo; Irena Jurman; Sonia Carulli; Frank Jacobsen; Thomas Luecke; Marcus Lehnhardt; Meike Fischer; Maximilian Kueckelhaus; Daniela Quaglino; Michele Morgante; Silvio Bicciato; Sergio Bondanza; Michele De Luca Journal: Nature Date: 2017-11-08 Impact factor: 49.962