Periodic Reporting for period 1 - SkinTERM (Skin Tissue Engineering and Regenerative Medicine: From skin repair to regeneration)
Okres sprawozdawczy: 2020-10-01 do 2022-09-30
Treating skin burns and large trauma using transplantation of autologous split-thickness skin carries a number of serious drawbacks, including pain, mobility-limiting contractures and disfiguring scars. The EU-funded SkinTERM project aims to address wound healing in a completely different way: reproducing embryonic skin development in adults by aiming for regeneration rather than repair. Skin organogenesis will be induced by key elements taken from the extracellular matrix of fetal skin and from skin of species that exhibit no scarring, and by employing (stem) cells from relevant cellular origins.
The general training objective is to deliver excellently, supradisciplinary and intersectorially trained, highly employable scientists with knowledge necessary to drive this research area further towards clinical translation in Europe. In addition, we pursue several research objectives 1) to identify important elements of the extracellular matrix involved in scarring and non-scarring systems, 2) to investigate the relevance of (stem) cells and cellular origin in scarring and non-scarring systems, 3) to investigate methodologies to regenerate skin appendages and sensory nerves in skin after wounding, as these are not restored during repair and 4) to take initial steps to translate the research results into clinical applications (medical devices and advanced therapy medicinal products).
The general training objective is to deliver excellently, supradisciplinary and intersectorially trained, highly employable scientists with knowledge necessary to drive this research area further towards clinical translation in Europe. In addition, we pursue several research objectives 1) to identify important elements of the extracellular matrix involved in scarring and non-scarring systems, 2) to investigate the relevance of (stem) cells and cellular origin in scarring and non-scarring systems, 3) to investigate methodologies to regenerate skin appendages and sensory nerves in skin after wounding, as these are not restored during repair and 4) to take initial steps to translate the research results into clinical applications (medical devices and advanced therapy medicinal products).
The SkinTERM project consists of seven different work packages (WPs). WP1 concerns the ethics requirements, which were fulfilled in time. Subsequently WP2-5 are related to the four different research objectives, WP6 concerns education and training and WP7 is dedicated to the management and dissemination of the project. The WP’s progress towards the objectives in the reporting period are summarized below.
Within WP2 “The regenerative matrix”, different components were prepared to be included in bioscaffolds, e.g. type III collagen and fractions of elastin hydrolysates. First prototypes of 3D collagen-based bioscaffolds were constructed and characterized. For extracellular matrix production by fibroblasts of different origins, protocols were optimized and initial characterizations performed. Three Early-stage researchers (ESRs) participated in a laboratory secondment at ADBC in Beverwijk to study the behaviour of various fibroblasts on biomaterials. Another ESR established a protocol to decellularize dermis of mouse and spiny mouse. During two distinct ESR secondments at HMGU, the basis was laid for collaborative proteomics studies. For this, the host ESR is setting up a histology-directed workflow for the molecular analysis of ECM proteins in an animal model for skin regeneration and in vitro cultured human foetal/adult fibroblasts while performing pilot experiments.
Within WP3 “Regenerative non-scarring cells”, one ESR has been trained spiny mouse handling and was able to adapt existing histological techniques protocols of tissue to the particularities of Acomys skin. For human foetal, adult, or eschar fibroblasts, protocols regarding e.g. RNA isolation, RT-qPCR and transcriptome sequencing were optimized for mesenchymal cell populations of different origins. To study the human fibroblast lineages accountable for scarless skin regeneration, another ESR successfully established a human induced pluripotent stem cells (iPSC)-derived skin organoid model. In addition, differentiation protocols for blood-derived monocytes to differentiate into pro-inflammatory or anti-inflammatory macrophage subsets were optimized.
Within WP4 “Introduction of skin appendages and sensory nerves”, focus lies on 1) human sweat gland stem cells, 2) human hair proto-follicles, and 3) human nerve cells. Human iPSC-derived skin organoids were used to isolate and propagate sweat gland stem cells, where sweat gland-specific markers proved that after long-time culture (day 120) the fate choice towards sweat gland formation was achieved. Human hair proto-follicle studies applied hair follicle dermal cell cultures in 2D and bilayered 3D spheroids. To investigate the neurovascular link, human endothelial cells and nerve cells are being included into collagen hydrogels to create a vascular link with nerve cells. For this purpose, human dermal microvascular endothelial cells were isolated and sorted from human skin biopsies and commercial human iPSCs cultures were established.
Within WP5 “Towards translation into medical devices and ATMPs”, prototypes of an intact three-dimensional collagen biomatrix were produced by different approaches and compounds and the most promising prototype was defined according to key opinion leader meetings, internal evaluation, and market needs. In addition, the first protocol for a melanocyte-containing tissue-engineered skin construct was designed and executed.
In WP6 “Education and training”, ESRs are trained in complementary and specific research skills. Nine training courses on personal skills and four workshops on interrelated fields such as biomaterials, skin tissue engineering, hair biology and regeneration were provided. The first SkinTERM annual meeting 2022 was held in Portugal and ESRs presented their research progress during seminars and poster session. A positive interaction and discussion of ESRs with the other consortium members were stimulated. Five ESRs have successfully completed their first secondments visiting SkinTERM partners to experience international, intersectoral and interdisciplinary exchange.
WP7 concerns management and dissemination of the SkinTERM project. Boards were established to monitor the project’s progress and to ensure that the objectives of the training and research programs are achieved. The first dissemination task was establishment of the SkinTERM website, where blogs have been posted about the ESRs’ experiences and training activities. In addition, a twitter account and a SkinTERM ESR Instagram page have been created. The ESR fellows attended and presented at several scientific conferences and meetings. Furthermore, SkinTERM attended the European Tissue Regeneration Society conference and ESRs had a stand with flyers. Both the general public and scientific community were reached by exhibition stands during local and national open days, such as the Scientifica Science Days in Zurich, Switzerland and Public Regenerative Medicine Day in Leiden, the Netherlands.
Within WP2 “The regenerative matrix”, different components were prepared to be included in bioscaffolds, e.g. type III collagen and fractions of elastin hydrolysates. First prototypes of 3D collagen-based bioscaffolds were constructed and characterized. For extracellular matrix production by fibroblasts of different origins, protocols were optimized and initial characterizations performed. Three Early-stage researchers (ESRs) participated in a laboratory secondment at ADBC in Beverwijk to study the behaviour of various fibroblasts on biomaterials. Another ESR established a protocol to decellularize dermis of mouse and spiny mouse. During two distinct ESR secondments at HMGU, the basis was laid for collaborative proteomics studies. For this, the host ESR is setting up a histology-directed workflow for the molecular analysis of ECM proteins in an animal model for skin regeneration and in vitro cultured human foetal/adult fibroblasts while performing pilot experiments.
Within WP3 “Regenerative non-scarring cells”, one ESR has been trained spiny mouse handling and was able to adapt existing histological techniques protocols of tissue to the particularities of Acomys skin. For human foetal, adult, or eschar fibroblasts, protocols regarding e.g. RNA isolation, RT-qPCR and transcriptome sequencing were optimized for mesenchymal cell populations of different origins. To study the human fibroblast lineages accountable for scarless skin regeneration, another ESR successfully established a human induced pluripotent stem cells (iPSC)-derived skin organoid model. In addition, differentiation protocols for blood-derived monocytes to differentiate into pro-inflammatory or anti-inflammatory macrophage subsets were optimized.
Within WP4 “Introduction of skin appendages and sensory nerves”, focus lies on 1) human sweat gland stem cells, 2) human hair proto-follicles, and 3) human nerve cells. Human iPSC-derived skin organoids were used to isolate and propagate sweat gland stem cells, where sweat gland-specific markers proved that after long-time culture (day 120) the fate choice towards sweat gland formation was achieved. Human hair proto-follicle studies applied hair follicle dermal cell cultures in 2D and bilayered 3D spheroids. To investigate the neurovascular link, human endothelial cells and nerve cells are being included into collagen hydrogels to create a vascular link with nerve cells. For this purpose, human dermal microvascular endothelial cells were isolated and sorted from human skin biopsies and commercial human iPSCs cultures were established.
Within WP5 “Towards translation into medical devices and ATMPs”, prototypes of an intact three-dimensional collagen biomatrix were produced by different approaches and compounds and the most promising prototype was defined according to key opinion leader meetings, internal evaluation, and market needs. In addition, the first protocol for a melanocyte-containing tissue-engineered skin construct was designed and executed.
In WP6 “Education and training”, ESRs are trained in complementary and specific research skills. Nine training courses on personal skills and four workshops on interrelated fields such as biomaterials, skin tissue engineering, hair biology and regeneration were provided. The first SkinTERM annual meeting 2022 was held in Portugal and ESRs presented their research progress during seminars and poster session. A positive interaction and discussion of ESRs with the other consortium members were stimulated. Five ESRs have successfully completed their first secondments visiting SkinTERM partners to experience international, intersectoral and interdisciplinary exchange.
WP7 concerns management and dissemination of the SkinTERM project. Boards were established to monitor the project’s progress and to ensure that the objectives of the training and research programs are achieved. The first dissemination task was establishment of the SkinTERM website, where blogs have been posted about the ESRs’ experiences and training activities. In addition, a twitter account and a SkinTERM ESR Instagram page have been created. The ESR fellows attended and presented at several scientific conferences and meetings. Furthermore, SkinTERM attended the European Tissue Regeneration Society conference and ESRs had a stand with flyers. Both the general public and scientific community were reached by exhibition stands during local and national open days, such as the Scientifica Science Days in Zurich, Switzerland and Public Regenerative Medicine Day in Leiden, the Netherlands.
The training of ESRs and cutting-edge research output from the SkinTERM ITN will deliver excellently, supradisciplinary and intersectorially trained, highly employable scientists with knowledge necessary to drive this research area further towards clinical translation in Europe. Early-stage researchers trained in this program will be able to take skin treatment to the next level. The individual consortium members in SkinTERM create a large synergy for the research projects. Cutting-edge research on regeneration in foetal and spiny mouse systems provides essential transferable knowledge about molecular mechanisms regulating regenerative processes. Discriminating proteomics and transcriptomics data lead to compounds involved in scar-free healing that can be incorporated in biomaterials and bio-engineered skin. Moreover, human bio-engineered skin grafts support the investigation on hair growth and regeneration, sweat gland regeneration, innervation and scarring properties of fibroblast subsets and modulatory influence of macrophages.