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miRNome and Proteome Profiling of Human Keratinocytes and Adipose Derived Stem Cells Proposed miRNA-Mediated Regulations of Epidermal Growth Factor and Interleukin 1-Alpha
Linköping University, Department of Biomedical and Clinical Sciences, Division of Surgery, Orthopedics and Oncology. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Anaesthetics, Operations and Specialty Surgery Center, Department of Hand and Plastic Surgery. Modern Sci & Arts Univ, Egypt.
Linköping University, Department of Biomedical and Clinical Sciences, Division of Surgery, Orthopedics and Oncology. Linköping University, Faculty of Medicine and Health Sciences.ORCID iD: 0000-0001-5717-9206
Linköping University, Department of Biomedical and Clinical Sciences, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences, Core Facility. (SciLifeLab)ORCID iD: 0000-0002-5649-4658
Linköping University, Department of Biomedical and Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences.
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2023 (English)In: International Journal of Molecular Sciences, ISSN 1661-6596, E-ISSN 1422-0067, Vol. 24, no 5, article id 4956Article in journal (Refereed) Published
Abstract [en]

Wound healing is regulated by complex crosstalk between keratinocytes and other cell types, including stem cells. In this study, a 7-day direct co-culture model of human keratinocytes and adipose-derived stem cells (ADSCs) was proposed to study the interaction between the two cell types, in order to identify regulators of ADSCs differentiation toward the epidermal lineage. As major mediators of cell communication, miRNome and proteome profiles in cell lysates of cultured human keratinocytes and ADSCs were explored through experimental and computational analyses. GeneChip(R) miRNA microarray, identified 378 differentially expressed miRNAs; of these, 114 miRNAs were upregulated and 264 miRNAs were downregulated in keratinocytes. According to miRNA target prediction databases and the Expression Atlas database, 109 skin-related genes were obtained. Pathway enrichment analysis revealed 14 pathways including vesicle-mediated transport, signaling by interleukin, and others. Proteome profiling showed a significant upregulation of the epidermal growth factor (EGF) and Interleukin 1-alpha (IL-1 alpha) compared to ADSCs. Integrated analysis through cross-matching the differentially expressed miRNA and proteins suggested two potential pathways for regulations of epidermal differentiation; the first is EGF-based through the downregulation of miR-485-5p and miR-6765-5p and/or the upregulation of miR-4459. The second is mediated by IL-1 alpha overexpression through four isomers of miR-30-5p and miR-181a-5p.

Place, publisher, year, edition, pages
MDPI , 2023. Vol. 24, no 5, article id 4956
Keywords [en]
keratinocytes; adipose-derived stem cells; direct co-culture; miRNA; proteome; epidermal growth factor; interleukin 1 alpha; stem cell differentiation
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
URN: urn:nbn:se:liu:diva-192940DOI: 10.3390/ijms24054956ISI: 000948184700001PubMedID: 36902387OAI: oai:DiVA.org:liu-192940DiVA, id: diva2:1749832
Note

Funding Agencies|Centre for Advanced Medical Product, Sweden; Hand and Plastic Surgery Department, Linkoeping University Hospital, Region OEstergoetland, Sweden

Available from: 2023-04-11 Created: 2023-04-11 Last updated: 2023-11-09
In thesis
1. Keratinocytes and Adipose-derived mesenchymal stem cells: The heir and the spare to regenerative cellular therapies for difficult-to-heal skin wounds
Open this publication in new window or tab >>Keratinocytes and Adipose-derived mesenchymal stem cells: The heir and the spare to regenerative cellular therapies for difficult-to-heal skin wounds
2023 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cell-based therapy is considered as Advanced Therapy Medicinal Product, (ATMP), which had increasingly stricter regulations in the last decade. The cells must be produced according to the ‘Guidelines on Good Manufacturing Practice (GMP) specific to Advanced Therapy Medicinal Products’, adopted by the European Medicines Agency (EMA). A fully compliant autologous keratinocyte-based ATMP certified for clinical use remains an unmet challenge in Europe. This necessitates the development of a comprehensive bio-production workflow to tackle key technical bottlenecks along this procedure. On the other hand, adipose-derived mesenchymal stem cells (AD-MSCs) hold promise as an effective alternative to primary keratinocytes in treating difficult-to-heal wounds, particularly for patients with extensive skin wounds. The overall aim of this thesis is to provide a bio-production workflow addressing the challenges associated with developing an autologous keratinocyte-based ATMP. Additionally, the thesis aims to elucidate the molecular and functional mechanisms that modulate the wound healing capabilities of keratinocytes and AD-MSCs. In papers I-III the bio-production procedure for an autologous keratinocyte-based ATMP to treat difficult-to-heal wounds was divided into 3 main stages; keratinocytes extraction, expansion, and transportation. Paper I validated the use of an animal-origin-free enzymatic workflow for the extraction of keratinocytes from the epidermis, compared to the classical workflow containing animal-derived products. Both workflows proved comparable in efficiency in terms of the final cell yield from skin samples, in addition to the purity and functionality of the keratinocytes following cultivation. This report confirms the feasibility of an entirely xeno-free workflow for acquiring GMP-compliant epidermal cells suitable for clinical application without altering key features of keratinocytes. Paper II evaluates an expansion approach for keratinocytes on three culture substrates (1) glass (2) conventional polystyrene (plastic) and (3) animal-derived collagen I ECM matrix. Keratinocytes cultured on glass showed better colonization and survival during the first 3 days of culture. Further molecular characterization revealed evidence of accelerated epidermal differentiation in keratinocytes cultured on glass. Henceforth, functional characterization revealed that glass enhanced the temporal angiogenic and migratory capabilities of keratinocytes. Our findings provided evidence that glass can be a promising substrate capable of supporting keratinocyte cultures, with enhanced wound repair characteristics favourable for transplantation applications. In paper III, we evaluated four candidate solutions for transporting keratinocytes in suspension at 4°C for 24h, namely (1) normal saline; (2) saline with 2.5% human serum albumin; (3) chemically defined, xenofree keratinocyte media; and (4) keratinocyte media with bovine pituitary extract. The tested conditions showed that 2.5% HSA preserved keratinocyte viability, colonization as well as phenotype. This study helped the research team to implement the use of human serum albumin as transportation solution for the proposed keratinocyte-ATMP approach. In paper IV, a direct co-culture model for human keratinocytes and AD-MSCs was proposed to investigate the ability of keratinocytes to enhance AD-MSCs’ differentiation toward the epidermal lineage. Furthermore, miRNA and protein content of human keratinocytes and AD-MSCs were analysed and bioinformatically analysed to identify possible regulations between differentially expressed miRNAs and proteins. This study predicted two potential miRNA-mediated gene regulations with strong implications in AD-MSCs-to-epidermal differentiation; the first was centred on epidermal growth factor (EGF) through miR-485-5p, miR-6765-5p and miR-4459. The second was the regulation of interleukin 1 alpha (IL-1α) by four isomers of miR-30-5p and miR-181a-5p. Paper V evaluates the regenerative potential of autologous AD-MSCs in-vivo using an excisional full-thickness porcine wound model. The data generated from miRNA and protein screening of AD-MSCs was re-analysed with a focus on possible regulations of AD-MCSs in wound healing. Our computational analyses predicted that miR-155 mediates multiple gene regulations of fibroblast growth factor 2 and 7, C-C motif chemokine ligand 2 and vascular cell adhesion molecule 1. The predicted model was verified experimentally and revealed a positive regulation between miR-155 and the identified four factors. Each of these factors carries out key functions within the wound healing process including vascularization, inflammation, proliferation, and remodelling. In summary, the core of the work presented in this thesis provides a complete, in-vitro validated, and EMA-compliant bio-production procedure for autologous keratinocyte as an ATMP. We also presented novel miRNA-mediated epigenetic regulations in human keratinocytes and AD-MSCs. These models can serve as a valuable tool to develop novel hypotheses aiming to elucidate the biology of stem cell differentiation and wound healing. 

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2023. p. 87
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1880
Keywords
Keratinocytes, ATMP, Bio-production, Adipose-derived mesenchymal stem cells, Wound healing
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:liu:diva-199061 (URN)10.3384/9789180753807 (DOI)9789180753791 (ISBN)9789180753807 (ISBN)
Public defence
2023-12-07, Berzeliussalen, building 463, Campus US, Linköping, 09:00 (English)
Opponent
Supervisors
Available from: 2023-11-09 Created: 2023-11-09 Last updated: 2024-05-08Bibliographically approved

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