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Keratinocytes and Adipose-derived mesenchymal stem cells: The heir and the spare to regenerative cellular therapies for difficult-to-heal skin wounds
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.ORCID iD: 0000-0003-3894-6849
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 [en]
Keratinocytes, ATMP, Bio-production, Adipose-derived mesenchymal stem cells, Wound healing
National Category
Cell and Molecular Biology
Identifiers
URN: urn:nbn:se:liu:diva-199061DOI: 10.3384/9789180753807ISBN: 9789180753791 (print)ISBN: 9789180753807 (electronic)OAI: oai:DiVA.org:liu-199061DiVA, id: diva2:1810734
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
List of papers
1. Xeno-free workflow exhibits comparable efficiency and quality of keratinocytes isolated from human skin biopsies
Open this publication in new window or tab >>Xeno-free workflow exhibits comparable efficiency and quality of keratinocytes isolated from human skin biopsies
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2021 (English)In: Regenerative Therapy, ISSN 2352-3204, Vol. 18, p. 401-407Article in journal (Refereed) Published
Abstract [en]

Introduction Regenerative solutions of the skin represent a hope for burn victims with extensive skin loss and chronic wound patients. The development of xeno-free workflow is crucial for clinical application in compliance with the directives of the European Medicines Agency. This study aimed at evaluating the outcome of the xeno-free isolation workflow of keratinocytes from human skin biopsy. Methods Skin biopsies were obtained from volunteers. The epidermis was digested with TrypLE™ Select, which was deactivated by dilution or with trypsin, deactivated by media with fetal bovine serum. Freshly isolated cells were compared for total cell number, viability, activity of caspase 3, gene expression and the presence of the keratinocyte surface markers cytokeratin 14. The cells were cultured in xeno-free conditions for one week and characterized regarding the number and viability as well as the metalloproteinase secretion. Results The number of obtained cells was similar in both workflows. The cell viability was less in the TrypLE group, with slight reduction of the cell surface marker cytokeratin 14. Caspase 3 activity was comparable as well as the gene expression of the apoptotic markers BAX, BCL2 and SLUG, as well as the keratinocyte markers cytokeratin 14, stratifin and filaggrin. Upon culture, the number of keratinocytes, their viability and secretion of matrix metalloproteinases 1 and 10 were equal in both groups. Conclusion This study reports the possibility of isolating functioning and viable keratinocytes through a xeno-free workflow for clinical application.

Place, publisher, year, edition, pages
Elsevier, 2021
Keywords
Keratinocytes, Regenerative medicine, European medicines agency, Xeno-free, TrypLE, Trypsin
National Category
Surgery
Identifiers
urn:nbn:se:liu:diva-179790 (URN)10.1016/j.reth.2021.09.005 (DOI)000703088900050 ()
Note

Funding agencies: Centre for Advanced Medical Product, Sweden; Hand and Plastic Surgery Department, Linköping University Hospital, Region Östergötland, Sweden.

Available from: 2021-10-01 Created: 2021-10-01 Last updated: 2023-11-09Bibliographically approved
2. Human serum albumin as a clinically accepted cell carrier solution for skin regenerative application
Open this publication in new window or tab >>Human serum albumin as a clinically accepted cell carrier solution for skin regenerative application
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2020 (English)In: Scientific Reports, E-ISSN 2045-2322, Vol. 10, no 1, article id 14486Article in journal (Refereed) Published
Abstract [en]

The rules governing Medicinal Products in the European Union necessitates the production of cell-based therapy in good manufacturing practice facilities. The produced cells may need several hours in transportation to reach the application sites. In this study, we investigated four candidate solutions for transporting human keratinocytes. The solutions are (1) normal saline, (2) saline with 2.5% human serum albumin (Saline + HSA), (3) chemically defined, xeno-free keratinocyte media and (4) keratinocyte media with pituitary bovine extract (PBE-media). One million keratinocytes from three donors were suspended in each solution and kept at 4 °C for up to 24 h. Cells kept in Saline + HSA showed higher viability after 1, 3 and 24 h. Then, equal number of viable cells were seeded on collagenous matrix and cultured for 48 h. The adhesion and colonization were higher in the cells kept in PBE-media, while the keratinocyte surface marker, cytokeratin 14, was present in all studied groups. These results confirmed the suitability of Saline + HSA as a cell transportation solution for clinical use, which will be the choice for the planned clinical trial. Keratinocyte PBE-media can be an alternative for cells transported for research purpose, if the same media type is going to be used in the following experiments.

Place, publisher, year, edition, pages
Nature Publishing Group, 2020
National Category
Cell and Molecular Biology Surgery
Identifiers
urn:nbn:se:liu:diva-168966 (URN)10.1038/s41598-020-71553-2 (DOI)000608582500022 ()32879384 (PubMedID)2-s2.0-85090141345 (Scopus ID)
Note

Funding: Open Access funding provided by Linköping University Library

Available from: 2020-09-04 Created: 2020-09-04 Last updated: 2023-11-09Bibliographically approved
3. miRNome and Proteome Profiling of Human Keratinocytes and Adipose Derived Stem Cells Proposed miRNA-Mediated Regulations of Epidermal Growth Factor and Interleukin 1-Alpha
Open this publication in new window or tab >>miRNome and Proteome Profiling of Human Keratinocytes and Adipose Derived Stem Cells Proposed miRNA-Mediated Regulations of Epidermal Growth Factor and Interleukin 1-Alpha
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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
Keywords
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:nbn:se:liu:diva-192940 (URN)10.3390/ijms24054956 (DOI)000948184700001 ()36902387 (PubMedID)
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

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Shahin, Hady

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