Long-term expansion of primary equine keratinocytes that maintain the ability to differentiate into stratified epidermis.
Abstract: Skin injuries in horses frequently lead to chronic wounds that lack a keratinocyte cover essential for healing. The limited proliferation of equine keratinocytes using current protocols has limited their use for regenerative medicine. Previously, equine induced pluripotent stem cells (eiPSCs) have been produced, and eiPSCs could be differentiated into equine keratinocytes suitable for stem cell-based skin constructs. However, the procedure is technically challenging and time-consuming. The present study was designed to evaluate whether conditional reprogramming (CR) could expand primary equine keratinocytes rapidly in an undifferentiated state but retain their ability to differentiate normally and form stratified epithelium. Conditional reprogramming was used to isolate and propagate two equine keratinocyte cultures. PCR and FISH were employed to evaluate the equine origin of the cells and karyotyping to perform a chromosomal count. FACS analysis and immunofluorescence were used to determine the purity of equine keratinocytes and their proliferative state. Three-dimensional air-liquid interphase method was used to test the ability of cells to differentiate and form stratified squamous epithelium. Conditional reprogramming was an efficient method to isolate and propagate two equine keratinocyte cultures. Cells were propagated at the rate of 2.39 days/doubling for more than 40 population doublings. A feeder-free culture method was also developed for long-term expansion. Rock-inhibitor is critical for both feeder and feeder-free conditions and for maintaining the proliferating cells in a stem-like state. PCR and FISH validated equine-specific markers in the cultures. Karyotyping showed normal equine 64, XY chromosomes. FACS using pan-cytokeratin antibodies showed a pure population of keratinocytes. When ROCK inhibitor was withdrawn and the cells were transferred to a three-dimensional air-liquid culture, they formed a well-differentiated stratified squamous epithelium, which was positive for terminal differentiation markers. Our results prove that conditional reprogramming is the first method that allows for the rapid and continued in vitro propagation of primary equine keratinocytes. These unlimited supplies of autologous cells could be used to generate transplants without the risk of immune rejection. This offers the opportunity for treating recalcitrant horse wounds using autologous transplantation.
Publication Date: 2018-07-04 PubMed ID: 29973296PubMed Central: PMC6032561DOI: 10.1186/s13287-018-0918-xGoogle Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The research describes a possible solution to chronic wound healing in equine species by developing a method for long-term expansion of primary equine keratinocytes (cells that constitute skin) in a lab setting. These expanded cells, when transplanted, can differentiate into skin layers, offering potential healing benefits.
Objective of the Study
- The study aims to test conditional reprogramming (CR) as a method to rapidly expand primary equine keratinocytes while ensuring the cells maintain their ability to differentiate into skin layers (stratified epidermis). This could potentially present a solution for chronic wound healing in horses.
Methods Used in the Study
- Conditional reprogramming was used to isolate and propagate equine keratinocyte cultures.
- To ensure the cells isolated were of equine origin, PCR (Polymerase Chain Reaction) and FISH (Fluorescent In-Situ Hybridization) were used.
- A chromosomal count was run using Karyotyping.
- FACS (Fluorescence-Activated Cell Sorting) analysis and immunofluorescence were used to confirm the quality of the cells and their proliferative state.
- A three-dimensional air-liquid interphase method was used to test the ability of the cells to differentiate and form a stratified epidermis.
Results of the Study
- The study found that conditional reprogramming method was efficient for isolating and propagating equine keratinocyte cultures.
- The cells were propagated at an impressive rate and a feeder-free culture method was developed that allowed their long-term expansion.
- Karyotyping showed normal equine chromosomes, validating that the cells maintained their genetic make-up.
- FACS results indicated a pure population of keratinocytes.
- When the cells were transferred to a three-dimensional air-liquid culture, they were successful in forming a well-differentiated stratified squamous epithelium, replicating the anatomy of skin layers.
Implications of the Study
- The results suggest that conditional reprogramming can be used to create large volumes of primary equine keratinocytes in the lab.
- When transplanted, these cells can effectively differentiate into skin layers, offering a potential solution for treating chronic wounds in horses.
- The use of autologous cells (cells from the same individual) reduces the risk of immune rejection when transplanted back into the horse.
Cite This Article
APA
Alkhilaiwi F, Wang L, Zhou D, Raudsepp T, Ghosh S, Paul S, Palechor-Ceron N, Brandt S, Luff J, Liu X, Schlegel R, Yuan H.
(2018).
Long-term expansion of primary equine keratinocytes that maintain the ability to differentiate into stratified epidermis.
Stem Cell Res Ther, 9(1), 181.
https://doi.org/10.1186/s13287-018-0918-x Publication
Researcher Affiliations
- Department of Pathology, Georgetown University Medical School, Washington, DC, 20057, USA. faa50@georgetown.edu.
- Department of Oncology, Georgetown University Medical School, Washington, DC, 20057, USA. faa50@georgetown.edu.
- Department of Biochemistry and Molecular Biology, Georgetown University Medical School, Washington, DC, 20057, USA. faa50@georgetown.edu.
- College of Pharmacy, King Abdul Aziz University, Jeddah, Saudi Arabia. faa50@georgetown.edu.
- Department of Pathology, Georgetown University Medical School, Washington, DC, 20057, USA.
- Department of Pathology, Georgetown University Medical School, Washington, DC, 20057, USA.
- Department of Veterinary Integrative Biosciences, Texas A&M University, College Station, TX, USA.
- Department of Veterinary Integrative Biosciences, Texas A&M University, College Station, TX, USA.
- Department of Pathology, Georgetown University Medical School, Washington, DC, 20057, USA.
- Department of Pathology, Georgetown University Medical School, Washington, DC, 20057, USA.
- Equine Clinic, VetOMICs Core Facility, Veterinary University Vienna, Vienna, Austria.
- Department of Population Health and Pathobiology, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA.
- Department of Pathology, Georgetown University Medical School, Washington, DC, 20057, USA.
- Department of Pathology, Georgetown University Medical School, Washington, DC, 20057, USA. richard.schlegel@georgetown.edu.
- Department of Pathology, Georgetown University Medical School, Washington, DC, 20057, USA. Hang.Yuan@georgetown.edu.
MeSH Terms
- Animals
- Cell Differentiation / physiology
- Cells, Cultured
- Epidermal Cells / cytology
- Epidermal Cells / metabolism
- Epidermis / metabolism
- Horses
- Keratinocytes / cytology
- Keratinocytes / metabolism
- Male
Grant Funding
- K01 OD023219 / NIH HHS
Conflict of Interest Statement
ETHICS APPROVAL AND CONSENT TO PARTICIPATE: All samples were collected after approval by the Ethics Review Committee, University of Georgetown. CONSENT FOR PUBLICATION: Not applicable. COMPETING INTERESTS: The authors declare that they have no competing interests. PUBLISHER’S NOTE: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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