New characterization and safety evaluation of human limbal stem cells used in clinical application: fidelity of mitotic process and mitotic spindle morphologies

Journal article

The limbal epithelial stem cell (LESC) niche is a vital stem cell pool which serves to replenish the cornea with competent stem cells for regrowth and repair throughout adult life. Injury and disease, such as limbal stem cell deficiency (LSCD), can damage and deplete this stem cell pool, resulting in aberrant growth across the cornea and can lead to blindness. The aims of this thesis are the investigation and replication of the LESC niche. Investigation of the niche provided valuable insight into the combined structural and mechanical properties of the niche. Meanwhile, the replication of the niche using smart material fabrication methods enabled active manipulation of primary isolated limbal cells to model niche conditions. These aims were achieved by focusing on the mechanical characterisation of human limbal tissues by non-destructive optical coherence elastography (OCE), combined with optical coherence tomography (OCT) and anatomic replication through the development of a novel bioreactor system. The first study, using OCT/OCE characterisation, revealed the limbal undulation architecture of palisades of Vogt with alteration (in dimension and modulus) in key anatomical features with ageing. To produce a biomimetic in vitro model of the limbus, polydimethylsiloxane (PDMS) based substrates with wrinkled topography were generated by plasma treatment, acid oxidation and dual treatments using a novel stretching frame. This system enabled the PDMS substrate to flatten and wrinkle in a reversible pattern when conducting cell culture, forming the culture surface of a new type of bioreactor. The crypt-like pattern’s dimensions resembled the topography of the LESC niche. The biocompatibility of the patterned substrate was markedly improved using a Gelatin Methacrylate (GelMa) gel coating. The limbal cells cultured on the wrinkled topography proved to retain stemness through the preservation of key stem cell markers such as ABCG2 and P63, whilst indicating the induction of epithelial change by increases in CK3 expression. It was also observed that these wrinkled PDMS surfaces were able to dictate cell growth patterns, showing alignment in motile cells and colony segregation in colony-forming cells in human and porcine limbal cells respectively. The biotechnology developed in this research has exciting potential applications as a disease model for conditions such as LSCD, ageing or injuries where substantial physical anatomical changes can be investigated for their effect on the native cell population. In translational terms, this benchtop application has the potential to reduce the dependency on patient study cases to investigate limbal and other optical surface diseases by providing an actively tuneable culture platform which has a small footprint. Additionally, this technology has scope to reduce the dependency on primary tissue isolations by providing a culture platform whose topographical features can be tailor-made to expansion conditions

Authors: Zekušić, M; Bujić Mihica, M; Skoko, M; Vukušić, K; Risteski, P

• Publication status: Published
• Peer review status: Peer reviewed
• Publisher: BioMed Central
• Journal: Stem Cell Research & Therapy
• Volume: 14
• Issue: 1
• Pages: 368-368
• Article number: 368
• Publication date: 2023-12-13
• DOI: 10.1186/s13287-023-03586-z
• EISSN: 1757-6512
• ISSN: 1757-6512
• Language: English
• Keywords: Genetics; Molecular biology; Biochemistry; Cell cycle; Population; Transplantation; Biology; Cell culture; Cell; Stem cell; Cell biology; Flow cytometry; Mitosis; Pathology; Surgery; Medicine