Postnatal periodontal ligament as a novel adult stem cell source for regenerative corneal cell therapy

Abstract

Corneal opacities are a leading cause of global blindness. They are conventionally treated by the transplantation of donor corneal tissue, which is, restricted by a worldwide donor material shortage and allograft rejection. Autologous adult stem cells with a potential to differentiate into corneal stromal keratocytes (CSKs) could offer a suitable choice of cells for regenerative cell therapy. Postnatal periodontal ligament (PDL) contains a population of adult stem cells, which has a similar embryological origin as CSK, that is cranial neural crest. We harvested PDL cells from young adult teeth extracted because of non‐functional or orthodontic reason and differentiated them towards CSK phenotype using a two‐step protocol with spheroid formation followed by growth factor and cytokine induction in a stromal environment (human amnion stroma and porcine corneal stroma). Our results showed that the PDL‐differentiated CSK‐like cells expressed CSK markers (CD34, ALDH3A1, keratocan, lumican, CHST6, B3GNT7 and Col8A2) and had minimal expression of genes related to fibrosis and other lineages (vasculogenesis, adipogenesis, myogenesis, epitheliogenesis, neurogenesis and hematogenesis). Introduction of PDL spheroids into the stroma of porcine corneas resulted in extensive migration of cells inside the host stroma after 14‐day organ culture. Their quiescent nature and uniform cell distribution resembled to that of mature CSKs inside the native stroma. Our results demonstrated the potential translation of PDL cells for regenerative corneal cell therapy for corneal opacities.

Safety and Feasibility of Intrastromal Injection of Cultivated Human Corneal Stromal Keratocytes as Cell-Based Therapy for Corneal Opacities

“…41,42 In vitro differentiation of human embryonic stem cells into CSK-like cells via neural crest progenitors has also been reported, 43 but a number of issues, such as induction efficiency and control of cell purity and risk of tumorigenesis, need to be managed before any clinical application. Human bone marrow-and adipose-derived mesenchymal stem cells (MSCs), dental pulp stem cells, and periodontal ligament stem cells, when intrastromally injected into animal corneas, have been shown to assume CSK phenotypes, 33,[44][45][46][47][48] suggesting that these cell types could potentially be used in stromal cell therapy. However, natural differentiation of stem cells in a particular niche cannot achieve specific cell fate determination; hence not all differentiated cells can display a consistent profile of CSK features.…”

“…Sections were treated with ice-cold 50 mM ammonium chloride (Sigma-Aldrich Corp.), saponin permeabilized, and blocked with 2% bovine serum albumin (BSA; Sigma-Aldrich Corp.) and 5% normal goat serum (NGS; Invitrogen, Carlsbad, CA, USA), followed by incubation with primary antibodies (Table 2) for 2 hours at room temperature. 33 For Kera and Lum immunostaining, sections were pretreated with endo-b-galactosidase (1.5 U/ mL in 10 mM phosphate buffer pH 7.4; Sigma-Aldrich Corp.) for 30 minutes at 378C prior to blocking and antibody incubation. After PBS washes, they were labelled with Red-X-or Alexa 488-conjugated IgG secondary antibody (Jackson ImmunoResearch Labs, West Grove, PA, USA) for 1 hour at room temperature, washed, and mounted with Fluoroshield with DAPI (4 0 ,6-diamidino-2-phenylindole; Santa Cruz Biotech, Santa Cruz, CA, USA) and viewed under fluorescence microscopy (Zeiss Axioplan 2; Carl Zeiss, Oberkochen, Germany).…”

“…The samples were incubated with rabbit anti-human ALDH3A1 antibody (Proteintech, Rosemont, IL, USA) and isotype-specific IgG (BD Biosciences, Singapore), respectively, followed by Alexa Fluor 488-conjugated IgG secondary antibody and propidium iodide, and the staining signal was analyzed by FACSVerse System (BD Biosciences) with a minimum of 10,000 events in each experiment. 33…”

Corneal Repair and Regeneration: Current Concepts and Future Directions

“…Several studies have been done to isolate and characterize multipotent stem cells from various tissues in order to use their great potential in regenerative medicine. Bone marrow-derived mesenchymal stem cells (Islam et al, 2018), human umbilical cord mesenchymal stem cells (Yamashita et al, 2018), postnatal periodontal ligament (Yam et al, 2018), and limbal stem cells (Inatomi et al, 2018; Sasamoto et al, 2018) are recently studied stem cells sources in corneal wound healing and regeneration. Saghizadeh et al (2017) have recently reviewed all major stem cells usage in corneal wound healing.…”

Current Trends and Future Perspective of Mesenchymal Stem Cells and Exosomes in Corneal Diseases

“…PDLSCs, on the other hand, express markers of MSC, embryonic and neural stem cells, and they exhibit multilineage potential in differentiating to adipocytes, chondrocytes, osteoblasts and neurons [78,80,81,82]. Our recent study has shown a robust two-step protocol involving spheroid formation and induction by growth factors and cytokines in a stromal niche, to differentiate human PDLSCs towards CSK-like cells with the expression of a broad spectrum of CSK markers [83]. Further work using animal models will delineate the functionality of these cells and assess their translational potential for stromal diseases.…”