Article

Experimental transplantation of corneal epithelium-like cells induced by Pax6 gene transfection of mouse embryonic stem cells.

Department of Ophthalmology, St Marianna University School of Medicine, Kawasaki, Kanagawa, Japan.
Cornea (Impact Factor: 2.36). 01/2008; 26(10):1220-7. DOI: 10.1097/ICO.0b013e31814fa814
Source: PubMed

ABSTRACT Corneal epithelial stem cells are deficient in cases of limbal disorders, leading to conjunctival epithelial ingrowth, vascularization, and eventually visual disturbance. We introduced the eye development-associated transcription factor pax6 to embryonic stem (ES) cells and tested whether pax6-transfected cells resembling purified corneal epithelial cells were applicable as a cell source for corneal transplantation.
pax6 cDNA with green fluorescence protein was electrotransfected to ES cells and the cells were cultured with G418 for 14 days. They were characterized by reverse transcription-polymerase chain reaction and immunohistochemistry. The cells were transplanted onto experimentally damaged mouse corneas. Histologic reconstitution of the corneal epithelium was assessed.
pax6-transfected cells formed a monolayer of epithelium-like cells in vitro. They expressed cytokeratin12, a specific keratin of corneal epithelial cells, E-cadherin, and CD44, which are important adhesion molecules of corneal epithelial cells on the cell membrane. They accumulated to make a colony that gave a staining pattern of reticular configuration for cytokeratin 12, E-cadherin, and CD44. When the cells were transplanted onto damaged cornea, they have been kept alive on the cornea.
The purified corneal epithelium-like cells derived from ES cells transfected with pax6 gene adapted to the injured cornea and were kept alive on it. These results suggested application of ES cell-derived corneal epithelial cells for treating corneal injuries.

1 Follower
 · 
151 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: Equine peripheral blood (ePB) can be used as a source of stem cells (SCs) in horses, both for research and for practical purposes. A relatively low volume of ePB is sufficient for the purification and expansion of the SCs. The identification of the SCs is performed by demonstrating the presence (CD34, CD90, CD105 and CD117) or absence (CD14) of specific markers on the cell surface by means of fluorescent staining, followed by Fluorescence Activated Cell Sorting (FACS) for sorting out the desired population of SCs. The entire process of SC isolation and enrichment from ePB typically takes three days, after which the enriched SC sample can be sent back to the patient for clinical application. The two most common clinical applications of SCs from ePB will be demonstrated with two field cases. The first case presents a lesion of the body of the suspensory ligament in a 13-year-old warmblood mare and the second case describes a bacterial ulcerative keratitis in a 20-year- old warmblood gelding.
    Vlaams Diergeneeskundig Tijdschrift 01/2011; · 0.23 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Limbal epithelial stem cell (LESC) deficiency (LSCD) leads to corneal abnormalities resulting in compromised vision and blindness. LSCD can be potentially treated by transplantation of appropriate cells, which should be easily expandable and bankable. Induced pluripotent stem cells (iPSCs) are a promising source of transplantable LESCs. The purpose of this study was to generate human iPSCs and direct them to timbal differentiation by maintaining them on natural substrata mimicking the native LESC niche, including feederless denuded human amniotic membrane (HAM) and de-epithelialized corneas. These iPSCs were generated with nonintegrating vectors from human primary timbal epithelial cells. This choice of parent cells was supposed to enhance timbal cell differentiation from iPSCs by partial retention of parental epigenetic signatures in iPSCs. When the gene methylation patterns were compared in iPSCs to parental LESCs using Illumina global methylation arrays, limbal-derived iPSCs had fewer unique methylation changes than fibroblast-derived iPSCs, suggesting retention of epigenetic memory during reprogramming. Limbal iPSCs cultured for 2 weeks on HAM developed markedly higher expression of putative LESC markers ABCG2, Delta Np63 alpha, keratins 14, 15, and 17, N-cadherin, and TrkA than did fibroblast iPSCs. On HAM culture, the methylation profiles of select timbal iPSC genes (including NTRK1, coding for TrkA protein) became closer to the parental cells, but fibroblast iPSCs remained closer to parental fibroblasts. On denuded air-lifted corneas, timbal iPSCs even upregulated differentiated corneal keratins 3 and 12. These data emphasize the importance of the natural niche and timbal tissue of origin in generating iPSCs as a LESC source with translational potential for LSCD treatment.
    Stem Cells 08/2014; 3(9). DOI:10.5966/sctm.2014-0076 · 7.13 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Several types of adult stem cells are capable of transdifferentiaton into other types of tissues. The hair follicle bulge area is an abundant and easily accessible source of pluripotent adult stem cells. We demonstrate that the bulge KSCs have the potential for transdifferentiation into corneal epithelial-like cells. Bulge KSCs isolated by collagen type IV adhesiveness possessed the highest colony formation efficiency (CFE), and expressed specific markers (CD34 and alpha6-integrin). The isolated cells transdifferentiate into corneal epithelial-like cells in conditioned medium containing corneal limbus soluble factors, including their specific marker, keratin12. The transdifferentiation depends on upregulation of pax6 and downregulation of beta-catenin and Lef-1. Furthermore, overexpression of pax6 in bulge KSCs induced their expression of k12. The expressions of beta-catenin and Lef-1 were not suppressed in the pax6-transfected bulge KSCs, but which were downregulated pax6-transfected cells cultured in the conditioned medium. Bulge KSCs may have potential therapeutic application as cell source for the construction of bioengineered corneas.
    Cell Biology International 08/2009; 33(8):861-6. DOI:10.1016/j.cellbi.2009.04.009 · 1.64 Impact Factor