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: 2.04).
01/2008; 26(10):1220-7. DOI: 10.1097/ICO.0b013e31814fa814
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.
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- "We have shown that k12 expression is accompanied by upregulation of pax6 expression during transdifferentiation. Overexpression of pax6 in bulge cells switched on k12 expression. A similar result is that pax6- transfected embryonic stem cells expressed k12 and differentiated into corneal epithelial-like cells (Ueno et al., 2007), suggesting that pax6 is necessary for k12 promoter n. Wnt signaling pathway is essential not only for embryogenesis , but also for stem cell differentiation in a range of epithelia. "
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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.
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ABSTRACT: Adult stem cell therapy is efficiently applied to severe skin burn patients but with some important limitations. A major challenge remains with regards to the maintenance of the stem cell's self-renewal and pluripotency ex vivo. Human embryonic stem (ES) cells that are derived from the inner cell mass of the developing blastocyst were successfully isolated for the first time in 1998. These cells can be passaged in culture as undifferentiated pluripotent colonies and may also enter lineage-specific differentiation under the appropriate signals. Thus, human ES cells are considered to be a promising source for future regenerative medicine. Herein, we review the most recent advances and achievements as well as important challenges and obstacles that must be resolved before using ES cells for regenerative medicine of cutaneous and corneal epithelium. Recently, ES cells have been successfully differentiated into pure progenitor cell populations of epidermal lineage but future attempts are still required for manipulating these cells into their correct functionality in vivo.
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ABSTRACT: It is theoretically possible to induce various cell types, including retinal neurons, from embryonic stem cells (ESCs). pax6 regulates early events in eye development, including the generation of retinal ganglion cells (RGCs). We previously reported the successful induction of corneal epithelial cells from ESCs transfected with the pax6 gene. Here, we attempted to establish cloned RGC-like cells from ESCs transfected with the pax6 gene.
Undifferentiated mouse ESCs were transfected with pax6 cDNA by electroporation, followed by selection with G418. We conducted limiting-dilution culture of pax6-transfected cells. We expanded the cloned pax6-transfected cells, which expressed nestin and musashi-1, for further characterization in culture media containing fibronectin. The cells were characterized using RT-PCR, immunostaining, electron microscopy, renal subcapsular transplantation assay and Ca imaging.
We obtained clonally expanding pax6-transfected cells, all of which were positive for six3, sonic hedgehog (shh), math5, brn3, thy1 and melanopsin, by using several ESCs. When transplanted into a mouse renal capsule, they differentiated into neurons with elongated axons, expressing betaIII tubulin and neurofilament middle chain, and were free from teratoma development. Electron-microscopic examination showed neurotubules and neurofilaments in the axon-like processes of the cloned pax6-transfected cells. High KCl stimulation increased free Ca influx on Ca2+ imaging.
ESCs were applicable for the induction of retinal progenitor cells, including RGC-like cells, by transfection with the pax6 gene and subsequent limiting-dilution culture. Cloned cell lines may be useful to analyze the requirements for retinal progenitor cell differentiation, and our study suggests the clinical application of this cell type.
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