Methods to generate epithelial cells
The invention provides in certain embodiments, a method of generating a re-programmed differentiated epithelial cell comprising (a) contacting a non-stem somatic cell obtained from a subject with an effective amount of a de-differentiation agent to form a de-differentiated cell, and (b) transfecting the de-differentiated cell with an expression cassette comprising a promoter operably linked to a nucleic acid encoding a conversion agent to form a re-programmed differentiated cell. The invention also provides in certain embodiments, a method of generating a re-programmed differentiated epithelial cell comprising (a) contacting a non-stem somatic cell obtained from a subject with an effective amount of a de-differentiation agent to form a de-differentiated cell, and (b) contacting the de-differentiated cell with a conversion agent to form a re-programmed differentiated cell. The invention provides in certain embodiments, re-programmed differentiated epithelial cells, and methods of using these re-programmed differentiated epithelial cells to repair or re-generate tissue in vivo.
1. A method of generating a re-programmed differentiated mammalian dental epithelial cell comprising
(a) transfecting a mammalian oral epithelial cell or a mammalian odontoblast mesenchymal cell via electroporation or Lipofectin with a vector comprising a nucleic acid encoding Pitx2 to form a de-differentiated cell, and
(b) transfecting the de-differentiated cell via electroporation or Lipofectin with a vector comprising a promoter operably linked to a nucleic acid encoding a miR-200a-3p to form a re-programmed differentiated epithelial cell, and
(c) culturing the re-programmed differentiated epithelial cell to generate a mammalian dental epithelial cell that expresses amelogenin.
2. The method of claim 1 , wherein the promoter is a polII or polIII promoter.
3. The method of claim 1 , wherein the promoter comprises at least one of a tissue-specific promoter and an inducible promoter.
4. The method of claim 1 , wherein the vector comprising the promoter operably linked to the nucleic acid encoding the miR-200a-3p further comprises a marker gene.
5. The method of claim 1 , wherein the vector of step (a) or step (b) is a viral vector.
6. The method of claim 1 , wherein the vector of step (a) or step (b) is a plasmid.
7. The method of claim 1 , further comprising
(d) growing the mammalian dental epithelial cell on a mesh in order to form a multi-cellular tissue.
8. The method of claim 7 , wherein the mesh comprises collagen matrix, sponge, nanoparticle mesh or scaffold, lipids, or fibers.
9. The method of claim 7 , wherein the growing is for 2 to 10 weeks.
10. The method of claim 7 , further comprising (e) implanting the multi-cellular tissue into the subject.
11. A mammalian dental epithelial cell that expresses amelogenin produced by the method of claim 1 .