Lens epithelial cells-induced pluripotent stem cells as a model to study epithelial-mesenchymal transition during posterior capsular opacification

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Abstract

The overall goal was to generate an epithelial-mesenchymal transition (EMT) model using lens epithelial cells-induced pluripotent stem cells to elucidate EMT-regulatory factors during posterior capsular opacification (PCO). For this purpose, the mouse lens epithelial cells-derived mesenchymal cells were reprogrammed to induced pluripotent stem cells (iPSC) and differentiated to lens epithelial cells to be used to determine regulatory factors during EMT. Lens epithelial cells from one-month-old C57BL/6 mice were transitioned to mesenchymal cells in culture, and were reprogrammed to iPSC by delivering reprogramming factors in a single polycistronic lentiviral vector (co-expressing four transcription factors, Oct 4, Sox2, Klf4, and Myc). iPSC were differentiated to epithelial cells by a three-step process using noggin, basic fibroblast growth factor (bFGF), bone morphogenetic protein 4 (BMP4) and Wnt-3. At various time points, the cells/clones were immunocytochemically analyzed for epithelial cell markers (Connexin-43 and E-cadherin), mesenchymal cell markers (Alpha-smooth muscle actin), stem cell markers (Sox1, Oct4, SSEA4 and Tra60) and lens-specific epithelial cell markers (αA- and βA3/A1-crystallins). By increasing the number of genetic transductions, the time needed for generating iPSC from lens mesenchymal cells was reduced, successfully reprogrammed epithelial/mesenchymal cells into iPSC, and retransformed iPSC into lens epithelial cells by the growth factors’ treatment. The epithelial cells could serve as a model system to elucidate regulatory factors involved during EMT to therapeutically stop it.

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In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state.

Marius Wernig, Alexander Meissner, Ruth Foreman … (2007)

Nuclear transplantation can reprogramme a somatic genome back into an embryonic epigenetic state, and the reprogrammed nucleus can create a cloned animal or produce pluripotent embryonic stem cells. One potential use of the nuclear cloning approach is the derivation of 'customized' embryonic stem (ES) cells for patient-specific cell treatment, but technical and ethical considerations impede the therapeutic application of this technology. Reprogramming of fibroblasts to a pluripotent state can be induced in vitro through ectopic expression of the four transcription factors Oct4 (also called Oct3/4 or Pou5f1), Sox2, c-Myc and Klf4. Here we show that DNA methylation, gene expression and chromatin state of such induced reprogrammed stem cells are similar to those of ES cells. Notably, the cells-derived from mouse fibroblasts-can form viable chimaeras, can contribute to the germ line and can generate live late-term embryos when injected into tetraploid blastocysts. Our results show that the biological potency and epigenetic state of in-vitro-reprogrammed induced pluripotent stem cells are indistinguishable from those of ES cells.

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Directly reprogrammed fibroblasts show global epigenetic remodeling and widespread tissue contribution.

Nimet Maherali, Rupa Sridharan, Wei Xie … (2007)

Ectopic expression of the four transcription factors Oct4, Sox2, c-Myc, and Klf4 is sufficient to confer a pluripotent state upon the fibroblast genome, generating induced pluripotent stem (iPS) cells. It remains unknown if nuclear reprogramming induced by these four factors globally resets epigenetic differences between differentiated and pluripotent cells. Here, using novel selection approaches, we have generated iPS cells from fibroblasts to characterize their epigenetic state. Female iPS cells showed reactivation of a somatically silenced X chromosome and underwent random X inactivation upon differentiation. Genome-wide analysis of two key histone modifications indicated that iPS cells are highly similar to ES cells. Consistent with these observations, iPS cells gave rise to viable high-degree chimeras with contribution to the germline. These data show that transcription factor-induced reprogramming leads to the global reversion of the somatic epigenome into an ES-like state. Our results provide a paradigm for studying the epigenetic modifications that accompany nuclear reprogramming and suggest that abnormal epigenetic reprogramming does not pose a problem for the potential therapeutic applications of iPS cells.

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Margaret Wheelock, Yasushi Shintani, Masato Maeda … (2008)

The cadherin molecules at adherens junctions have multiple isoforms. Cadherin isoform switching (cadherin switching) occurs during normal developmental processes to allow cell types to segregate from one another. Tumor cells often recapitulate this activity and the result is an aggressive tumor cell that gains the ability to leave the site of the tumor and metastasize. At present, we understand some of the mechanisms that promote cadherin switching and some of the pathways downstream of this process that influence cell behavior. Specific cadherin family members influence growth-factor-receptor signaling and Rho GTPases to promote cell motility and invasion. In addition, p120-catenin probably plays multiple roles in cadherin switching, regulating Rho GTPases and stabilizing cadherins.