Generation of Stratified Squamous Epithelial Progenitor Cells from Mouse Induced Pluripotent Stem Cells

Department of Ophthalmology, Keio University School of Medicine, Tokyo, Japan.
PLoS ONE (Impact Factor: 3.23). 12/2011; 6(12):e28856. DOI: 10.1371/journal.pone.0028856
Source: PubMed


Application of induced pluripotent stem (iPS) cells in regenerative medicine will bypass ethical issues associated with use of embryonic stem cells. In addition, patient-specific IPS cells can be useful to elucidate the pathophysiology of genetic disorders, drug screening, and tailor-made medicine. However, in order to apply iPS cells to mitotic tissue, induction of tissue stem cells that give rise to progeny of the target organ is required.
We induced stratified epithelial cells from mouse iPS cells by co-culture with PA6 feeder cells (SDIA-method) with use of BMP4. Clusters of cells positive for the differentiation markers KRT1 or KRT12 were observed in KRT14-positive colonies. We successfully cloned KRT14 and p63 double-positive stratified epithelial progenitor cells from iPS-derived epithelial cells, which formed stratified epithelial sheets consisting of five- to six-polarized epithelial cells in vitro. When these clonal cells were cultured on denuded mouse corneas, a robust stratified epithelial layer was observed with physiological cell polarity including high levels of E-cadherin, p63 and K15 expression in the basal layer and ZO-1 in the superficial layer, recapitulating the apico-basal polarity of the epithelium in vivo.
These results suggest that KRT14 and p63 double-positive epithelial progenitor cells can be cloned from iPS cells in order to produce polarized multilayer epithelial cell sheets.

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    • "K-cES cell populations all displayed the typical morphology of chicken keratinocytes, with large intracellular lipid droplet, desmosomes and tight junctions with abundant (keratin) tonofilaments (Vanhoutteghem et al., 2004). As in CPK, corneocytes could be observed in our cell culture system; however optimization of cell culture conditions will have to be conducted toward the generation of pluristratified reconstituted epidermis, possibly at the air-to-liquid interphase as reported for keratinocytes derived from mammalian ES cells (Aberdam, 2004; Chavez-Munoz et al., 2013; Coraux et al., 2003; Guenou et al., 2009; Lamb and Ambler, 2013; Metallo et al., 2010; Yoshida et al., 2011). "
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    ABSTRACT: A common challenge in avian cell biology is the generation of differentiated cell-lines, especially in the keratinocyte lineage. Only a few avian cell-lines are available and very few of them show an interesting differentiation profile. During the last decade, mammalian embryonic stem cell-lines were shown to differentiate into almost all lineages, including keratinocytes. Although chicken embryonic stem cells had been obtained in the 1990s, few differentiation studies toward the ectodermal lineage were reported. Consequently, we explored the differentiation of chicken embryonic stem cells toward the keratinocyte lineage by using a combination of stromal induction, ascorbic acid, BMP4 and chicken serum. During the induction period, we observed a downregulation of pluripotency markers and an upregulation of epidermal markers. Three homogenous cell populations were derived, which were morphologically similar to chicken primary keratinocytes, displaying intracellular lipid droplets in almost every pavimentous cell. These cells could be serially passaged without alteration of their morphology and showed gene and protein expression profiles of epidermal markers similar to chicken primary keratinocytes. These cells represent an alternative to the isolation of chicken primary keratinocytes, being less cumbersome to handle and reducing the number of experimental animals used for the preparation of primary cells. Copyright © 2015. Published by Elsevier B.V.
    Full-text · Article · Jan 2015 · Stem Cell Research
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    • "It has been reported that early BMP4 stimulation of ES/PA6 co-cultures leads to the appearance of Keratin 14 (Krt14) positive cells after switching to serum containing medium [3], [17]. Although some cells showed Krt14 staining after 9 days of differentiation, there was no effect of BMP4 in inducing Krt14 expression (Fig. 1C). "
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    ABSTRACT: The ability to direct differentiation of mouse embryonic stem (ES) cells into specific lineages not only provides new insights into the pathways that regulate lineage selection but also has translational applications, for example in drug discovery. We set out to develop a method of differentiating ES cells into mesodermal cells at high efficiency without first having to induce embryoid body formation. ES cells were plated on a feeder layer of PA6 cells, which have membrane-associated stromal-derived inducing activity (SDIA), the molecular basis of which is currently unknown. Stimulation of ES/PA6 co-cultures with Bone Morphogenetic Protein 4 (BMP4) both favoured self-renewal of ES cells and induced differentiation into a Desmin and Nestin double positive cell population. Combined stimulation with BMP4 and all-trans Retinoic Acid (RA) inhibited self-renewal and resulted in 90% of cells expressing Desmin and Nestin. Quantitative reverse transcription-polymerase chain reaction (qPCR) analysis confirmed that the cells were of mesodermal origin and expressed markers of mesenchymal and smooth muscle cells. BMP4 activation of a MAD-homolog (Smad)-dependent reporter in undifferentiated ES cells was attenuated by co-stimulation with RA and co-culture with PA6 cells. The Notch ligand Jag1 was expressed in PA6 cells and inhibition of Notch signalling blocked the differentiation inducing activity of PA6 cells. Our data suggest that mesodermal differentiation is regulated by the level of Smad activity as a result of inputs from BMP4, RA and the Notch pathway.
    Full-text · Article · Apr 2012 · PLoS ONE
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    ABSTRACT: We determined the feasibility and optimization of differentiating human induced pluripotent stem cells (hiPS) into nonkeratinized stratified squamous epithelial cells for vocal fold engineering. hiPS were cultured and assessed for differentiation in 3 conditions: a 3-dimensional (3D) hyaluronic acid (HA) hydrogel scaffold, a 3D HA hydrogel scaffold with epidermal growth factor (EGF), and a 3D HA hydrogel scaffold cocultured with human vocal fold fibroblasts (hVFF). After 1, 2, and 4 weeks of cultivation, hiPS were selected for histology, immunohistochemistry, and/or transcript expression analysis. At 4 weeks, hiPS cultivated with hVFF or with EGF had significantly decreased levels of Oct 3/4, indicating loss of pluripotency. Immunofluorescence revealed the presence of pancytokeratin and of cytokeratin (CK) 13 and 14 epithelial-associated proteins at 4 weeks after cultivation in hiPS EGF and hiPS hVFF cultures. The transcript expression level of CK14 was significantly increased for hiPS hVFF cultures only and was measured concomitantly with cell morphology that was clearly cohesive and displayed a degree of nuclear polarity suggestive of epithelial differentiation. We found that hiPS cultivated in 3D HA hydrogel with hVFF demonstrated the most robust conversion evidence to date of epithelial differentiation. Further work is necessary to focus on amplification of these progenitors for application in vocal fold regenerative biology.
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