We show that iris tissue in the adult rat eye, which is embryonically related to the neural retina, can generate cells expressing differentiated neuronal antigens. In addition, the Crx gene transfer induced the specific antigens for rod photoreceptors in the iris-derived cells, which was not seen in the adult hippocampus-derived neural stem cells. Our findings demonstrate a remarkable plasticity of adult iris tissue with potential clinical applications, as autologous iris tissue can be feasibly obtained with peripheral iridectomy.

The Wnt signaling pathway is essential for the development of diverse tissues during embryogenesis. Signal transduction is activated by the binding of Wnt proteins to the type I receptor lowdensity lipoprotein receptor-related protein 5/6 and the seven-pass transmembrane protein Frizzled (Fzd), which contains a Wntbinding site in the form of a cysteine-rich domain. Known extracellular antagonists of the Wnt signaling pathway can be subdivided into two broad classes depending on whether they bind primarily to Wnt or to low-density lipoprotein receptor-related protein 5/6. We show that the secreted protein Tsukushi (TSK) functions as a Wnt signaling inhibitor by binding directly to the cysteine-rich domain of Fzd4 with an affinity of 2.3 × 10 −10 M and competing with Wnt2b. In the developing chick eye, TSK is expressed in the ciliary/iris epithelium, whereas Wnt2b is expressed in the adjacent anterior rim of the optic vesicle, where it controls the differentiation of peripheral eye structures, such as the ciliary body and iris. TSK overexpression effectively antagonizes Wnt2b signaling in chicken embryonic retinal cells both in vivo and in vitro and represses Wnt-dependent specification of peripheral eye fates. Conversely, targeted inactivation of the TSK gene in mice causes expansion of the ciliary body and upregulation of Wnt2b and Fzd4 expression in the developing peripheral eye. Thus, we uncover a crucial role for TSK as a Wnt signaling inhibitor that regulates peripheral eye formation. eye development | signaling modulator | small leucine-rich proteoglycan

Neural stem cells/progenitors that give rise to neurons and glia have been identified in different regions of the brain, including the embryonic retina and ciliary epithelium of the adult eye. Here, we first demonstrate the characterization of neural stem/progenitors in postnatal iris pigment epithelial (IPE) cells. Pure isolated IPE cells could form spheres that contained cells expressing retinal progenitor markers in non-adherent culture. The spheres grew by cell proliferation, as indicated by bromodeoxyuridine incorporation. When attached to laminin, the spheres forming IPE derived cells were able to exhibit neural phenotypes, including retinal-specific neurons. When co-cultured with embryonic retinal cells, or grafted into embryonic retina in vivo, the IPE cells could also display the phenotypes of photoreceptor neurons and Muller glia. Our results suggest that the IPE derived cells have retinal stem/progenitor properties and neurogenic potential without gene transfer, thereby providing a novel potential source for both basic stem cell biology and therapeutic applications for retinal diseases.

The regeneration of lens tissue from the iris of newts has become a classical model of developmental plasticity, although little is known about the corresponding plasticity of the mammalian iris. We here demonstrate and characterize multipotent cells within the iris pigment epithelium (IPE) of postnatal and adult rodents. Acutely-isolated IPE cells were morphologically homogeneous and highly pigmented, but some produced neurospheres which expressed markers characteristic of neural stem/progenitor cells. Stem/progenitor cell markers were also expressed in the IPE in vivo both neonatally and into adulthood. Inner and outer IPE layers differentially expressed Nestin (Nes) in a manner suggesting that they respectively shared origins with neural retina (NR) and pigmented epithelial (RPE) layers. Transgenic marking enabled the enrichment of Nes-expressing IPE cells ex vivo, revealing a pronounced capacity to form neurospheres and differentiate into photoreceptor cells. IPE cells that did not express Nes were less able to form neurospheres, but a subset initiated the expression of pan-neural markers in primary adherent culture. These data collectively suggest that discrete populations of highly-pigmented cells with heterogeneous developmental potencies exist postnatally within the IPE, and that some of them are able to differentiate into multiple neuronal cell types.

It has been suggested that Oct-3/4 may regulate self-renewal in somatic stem cells, as it does in embryonic stem cells. However, recent reports raise the possibility that detection of human Oct-3/4 expression by RT-PCR is prone to artifacts generated by pseudogene transcripts and argue against a role for Oct-3/4 in somatic cells. In this study, we clarified Oct-3/4 expression in mouse somatic tissues using designed PCR primers, which can exclude amplification of its pseudogenes. We found that novel alternative transcripts are indeed expressed in somatic tissues, rather than the normal length transcripts in germline and ES cells. The alternative transcripts indicate the expression of two kinds of truncated proteins. Furthermore, we determined novel promoter regions that are sufficient for the expression of Oct-3/4 transcript variants in somatic cells. These findings provide new insights into the postnatal role of Oct-3/4 in somatic tissues.Oct-3/4 is one of the earliest transcription factors expressed in the embryo, and its orthologs have been found in many different animal species. The nuclear protein is encoded by a homeobox-containing gene Pou5f1, which belongs to the family of POU (Pit Oct Unc) genes (1-3). It regulates gene transcription by binding DNA to octamer motifs ATGCA/TAAT. For many years, Oct-3/4 has been recognized as a gatekeeper for maintaining pluripotency in embryonic stem (ES) 2 cells and pre-implantation embryos. Pluripotent state of ES cells is influenced by expression levels of Oct-3/4 gene (4, 5). More recently, it was demonstrated that mature adult cells could be "reprogrammed" to a very primitive embryonic state via forced expression of four genes (Oct-3/4, c-Myc, Klf-4, and Sox-2) (6); Oct-3/4 is an essential reprogramming factor in this case.Another line of studies have shown Oct-3/4 expression in somatic stem cells and cancer cells (refer to supplement data in Ref. 7). In mouse epithelial tissues, ectopic expression of Oct-3/4 blocks progenitor cell differentiation and causes dysplasia (8). Another report suggests that Oct-3/4 plays a critical role in the genesis of germ cell tumors (9). In addition, Oct-3/4 expression is reported in adult normal tissues (e.g. hematopoietic and mesenchymal stem cells, pancreatic islets, kidney, brain, etc.). These results suggest that Oct-3/4 may not be only crucial for maintaining pluripotency in pre-implantation embryos and germline cells.Although many investigators have reported Oct-3/4 expression in a number of somatic stem cells and cancer cells, this remains a controversial and unsolved issue because of the existence of Oct-3/4 pseudogenes. A number of Oct-3/4 pseudogenes have been identified in humans, and some Oct-3/4 pseudogenes have also been suggested in mouse (10 -14). Many of the results obtained from previous studies for Oct-3/4 expression in somatic tissues using RT-PCR analysis were confusing because PCR primer sets used in the analysis were unable to clearly distinguish Oct-3/4 from its pseudogenes. In fact, Cantz et al. (14) have r...

We investigated the ability of adult human RPE cells to differentiate into neurons. Two cell lines were evaluated. The cells were cultured in medium with 8% serum, transferred to a neural stem cell maintenance culture, and induced to differentiate with retinoic acid. The cells were immunocytochemically examined at each step. The cells showed epithelial-like morphology with 8% serum and all were immunoreactive for beta-III tubulin. After transfer to the stem cell maintenance culture, they changed morphologically and became immunoreactive for MAP5 and neurofilament200 after inducement with retinoic acid. The ratio of MAP5 positive cells was higher in the young adult RPE cell line. No GFAP or rod-opsin immunoreactive cells were observed. Adult human RPE cells even from old person are capable of differentiating into neurons, although the ratio of mature neurons was greater in the young than in the old cell line in this condition.

The success rate is generally higher when cloning mice from embryonic stem (ES) cell nuclei than from somatic cell nuclei, suggesting that the embryonic nature or the undifferentiated state of the donor cell increases cloning efficiency. We assessed the developmental ability of cloned embryos derived from cultured neural stem cell (NSC) nuclei and compared the success rate with that of embryos cloned from other donor cells such as differentiated NSCs, cumulus cells, Sertoli cells and ES cells in the mouse. The transfer of two-cell cloned embryos derived from cultured NSC nuclei into surrogate mothers produced five live cloned mice. However, the success rate (0.5%) was higher in embryos cloned from cultured NSC nuclei than from differentiated NSCs (0%), but lower than that obtained by cloning mice from other cell nuclei (2.2-3.5%). Although the in vitro developmental potential to the two-cell stage of the cloned embryos derived from NSC nuclei (73%) was similar to that of the cloned embryos derived from other somatic cell nuclei (e.g., 85% in Sertoli cells and 75% in cumulus cells), the developmental rate to the morula-blastocyst stage was only 7%. This rate is remarkably lower than that produced from other somatic cells (e.g., 50% in Sertoli cells and 54% in cumulus cells). These results indicate that the undifferentiated state of neural cells does not enhance the cloning efficiency in mice and that the arrest point for in vitro development of cloned embryos depends on the donor cell type.

The role of octamer-binding transcription factor 4 (OCT4) in human cancer is still debated. Although many studies have been published on human OCT4, determining which of the findings are accurate or which are false-positives is currently challenging. We thus developed the most reliable method to date for highly specific and comprehensive detection of genuine OCT4-transcript variants without false-positive results. Our results provided clear evidence that the transcripts of OCT4A, OCT4B, OCT4B1, and other novel splicing variants are indeed present in many cancer cell lines, but are rarely detected in normal tissue-derived differentiated cells. Using the tagged genomic transgene, we then verified endogenous OCT4A translation in cancer cell subpopulations. Moreover, analysis of possible other protein isoforms by enforced expression of OCT4B variants showed that the B164 isoform, designated human OCT4C, is preferentially produced in a cap-dependent manner. We confirmed that the OCT4C isoform, similar to OCT4A, can transform non-tumorigenic fibroblasts in vitro. Finally, ablation of OCT4-positive cells using promoter-driven diphtheria toxin A in high malignant cancer cells caused a significant decrease in migration and Matrigel invasion. These findings strongly suggest a significant contribution of OCT4 to the phenotype of human cancer cells. Stem Cells 2018.