Epithelial Cell Adhesion Molecule Regulates Tumor Initiation and Tumorigenesis via Activating Reprogramming Factors and Epithelial-Mesenchymal Transition Gene Expression in Colon Cancer

Epithelial cell adhesion molecule (EpCAM) is highly expressed in epithelial-transformed neoplasia and tumor-initiated cells (TICs), but the role that EpCAM plays in the stemness properties of TICs is still unclear. Here we show that EpCAM and reprogramming factors (c-Myc, Oct4, Nanog, and Sox2) were concomitantly elevated in TICs, which were shown to have superior self-renewal, invasiveness, and tumor-initiating abilities. Elevation of EpCAM enhanced tumorsphere formation and tumor initiation. Knockdown of EpCAM inhibited the expressions of reprogramming factors and epithelial-mesenchymal transition genes, thereby suppressing tumor initiation, self-renewal, and invasiveness. In addition, EpCAM, especially intracellular domain of EpCAM (EpICD), bound to and activated the promoter of reprogramming factors. Treatment with the inhibitor of γ-secretase (DAPT) led to the blockage of the expressions of reprogramming factors and epithelial-mesenchymal transition genes, which was accompanied by the reduction of tumor self-renewal and invasion. Furthermore, the increased release of EpEX enhanced production of EpICD and regulated the expression of reprogramming factors. Together, these findings suggest that EpCAM plays an important role in regulating cancer-initiating abilities in TICs of colon cancer. This discovery can be used in the development of new strategies for cancer therapy.     

A germ cell-specific gene, Prmt5, works in somatic cell reprogramming

Germ cells possess the unique ability to acquire totipotency during development in vivo as well as give rise to pluripotent stem cells under the appropriate conditions in vitro. Recent studies in which somatic cells were experimentally converted into pluripotent stem cells revealed that genes expressed in primordial germ cells (PGCs), such as Oct3/4, Sox2, and Lin28, are involved in this reprogramming. These findings suggest that PGCs may be useful for identifying factors that successfully and efficiently reprogram somatic cells into toti- and/or pluripotent stem cells. Here, we show that Blimp-1, Prdm14, and Prmt5, each of which is crucial for PGC development, have the potential to reprogram somatic cells into pluripotent stem cells. Among them, Prmt5 exhibited remarkable reprogramming of mouse embryonic fibroblasts into which Prmt5, Klf4, and Oct3/4 were introduced. The resulting cells exhibited pluripotent gene expression, teratoma formation, and germline transmission in chimeric mice, all of which were indistinguishable from those induced with embryonic stem cells. These data indicate that some of the factors that play essential roles in germ cell development are also active in somatic cell reprogramming.     

Cytoskeletal Expression and Remodeling in Pluripotent Stem Cells

Many emerging cell-based therapies are based on pluripotent stem cells, though complete understanding of the properties of these cells is lacking. In these cells, much is still unknown about the cytoskeletal network, which governs the mechanoresponse. The objective of this study was to determine the cytoskeletal state in undifferentiated pluripotent stem cells and remodeling with differentiation. Mouse embryonic stem cells (ESCs) and reprogrammed induced pluripotent stem cells (iPSCs), as well as the original un-reprogrammed embryonic fibroblasts (MEFs), were evaluated for expression of cytoskeletal markers. We found that pluripotent stem cells overall have a less developed cytoskeleton compared to fibroblasts. Gene and protein expression of smooth muscle cell actin, vimentin, lamin A, and nestin were markedly lower for ESCs than MEFs. Whereas, iPSC samples were heterogeneous with most cells expressing patterns of cytoskeletal proteins similar to ESCs with a small subpopulation similar to MEFs. This indicates that dedifferentiation during reprogramming is associated with cytoskeletal remodeling to a less developed state. In differentiation studies, it was found that shear stress-mediated differentiation resulted in an increase in expression of cytoskeletal intermediate filaments in ESCs, but not in iPSC samples. In the embryoid body model of spontaneous differentiation of pluripotent stem cells, however, both ESCs and iPSCs had similar gene expression for cytoskeletal proteins during early differentiation. With further differentiation, however, gene levels were significantly higher for iPSCs compared to ESCs. These results indicate that reprogrammed iPSCs more readily reacquire cytoskeletal proteins compared to the ESCs that need to form the network de novo. The strategic selection of the parental phenotype is thus critical not only in the context of reprogramming but also the ultimate functionality of the iPSC-differentiated cell population. Overall, this increased characterization of the cytoskeleton in pluripotent stem cells will allow for the better understanding and design of stem cell-based therapies.     

Regulation of embryonic and induced pluripotency by aurora kinase-p53 signaling

Many signals must be integrated to maintain self-renewal and pluripotency in embryonic stem cells (ESCs) and to enable induced pluripotent stem cell (iPSC) reprogramming. However, the exact molecular regulatory mechanisms remain elusive. To unravel the essential internal and external signals required for sustaining the ESC state, we conducted a short hairpin (sh) RNA screen of 104 ESC-associated phosphoregulators. Depletion of one such molecule, aurora kinase A?(Aurka), resulted in compromised self-renewal and consequent differentiation. By integrating global gene expression and computational analyses, we discovered that loss of Aurka leads to upregulated p53 activity?that triggers ESC differentiation. Specifically, Aurka regulates pluripotency through phosphorylation-mediated inhibition of p53-directed ectodermal and mesodermal gene expression. Phosphorylation of p53 not only impairs p53-induced ESC differentiation but also p53-mediated suppression of iPSC reprogramming. Our studies demonstrate an essential role for Aurka-p53 signaling in the regulation of self-renewal, differentiation, and somatic cell reprogramming.     

Cyclin-dependent Kinase-mediated Sox2 Phosphorylation Enhances the Ability of Sox2 to Establish the Pluripotent State

Sox2 is a key factor in maintaining self-renewal of embryonic stem cells (ESCs) and adult stem cells as well as in reprogramming differentiated cells back into pluripotent or multipotent stem cells. Although previous studies have shown that Sox2 is phosphorylated in human ESCs, the biological significance of Sox2 phosphorylation in ESC maintenance and reprogramming has not been well understood. In this study we have identified new phosphorylation sites on Sox2 and have further demonstrated that Cdk2-mediated Sox2 phosphorylation at Ser-39 and Ser-253 is required for establishing the pluripotent state during reprogramming but is dispensable for ESC maintenance. Mass spectrometry analysis of purified Sox2 protein has identified new phosphorylation sites on two tyrosine and six serine/Threonine residues. Cdk2 physically interacts with Sox2 and phosphorylates Sox2 at Ser-39 and Ser-253 in vitro. Surprisingly, Sox2 phosphorylation at Ser-39 and Ser-253 is dispensable for ESC self-renewal and cell cycle progression. In addition, Sox2 phosphorylation enhances its ability to establish the pluripotent state during reprogramming by working with Oct4 and Klf4. Finally, Cdk2 can also modulate the ability of Oct4, Sox2, and Klf4 in reprogramming fibroblasts back into pluripotent stem cells. Therefore, this study has for the first time demonstrated that Sox2 phosphorylation by Cdk2 promotes the establishment but not the maintenance of the pluripotent state. It might also help explain why the inactivation of CDK inhibitors such as p53, p21, and Arf/Ink4 promotes the induction of pluripotent stem cells.     

Conversion of mouse fibroblasts into cardiomyocytes using a direct reprogramming strategy

Here we show that conventional reprogramming towards pluripotency through overexpression of Oct4, Sox2, Klf4 and c-Myc can be shortcut and directed towards cardiogenesis in a fast and efficient manner. With as little as 4 days of transgenic expression of these factors, mouse embryonic fibroblasts (MEFs) can be directly reprogrammed to spontaneously contracting patches of differentiated cardiomyocytes over a period of 11-12 days. Several lines of evidence suggest that a pluripotent intermediate is not involved. Our method represents a unique strategy that allows a transient, plastic developmental state established early in reprogramming to effectively function as a cellular transdifferentiation platform, the use of which could extend beyond cardiogenesis. Our study has potentially wide-ranging implications for induced pluripotent stem cell (iPSC)-factor-based reprogramming and broadens the existing paradigm.     

Cell growth arrest and apoptosis induced by Oct4 or Nanog knockdown in mouse embryonic stem cells: a possible role of Trp53

It has been clear that both Oct4 and Nanog play essential roles in maintaining embryonic stem cells (ESCs) undifferentiation. However, the roles of Oct4 and Nanog in ESCs growth and apoptosis have been much less explored. In this study, we systematically examined the effects of Oct4 or Nanog knockdown on mouse ESCs (mESCs) growth and apoptosis as well as potential mechanisms. Our results show that Oct4 or Nanog knockdown induces growth arrest and apoptosis in mESCs, indicating that the two genes also play important roles in mESCs survival and growth. Moreover, upregulation in Trp53 and its downstream genes expression was detected in Oct4 or Nanog knockdown mESCs, suggesting a possible role of Trp53 in Oct4 or Nanog knockdown induced mESCs growth arrest and apoptosis.     

Derivation, characterization and retinal differentiation of induced pluripotent stem cells

Millions of people world over suffer visual disability due to retinal dystrophies which can be age-related or a genetic disorder resulting in gradual degeneration of the retinal pigmented epithelial (RPE) cells and photoreceptors. Therefore, cell replacement therapy offers a great promise in treating such diseases. Since the adult retina does not harbour any stem cells, alternative stem cell sources like the embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) offer a great promise for generating different cell types of the retina. Here, we report the derivation of four iPSC lines from mouse embryonic fibroblasts (MEFs) using a cocktail of recombinant retroviruses carrying the genes for Oct4, Sox2, Klf4 and cMyc. The iPS clone MEF-4F3 was further characterized for stemness marker expression and stable reprogramming by immunocytochemistry, FACS and RT-PCR analysis. Methylation analysis of the nanog promoter confirmed the reprogrammed epigenetic state. Pluripotency was confirmed by embryoid body (EB) formation and lineage-specific marker expression. Also, upon retinal differentiation, patches of pigmented cells with typical cobble-stone phenotype similar to RPE cells are generated within 6 weeks and they expressed ZO-1 (tight junction protein), RPE65 and bestrophin (mature RPE markers) and showed phagocytic activity by the uptake of fluorescent latex beads.