EPHA2 is a novel cell surface marker of OCT4-positive undifferentiated cells during the differentiation of mouse and human pluripotent stem cells.

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) possess the intrinsic ability to differentiate into diverse cellular lineages, marking them as potent instruments in regenerative medicine. Nonetheless, the proclivity of these stem cells to generate teratomas post-transplantation presents a formidable obstacle to their therapeutic utility. In previous studies, we identified an array of cell surface proteins specifically expressed in the pluripotent state, as revealed through proteomic analysis. Here we focused on EPHA2, a protein found to be abundantly present on the surface of undifferentiated mouse ESCs and is diminished upon differentiation. Knock-down of Epha2 led to the spontaneous differentiation of mouse ESCs, underscoring a pivotal role of EPHA2 in maintaining an undifferentiated cell state. Further investigations revealed a strong correlation between EPHA2 and OCT4 expression during the differentiation of both mouse and human PSCs. Notably, removing EPHA2+ cells from mouse ESC-derived hepatic lineage reduced tumor formation after transplanting them into immune-deficient mice. Similarly, in human iPSCs, a larger proportion of EPHA2+ cells correlated with higher OCT4 expression, reflecting the pattern observed in mouse ESCs. Conclusively, EPHA2 emerges as a potential marker for selecting undifferentiated stem cells, providing a valuable method to decrease tumorigenesis risks after stem-cell transplantation in regenerative treatments.

Identification of novel proteins differentially expressed in pluripotent embryonic stem cells and differentiated cells.

Mammalian pluripotent stem cells possess properties of self-renewal and pluripotency. These abilities are maintained by the strict regulation of pluripotent stem cell-specific transcription factor network and unique properties of chromatin in the stem cells. Although these major signaling pathways robustly control the characteristics of stem cells, other regulatory factors, such as metabolic pathways, are also known to modulate stem cell proliferation and differentiation. In this study, we fractionated protein samples from mouse embryonic stem (ES) cells cultured with or without the leukemia inhibitory factor (LIF). Protein expression was quantified by 2-dimensional differential gel electrophoresis (2D-DIGE). In total, 44 proteins were identified as being differentially expressed in the pluripotent stem cells and the differentiated cells. Surprisingly, half of the identified proteins were the proteins localized in mitochondria, which supply cellular energy and regulate cell cycle, development, and cell death. Some of these identified proteins are involved in the metabolic function and the regulation of pluripotency. Further analysis of the identified proteins could provide new information for the manipulation of pluripotency in ES cells.

Biosynthesis of ribosomal RNA in nucleoli regulates pluripotency and differentiation ability of pluripotent stem cells.

Pluripotent stem cells have been shown to have unique nuclear properties, for example, hyperdynamic chromatin and large, condensed nucleoli. However, the contribution of the latter unique nucleolar character to pluripotency has not been well understood. Here, we show that fibrillarin (FBL), a critical methyltransferase for ribosomal RNA (rRNA) processing in nucleoli, is one of the proteins highly expressed in pluripotent embryonic stem (ES) cells. Stable expression of FBL in ES cells prolonged the pluripotent state of mouse ES cells cultured in the absence of leukemia inhibitory factor (LIF). Analyses using deletion mutants and a point mutant revealed that the methyltransferase activity of FBL regulates stem cell pluripotency. Knockdown of this gene led to significant delays in rRNA processing, growth inhibition, and apoptosis in mouse ES cells. Interestingly, both partial knockdown of FBL and treatment with actinomycin D, an inhibitor of rRNA synthesis, induced the expression of differentiation markers in the presence of LIF and promoted stem cell differentiation into neuronal lineages. Moreover, we identified p53 signaling as the regulatory pathway for pluripotency and differentiation of ES cells. These results suggest that proper activity of rRNA production in nucleoli is a novel factor for the regulation of pluripotency and differentiation ability of ES cells.

Prohibitin 2 regulates the proliferation and lineage-specific differentiation of mouse embryonic stem cells in mitochondria.

BACKGROUND: The pluripotent state of embryonic stem (ES) cells is controlled by a network of specific transcription factors. Recent studies also suggested the significant contribution of mitochondria on the regulation of pluripotent stem cells. However, the molecules involved in these regulations are still unknown. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we found that prohibitin 2 (PHB2), a pleiotrophic factor mainly localized in mitochondria, is a crucial regulatory factor for the homeostasis and differentiation of ES cells. PHB2 was highly expressed in undifferentiated mouse ES cells, and the expression was decreased during the differentiation of ES cells. Knockdown of PHB2 induced significant apoptosis in pluripotent ES cells, whereas enhanced expression of PHB2 contributed to the proliferation of ES cells. However, enhanced expression of PHB2 strongly inhibited ES cell differentiation into neuronal and endodermal cells. Interestingly, only PHB2 with intact mitochondrial targeting signal showed these specific effects on ES cells. Moreover, overexpression of PHB2 enhanced the processing of a dynamin-like GTPase (OPA1) that regulates mitochondrial fusion and cristae remodeling, which could induce partial dysfunction of mitochondria. CONCLUSIONS/SIGNIFICANCE: Our results suggest that PHB2 is a crucial mitochondrial regulator for homeostasis and lineage-specific differentiation of ES cells.

Characterization of human adipose tissue-resident hematopoietic cell populations reveals a novel macrophage subpopulation with CD34 expression and mesenchymal multipotency.

Adipose tissue (AT) is composed of mature adipocytes and stromal vascular fraction (SVF) cells, including adipose stem/stromal cells (ASCs). We characterized hematopoietic cells residing in human nonobese AT by analyzing the SVF isolated from human lipoaspirates and peripheral blood (PB). Flow cytometry revealed that AT-resident hematopoietic cells consisted of AT-resident macrophages (ATMs) or lymphocytes with a negligible number of granulocytes. AT-resident lymphocytes were composed of helper T cells and natural killer cells. Almost no B cells and few cytotoxic T cells were observed in nonobese AT. More than 90% of ATMs were M2 state CD206(+) macrophages (CD45(+)/CD14(+)) that were located in the periendothelium or interstitial spaces between adipocytes. We also discovered a novel subpopulation of CD34(+)/CD206(+) ATMs (11.1% of CD206(+)ATMs) that localized in the perivascular region. Microarray of noncultured CD34(+)/CD206(+) ATMs, CD34(-)/CD206(+) ATMs, CD45(-)/CD31(-)/CD34(+) ASCs, and PB-derived circulating monocytes revealed that CD34(+)/CD206(+) ATMs shared characteristics with ASCs and circulating monocytes. Unlike CD34(-)/CD206(+) ATMs, CD34(+)/CD206(+) ATMs could grow in adherent culture and were capable of differentiating into multiple mesenchymal (adipogenic, osteogenic, and chondrogenic) lineages, similar to ASCs. CD34(+)/CD206(+) ATMs grew rapidly and lost expression of CD45, CD14, and CD206 by passage 3, which resulted in a similar expression profile to ASCs. Thus, this novel ATM subpopulation (CD45(+)/CD14(+)/CD34(+)/CD206(+)) showed distinct biological properties from other ATMs and circulating monocytes/macrophages. The CD34(+)/CD206(+) ATMs possessed characteristics similar to ASCs, including adherence, localization, morphology, and mesenchymal multipotency. This AT-resident subpopulation may have migrated from the bone marrow and may be important to tissue maintenance and remolding.

Novel cell surface genes expressed in the stomach primordium during gastrointestinal morphogenesis of mouse embryos.

The mechanisms of gastrointestinal morphogenesis in mammals are not well understood. This is partly due to the lack of appropriate markers that are expressed with spatiotemporal specificity in the gastrointestinal tract during development. Using mouse embryos, we surveyed markers of the prospective stomach region during gastrointestinal morphogenesis. The initiation of organ bud formation occurs at E10.5 in mice. These primordia for the digestive organs protrude from a tube-like structured endoderm and have their own distinct morphogenesis. We identified 3 cell surface genes -Adra2a, Fzd5, and Trpv6 - that are expressed in the developing stomach region during gastrointestinal morphogenesis using a microarray-based screening. These novel genes will be useful in expanding our understanding of the mechanisms of gastrointestinal development.

TIF1beta regulates the pluripotency of embryonic stem cells in a phosphorylation-dependent manner.

Transcription networks composed of various transcriptional factors specifically expressed in undifferentiated embryonic stem (ES) cells have been implicated in the regulation of pluripotency in ES cells. However, the molecular mechanisms responsible for self-renewal, maintenance of pluripotency, and lineage specification during differentiation of ES cells are still unclear. The results of this study demonstrate that a phosphorylation-dependent chromatin relaxation factor, transcriptional intermediary factor-1beta (TIF1beta), is a unique regulator of the pluripotency of ES cells and regulates Oct3/4-dependent transcription in a phosphorylation-dependent manner. TIF1beta is specifically phosphorylated in pluripotent mouse ES cells at the C-terminal serine 824, which has been previously shown to induce chromatin relaxation. Phosphorylated TIF1beta is partially colocalized at the activated chromatin markers, and forms a complex with the pluripotency-specific transcription factor Oct3/4 and subunits of the switching defective/sucrose nonfermenting, ATP-dependent chromatin remodeling complex, Smarcd1 [corrected], Brg-1, and BAF155, all of which are components of an ES-specific chromatin remodeling complex, esBAF. Phosphorylated TIF1beta specifically induces ES cell-specific genes and enables prolonged maintenance of an undifferentiated state in mouse ES cells. Moreover, TIF1beta regulates the reprogramming process of somatic cells in a phosphorylation-dependent manner. Our results suggest that TIF1beta provides a phosphorylation-dependent, bidirectional platform for specific transcriptional factors and chromatin remodeling enzymes that regulate the cell differentiation process and the pluripotency of stem cells.

Musashi-1, an RNA-binding protein, is indispensable for survival of photoreceptors.

Musashi-1 (Msi1), an RNA-binding protein (RBP), has been postulated to play important roles in the maintenance of the stem-cell state, differentiation, and tumorigenesis. However, the expression and function of Msi1 in differentiated cells remain obscure. Here we show that Msi1 is expressed in mature photoreceptors and retinal pigment epithelium (RPE) cells, and is indispensable for the survival of photoreceptors. We found in the adult newt eye that Msi1 is expressed in all photoreceptors and RPE cells as well as in the retinal stem/progenitor cells in the ciliary marginal zone (CMZ). We found in the analyses of the newt normal and regenerating retinas that the expression profiles of the Msi1 transcripts and protein isoforms in the photoreceptors are different from those in the retinal stem/progenitor cells. Furthermore, we found that all photoreceptors and RPE cells of the adult mice also express Msi1, and that Msi1 knockout (Msi1-KO) results in degeneration of photoreceptors and a lack of a visual cycle protein RPE65 in the microvilli of RPE cells. Taken together, our current results demonstrate that the expression of Msi1 in mature photoreceptors and RPE cells is evolutionarily conserved, and that Msi1 bears essential functions for vision. Considering such an Msi1-KO phenotype in the retina, it is now reasonable to address whether defects of the Msi1 functions are responsible for inherited retinal diseases. Studying the regulation of Msi1 and the target RNAs of Msi1 in photoreceptors and RPE cells might contribute to fundamental and clinical studies of retinal degeneration.

MEK mediates in vitro neural transdifferentiation of the adult newt retinal pigment epithelium cells: Is FGF2 an induction factor?

Adult newts can regenerate their entire retinas through transdifferentiation of the retinal pigment epithelium (RPE) cells. As yet, however, underlying molecular mechanisms remain virtually unknown. On the other hand, in embryonic/larval vertebrates, an MEK [mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase] pathway activated by fibroblast growth factor-2 (FGF2) is suggested to be involved in the induction of transdifferentiation of the RPE into a neural retina. Therefore, we examined using culture systems whether the FGF2/MEK pathway is also involved in the adult newt RPE transdifferentiation. Here we show that the adult newt RPE cells can switch to neural cells expressing pan-retinal-neuron (PRN) markers such as acetylated tubulin, and that an MEK pathway is essential for the induction of this process, whereas FGF2 seems an unlikely primary induction factor. In addition, we show by immunohistochemistry that the PRN markers are not expressed until the 1-3 cells thick regenerating retina, which contains retinal progenitor cells, appears. Our current results suggest that the activation of an MEK pathway in RPE cells might be involved in the induction process of retinal regeneration in the adult newt, however if this is the case, we must assume complementary mechanisms that repress the MEK-mediated misexpression of PRN markers in the initial process of transdifferentiation.

Visual cycle protein RPE65 persists in new retinal cells during retinal regeneration of adult newt.

Adult newts can regenerate their entire retina through transdifferentiation of the retinal pigment epithelium (RPE). The objective of this study was to redescribe the retina regeneration process by means of modern biological techniques. We report two different antibodies (RPE-No.112 and MAB5428) that recognize the newt homolog of RPE65, which is involved in the visual cycle and exclusively label the RPE cell-layer in the adult newt eye. We analyzed the process of retinal regeneration by immunohistochemistry and immunoblotting and propose that this process should be divided into nine stages. We found that the RPE65 protein is present in the RPE-derived new retinal rudiment at 14 days postoperative (po) and in the regenerating retinas at the 3-4 cell stage (19 days po). These observations suggest that certain characteristics of RPE cells overlap with those of retinal stem/progenitor cells during the period of transdifferentiation. However, RPE65 protein was not detected in either retinal stem/progenitor cells in the ciliary marginal zone (CMZ) of adult eyes or in neuroepithelium present during retina development, where it was first detected in differentiated RPE. Moreover, the gene expression of RPE65 was drastically downregulated in the early phase of transdifferentiation (by 10 days po), while those of Connexin43 and Pax-6, both expressed in regenerating retinas, were differently upregulated. These observations suggest that the RPE65 protein in the RPE-derived retinal rudiment may represent the remainder after protein degradation or discharge rather than newly synthesized protein.