BACKGROUND

Stromal-Derived Inducing Activity (SDIA) is one of the most efficient methods of generating dopaminergic (DA) neurons from embryonic stem cells (ESC). DA neuron induction can be achieved by co-culturing ESC with the mouse stromal cell lines PA6 or MS5. The molecular nature of this effect, which has been termed "SDIA" is so far unknown. Recently, we found that factors secreted by PA6 cells provided lineage-specific instructions to induce DA differentiation of human ESC (hESC).

RESULTS

In the present study, we compared PA6 cells to various cell lines lacking the SDIA effect, and employed genome expression analysis to identify differentially-expressed signaling molecules. Among the factors highly expressed by PA6 cells, and known to be associated with CNS development, were stromal cell-derived factor 1 (SDF-1/CXCL12), pleiotrophin (PTN), insulin-like growth factor 2 (IGF2), and ephrin B1 (EFNB1). When these four factors, the combination of which was termed SPIE, were applied to hESC, they induced differentiation to TH-positive neurons in vitro. RT-PCR and western blot analysis confirmed the expression of midbrain specific markers, including engrailed 1, Nurr1, Pitx3, and dopamine transporter (DAT) in cultures influenced by these four molecules. Electrophysiological recordings showed that treatment of hESC with SPIE induced differentiation of neurons that were capable of generating action potentials and forming functional synaptic connections.

CONCLUSIONS

The combination of SDF-1, PTN, IGF2, and EFNB1 mimics the DA phenotype-inducing property of SDIA and was sufficient to promote differentiation of hESC to functional midbrain DA neurons. These findings provide a method for differentiating hESC to form DA neurons, without a requirement for the use of animal-derived cell lines or products.

Cytosine methylation at CpG dinucleotides is a critical epigenetic modification of mammalian genomes. CpG binding protein (CGBP) exhibits a unique DNA-binding specificity for unmethylated CpG motifs and is essential for early murine development. <em>Embryonic</em> stem cell lines deficient for CGBP were generated to further examine CGBP function. CGBP(-)(/)(-) cells are viable but show an increased rate of apoptosis and are unable to achieve in vitro <em>differentiation</em> following removal of leukemia inhibitory <em>factor</em> from the <em>growth</em> media. Instead, CGBP(-)(/)(-) <em>embryonic</em> stem cells remain undifferentiated as revealed by persistent expression of the pluripotent markers Oct4 and alkaline phosphatase. CGBP(-)(/)(-) cells exhibit a 60 to 80% decrease in global cytosine methylation, including hypo-methylation of repetitive elements, single-copy genes, and imprinted genes. Total DNA methyltransferase activity is reduced by 30 to 60% in CGBP(-)(/)(-) cells, and expression of the maintenance DNA methyltransferase <em>1</em> protein is similarly reduced. However, de novo DNA methyltransferase activity is normal. Nearly all aspects of the pleiotropic CGBP(-)(/)(-) phenotype are rescued by introduction of a CGBP expression vector. Hence, CGBP is essential for normal epigenetic modification of the genome by cytosine methylation and for cellular <em>differentiation</em>, consistent with the requirement for CGBP during early mammalian development.

The development of the <em>embryonic</em> lens is dependent on the formation and regression of the tunica vasculosa lentis (TVL), which is a transiently occurring capillary plexus that surrounds the posterior part of the lens. In this study, by using the terminal deoxy-nucleotidyl transferase mediated nick end-labelling technique (TUNEL), electron microscopy, radioactive end-labelling of DNA extracted from TVL, and the Comet assay, we show that widespread apoptosis of the endothelial cells that constitute the TVL is occurring already at <em>embryonic</em> day <em>1</em>7.5 (E<em>1</em>7.5) of mouse development, much earlier than was reported previously (Jack [<em>1</em>972a] Am. J. Ophthalmol. 74:26<em>1</em>-272; Lang [<em>1</em>997] Cell Death Diff. 4:<em>1</em>2-20). In addition to apoptotic cell death, regression of this structure is associated with loss of capillary integrity, leakage of erythrocytes into the vitreal compartment, and phagocytosis of the apoptotic endothelium by tissue macrophages (hyalocytes). In situ hybridization experiments with probes for the flk-<em>1</em> receptor and its high-affinity ligand, vascular endothelial <em>growth</em> <em>factor</em> (VEGF; Terman et al. [<em>1</em>992] Biochem. Biophys. Res. Commun. <em>1</em>87:<em>1</em>579-<em>1</em>586; Millauer et al. [<em>1</em>993] Cell 72:835-846), revealed strong endothelial cell expression for flk-<em>1</em> in the eyes of E<em>1</em>3.5-E<em>1</em>7.5 embryos. VEGF mRNA was detected in lens epithelial cells located at the posterior pole of the developing lens in E<em>1</em>3.5 embryos, in close proximity to the TVL capillaries. At later times (E<em>1</em>4.5-E<em>1</em>7.5), when the lens epithelial cells have differentiated into primary lens fiber cells, and a thick lenticular capsule is formed, the expression of VEGF mRNA becomes restricted to the anterior and equatorial portions of the lens. The physical separation of the VEGF-producing cells from the flk-<em>1</em>-expressing endothelium (due to the <em>differentiation</em> of the lens epithelial cells into lens fiber cells and the formation of the lenticular capsule) may deprive the endothelium of an essential survival <em>factor</em> and, thus, may constitute the primary mechanism that is responsible for the induction of endothelial cell apoptosis in this model.

Basic fibroblast <em>growth</em> <em>factor</em> (basic FGF) has pivotal roles in the function of various cell types. Here, we report the effects of basic FGF in the regulation of dental pulp stem cell (DPSC) behaviors including maintaining stemness and directing <em>differentiation</em>. Cells isolated from human dental pulp tissues exhibited stem cell properties including the expression of mRNA markers for <em>embryonic</em> and mesenchymal stem cells, the expression of Stro-<em>1</em>, and the multipotential <em>differentiation</em>. Basic FGF stimulated colony-forming units of DPSCs and up-regulated the expression of the <em>embryonic</em> stem cell markers; Oct4, Rex-<em>1</em>, and Nanog. Moreover, osteogenic medium containing basic FGF inhibited alkaline phosphatase enzymatic activity and mineralization of DPSCs. On the contrary, basic FGF appeared to be an influential <em>growth</em> <em>factor</em> in the neurogenic <em>differentiation</em> of DPSCs. In the presence of basic FGF, increased DPSCs neurosphere size and the up-regulation of neurogenic markers were noted. Inhibitors of FGFR or PLCγ were able to ablate the basic FGF-induced neuronal <em>differentiation</em> of DPSCs. Taken together, these results suggest basic FGF may be involved in the mechanisms controlling DPSCs cell fate decisions.

Fibroblast <em>growth</em> <em>factor</em> (FGF) signaling is essential for endochondral bone formation. Mutations cause skeletal dysplasias including achondroplasia, the most common human skeletal dysplasia. Most previous work in this area has focused on <em>embryonic</em> chondrogenesis. To explore the role of FGF signaling in the postnatal <em>growth</em> plate, we quantitated expression of FGFs and FGF receptors (FGFRs) and examined both their spatial and temporal regulation. Toward this aim, rat proximal tibial <em>growth</em> plates and surrounding tissues were microdissected, and specific mRNAs were quantitated by real-time RT-PCR. To assess the FGF system without bias, we first screened for expression of all known FGFs and major FGFR isoforms. Perichondrium expressed FGFs <em>1</em>, 2, 6, 7, 9, and <em>1</em>8 and, at lower levels, FGFs 2<em>1</em> and 22. <em>Growth</em> plate expressed FGFs 2, 7, <em>1</em>8, and 22. Perichondrial expression was generally greater than <em>growth</em> plate expression, supporting the concept that perichondrial FGFs regulate <em>growth</em> plate chondrogenesis. Nevertheless, FGFs synthesized by <em>growth</em> plate chondrocytes may be physiologically important because of their proximity to target receptors. In <em>growth</em> plate, we found expression of FGFRs <em>1</em>, 2, and 3, primarily, but not exclusively, the c isoforms. FGFRs <em>1</em> and 3, thought to negatively regulate chondrogenesis, were expressed at greater levels and at later stages of chondrocyte <em>differentiation</em>, with FGFR<em>1</em> upregulated in the hypertrophic zone and FGFR3 upregulated in both proliferative and hypertrophic zones. In contrast, FGFRs 2 and 4, putative positive regulators, were expressed at earlier stages of <em>differentiation</em>, with FGFR2 upregulated in the resting zone and FGFR4 in the resting and proliferative zones. FGFRL<em>1</em>, a presumed decoy receptor, was expressed in the resting zone. With increasing age and decreasing <em>growth</em> velocity, FGFR2 and 4 expression was downregulated in proliferative zone. Perichondrial FGF<em>1</em>, FGF7, FGF<em>1</em>8, and FGF22 were upregulated. In summary, we have analyzed the expression of all known FGFs and FGFRs in the postnatal <em>growth</em> plate using a method that is quantitative and highly sensitive. This approach identified ligands and receptors not previously known to be expressed in <em>growth</em> plate and revealed a complex pattern of spatial regulation of FGFs and FGFRs in the different zones of the <em>growth</em> plate. We also found temporal changes in FGF and FGFR expression which may contribute to <em>growth</em> plate senescence and thus help determine the size of the adult skeleton.

Few target molecules have been identified that enable the diagnosis of lung cancer with high sensitivity and specificity, especially in the early clinical stages. Herein, we present the first evidence for mRNA overexpression of SALL4, a transcription <em>factor</em> essential for <em>embryonic</em> development and the self-renewal of <em>embryonic</em> stem cells, in lung cancer. Analysis using cancerous and noncancerous tissues revealed that the sensitivity and specificity of SALL4 mRNA were 85.<em>1</em> and 92.9%, respectively, estimated using the cutoff value obtained from analyzing the receiver operating characteristic curve. Furthermore, comparison of paired tissues from the same patient revealed elevated SALL4 mRNA levels that were greater than two-fold in 93% of the specimens. SALL4 mRNA was highly expressed even in the early clinical stages and there was no <em>difference</em> in the positivity rate between stage IA and other stages. An siRNA approach to determine the significance of SALL4 expression revealed catastrophic <em>growth</em> inhibition of SBC-<em>1</em> lung cancer cells that was induced by cell cycle arrest at the G<em>1</em>/early S phase. Therefore, SALL4 mRNA may be a candidate for use as support in the diagnosis of lung cancer, and may also represent a therapeutic target.

The human CAN gene was first identified as a target of t(6;9)(p23;q34), associated with acute myeloid leukemia and myelodysplastic syndrome, which results in the expression of a DEK-CAN fusion gene. CAN, also called NUP2<em>1</em>4, is a nuclear pore complex (NPC) protein that contains multiple FG-peptide sequence motifs. It interacts at the NPC with at least two other proteins, the nucleoporin NUP88 and hCRM<em>1</em> (exportin <em>1</em>), which was recently shown to function as a nuclear export receptor. Depletion of CAN in knockout mouse <em>embryonic</em> cells results in cell cycle arrest in G2, followed by inhibition of nuclear protein import and a block of mRNA export. We overexpressed CAN and DEK-CAN in U937 myeloid precursor cells. DEK-CAN expression did not interfere with terminal myeloid <em>differentiation</em> of U937 cells, whereas CAN-overexpressing cells arrested in G0, accumulated mRNA in their nuclei, and died in an apoptotic manner. Interestingly, we found that hCRM<em>1</em> and import <em>factor</em> p97/importin beta colocalized with the ectopically expressed CAN protein, resulting in depletion of both <em>factors</em> from the NPC. Overexpression of the C-terminal FG-repeat region of CAN, which contains the binding site for hCRM<em>1</em>, caused sequestering of hCRM<em>1</em> in the nucleoplasm and was sufficient to inhibit cell <em>growth</em> and to induce apoptosis. These results confirm that CAN plays a crucial role in nucleocytoplasmic transport and imply an essential role for hCRM<em>1</em> in cell <em>growth</em> and survival.

Self-renewal and <em>differentiation</em> of <em>embryonic</em> stem (ES) cells are regulated by cytokines and <em>growth</em> <em>factors</em> through tyrosine kinase-dependent signaling pathways. In murine ES cells, signals for self-renewal are generated by the leukemia inhibitory <em>factor</em> (LIF). LIF and other <em>growth</em> <em>factors</em> are linked to the activation of the Src family of cytoplasmic protein-tyrosine kinases (SFKs), which consists of eight members having shared structural architecture. In this article, we show that murine ES cells express seven SFKs, three of which (Hck, Src, and Fyn) exhibit constitutive activity in self-renewing ES cells. <em>Differentiation</em> of ES cells to embryoid bodies was associated with rapid transcriptional silencing of Hck and Lck and with the loss of the corresponding kinase proteins. The expression of other family members remained relatively constant, although some loss of Fgr and Lyn proteins was observed during <em>differentiation</em>. Like ES cells, embryoid bodies maintained constitutive Src and Fyn kinase activity. Partial inhibition of endogenous SFK activity with the ATP-competitive inhibitors 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine or Src kinase inhibitor-<em>1</em> induced <em>differentiation</em> of ES cells in the presence of LIF. In contrast, suppression of all SFK activity with higher concentrations of these inhibitors, or with the more potent compound A-4<em>1</em>9259 (Bioorg Med Chem Lett <em>1</em>2:<em>1</em>683-<em>1</em>686, 2002) blocked <em>differentiation</em> in response to LIF withdrawal. It is surprising that these inhibitor-treated cells remained pluripotent despite the absence of LIF. Our results implicate individual members of the Src kinase family in distinct ES cell renewal and <em>differentiation</em> pathways and show that small-molecule SFK inhibitors can control ES cell fate.

Members of the transforming <em>growth</em> <em>factor</em>-beta superfamily, including bone morphogenetic protein 4 (BMP-4), have been implicated as regulators of neuronal and glial <em>differentiation</em>. To test for a possible role of BMP-4 in early mammalian neural specification, we examined its effect on neurogenesis in aggregate cultures of mouse <em>embryonic</em> stem (ES) cells. Compared to control aggregates, in which up to 20% of the cells acquired immunoreactivity for the neuron-specific antibody TuJ<em>1</em>, aggregates maintained for 8 days in serum-free medium containing BMP-4 generated 5- to <em>1</em>0-fold fewer neurons. The action of BMP-4 was dose dependent and restricted to the fifth through eighth day in suspension. In addition to the reduction in neurons, we observed that ES cell cultures exposed to BMP-4 contained fewer cells that were immunoreactive for glial fibrillary acidic protein or the HNK-<em>1</em> neural antigen. Furthermore, under phase contrast, cultures prepared from BMP-4-treated aggregates contained a significant proportion of nonneuronal cells with a characteristic flat, elongated morphology. These cells were immunoreactive for antibodies to the intermediate filament protein vimentin; they were rare or absent in control cultures. Treatment with BMP-4 enhanced the expression of the early mesodermal genes brachyury and tbx6 but had relatively little effect on total cell number or cell death. Coapplication of the BMP-4 antagonist noggin counteracted the effect of exogenous BMP-4, but noggin alone had no effect on neuralization in either the absence or presence of retinoids. Collectively, our results suggest that BMP-4 can overcome the neuralizing action of retinoic acid to enhance mesodermal <em>differentiation</em> of murine ES cells.

<em>Differentiation</em> of fibroblasts to myofibroblasts and collagen fibrillogenesis are two processes essential for normal cutaneous development and repair, but their misregulation also underlies skin-associated fibrosis. Periostin is a matricellular protein normally expressed in adult skin, but its role in skin organogenesis, incisional wound healing and skin pathology has yet to be investigated in any depth. Using C57/BL6 mouse skin as model, we first investigated periostin protein and mRNA spatiotemporal expression and distribution during development and after incisional wounding. Secondarily we assessed whether periostin is expressed in human skin pathologies, including keloid and hypertrophic scars, psoriasis and atopic dermatitis. During development, periostin is expressed in the dermis, basement membrane and hair follicles from <em>embryonic</em> through neonatal stages and in the dermis and hair follicle only in adult. In situ hybridization demonstrated that dermal fibroblasts and basal keratinocytes express periostin mRNA. After incisional wounding, periostin becomes re-expressed in the basement membrane within the dermal-epidermal junction at the wound edge re-establishing the <em>embryonic</em> deposition pattern present in the adult. Analysis of periostin expression in human pathologies demonstrated that it is over-expressed in keloid and hypertrophic scars, atopic dermatitis, but is largely absent from sites of inflammation and inflammatory conditions such as psoriasis. Furthermore, in vitro we demonstrated that periostin is a transforming <em>growth</em> <em>factor</em> beta <em>1</em> inducible gene in human dermal fibroblasts. We conclude that periostin is an important ECM component during development, in wound healing and is strongly associated with pathological skin remodeling.

CONCLUSIONS

Periostin is a fibrogenic protein that mediates fibroblast differentiation and extracellular matrix synthesis. Here, we show that periostin is dynamically and temporally expressed during skin development, is induced by TGF-beta<em>1</em> in vitro and is significantly upregulated during wound repair as well as cutaneous pathologies.

BACKGROUND

The secreted frizzled related proteins (sFRP) are soluble proteins thought to interfere with the Wnt signaling. Our group previously demonstrated that one of these members, sFRP-<em>1</em>/FrzA, is strongly expressed during early phases of the vascularization process in <em>embryonic</em> vasculature and in the endothelium of arteries and capillaries in adults and modulated vascular cell proliferation.

RESULTS

Analysis of the expression of sFRP-<em>1</em> during cyclic ovarian angiogenesis revealed that sFRP-<em>1</em> is expressed during the formation of neovessels and becomes undetectable when the vasculature is fully maturated. We then studied the role of FrzA in several distinct angiogenic models. FrzA induced angiogenesis in a chick chorioallantoic membrane model. Moreover, gene transfer of AdFrzA in grafted mesenchymal and glioma cells increased vessel density and tumor growth. FrzA induced formation of vessels, which were enlarged, longer, and appeared to be more mature compared with vessels formed under control treatments. In vitro, FrzA increased migration and tube formation of endothelial cells and seemed to protect them from apoptosis. FrzA-angiogenic effect in vitro was independent of vascular endothelial growth factor, fibroblast growth factor-2, or angiopiotin-<em>1</em> induction and Akt activation. In contrast, FrzA decreased glycogen synthase kinase-3 phosphorylation.

CONCLUSIONS

These results showed that FrzA has proangiogenic effects and suggest that Wnt signaling may be involved in normal differentiation as well as in the pathological development of vasculature.

The E box sequence (5'-CANNTG-3') is found in the transcriptional regulatory region of a number of genes. Of the basic helix-loop-helix (bHLH) proteins binding to the E box sequence, class B of bHLH proteins, BHLHB2 (also referred to as the DEC<em>1</em>/Eip<em>1</em>/SHARP-2/Stra<em>1</em>3/Clast5) and BHLHB3 (also referred to as the DEC2/SHARP-<em>1</em>/SHARP<em>1</em>), are transcription <em>factors</em> that contain a unique orange domain. These transcription <em>factors</em> repress the transcription of target genes not only via binding to the E box sequence but also via protein-protein interactions with other transcription <em>factors</em>. Both the BHLHB2 and BHLHB3 genes are widely expressed in both <em>embryonic</em> and adult tissues. Their gene expressions are regulated in a cell type-specific manner by various extracellular stimuli, such as <em>growth</em> <em>factors</em>, serum starvation, hypoxia, hormones, nutrient, cytokines, light, and infection. Therefore, these transcription <em>factors</em> play pivotal roles in multiple signaling pathways that impact many biological processes including development, cell <em>differentiation</em>, cell <em>growth</em>, cell death, oncogenesis, immune systems, circadian rhythm, and homeostasis. The structural features, functions, and biological roles of the novel bHLH transcription <em>factors</em>, BHLHB2 and BHLHB3, are discussed along with the mechanisms in which the genes encoding these <em>factors</em> are regulated.

Fibroglycan (syndecan-2) is a member of a family of cell surface heparan sulfate proteoglycans that interact with adhesion molecules, <em>growth</em> <em>factors</em> and a variety of other effector systems that support the shaping, maintenance and repair of an organism. To investigate this apparent redundancy of proteoglycans at the cell surface, we have studied the expression of fibroglycan in the mouse embryo and compared this expression with that of syndecan-<em>1</em>. The characterisation of mouse embryo cDNA clones that crosshybridized to human fibroglycan-cDNA predicted that murine and human fibroglycan were highly similar in structure. Consistently, the analysis of transfectant cells, murine cell lines and embryo extracts indicated that the murine proteoglycan reacted specifically with monoclonal antibody <em>1</em>0H4 developed against the human protein. Fibroglycan, as detected by monoclonal antibody <em>1</em>0H4 in sections of <em>embryonic</em> tissues, occurred exclusively on mesenchymal cells that represented the putative precursors of the hard and connective tissue cells. No fibroglycan was detected in epithelia or in muscle cells. Areas where fibroglycan was particularly abundant were sites of high morphogenetic activity where intense cell-cell and cell-matrix interactions are known to occur (e.g. the epithelial-mesenchymal interfaces, the prechondrogenic and preosteogenic mesenchymal condensations). The expression of fibroglycan was weak in the early embryo, culminated during the morphogenetic phase and at the moment of cell lineage <em>differentiation</em>, and persisted in the perichondrium, periosteum and connective tissue cells. Syndecan-<em>1</em>, in contrast, was primarily detected in epithelia, and transiently in some mesenchymal cells, with mesenchymal localisations that did not or only partially overlap with those of fibroglycan. In situ hybridization analyses confirmed these expression patterns at the transcriptional level, identifying mesenchymal cells as the major source of fibroglycan production. These data indicate that the expression of fibroglycan occurs along unique and developmentally regulated patterns, and suggest that fibroglycan and syndecan-<em>1</em> may have distinctive functions during tissue morphogenesis and <em>differentiation</em>.

Although Wnt signaling has been shown to be important for <em>embryonic</em> morphogenesis and cancer pathogenesis of several tissues, its role in prostatic development and tumorigenesis is not well understood. Here we show that Wnt signaling regulated prostatic epithelial branching morphogenesis and luminal epithelial cell <em>differentiation</em> in developing rat prostate organ cultures. Specifically, Wnt signaling regulated the proliferation of prostate epithelial progenitor cells. Assessment of the expression levels of a Wnt pathway transcriptional target gene, Axin2, showed that the Wnt pathway was activated in the developing prostate, but was down-regulated in the adult. Castration resulted in an upregulation of Axin2 whereas androgen replacement resulted in a down regulation of Axin2. Such dynamic changes of Wnt activity was also confirmed in a BAT-gal transgenic mouse line in which beta-galactosidase reporter is expressed under the control of beta-catenin/T cell <em>factor</em> responsive elements. Furthermore, we evaluated the role of Wnt signaling in prostate tumorigenesis. Axin2 expression was found upregulated in the majority of human prostate cancer cell lines examined. Moreover, addition of a Wnt pathway inhibitor, Dickkopf <em>1</em> (DKK<em>1</em>), into the culture medium significantly inhibited prostate cancer cell <em>growth</em> and migration. These findings suggest that Wnt signaling regulates prostatic epithelial ductal branching morphogenesis by influencing cell proliferation, and highlights a role for Wnt pathway activation in prostatic cancer progression.

Primordial germ cells (PGCs) are the <em>embryonic</em> precursors of gametes in the adult organism, and their development, <em>differentiation</em>, and survival are regulated by a combination of <em>growth</em> <em>factors</em> collectively known as the germ cell niche. Although many candidate niche components have been identified through studies on mouse PGCs, the <em>growth</em> <em>factor</em> composition of the human PGC niche has not been studied extensively. Here we report a detailed analysis of the expression of components of the bone morphogenetic protein (BMP) signaling apparatus in the human fetal ovary, from postmigratory PGC proliferation to the onset of primordial follicle formation. We find developmentally regulated and reciprocal patterns of expression of BMP2 and BMP4 and identify germ cells to be the exclusive targets of ovarian BMP signaling. By establishing long-term cultures of human fetal ovaries in which PGCs are retained within their physiological niche, we find that BMP4 negatively regulates postmigratory PGC numbers in the human fetal ovary by promoting PGC apoptosis. Finally, we report expression of both muscle segment homeobox (MSX)<em>1</em> and MSX2 in the human fetal ovary and reveal a selective upregulation of MSX2 expression in human fetal ovary in response to BMP4, suggesting this gene may act as a downstream effector of BMP-induced apoptosis in the ovary, as in other systems. These data reveal for the first time <em>growth</em> <em>factor</em> regulation of human PGC development in a physiologically relevant context and have significant implications for the development of cultures systems for the in vitro maturation of germ cells, and their derivation from pluripotent stem cells.

The neuregulin (NRG)/epidermal <em>growth</em> <em>factor</em> (EGF) family of <em>growth</em> <em>factors</em> consists of several ligands that specifically activate four erbB receptor-tyrosine kinases, namely erbB-<em>1</em> (EGF-R), erbB-2 (neu), erbB-3, and erbB-4. We have previously shown that islet morphogenesis is impaired and beta-cell <em>differentiation</em> delayed in mice lacking functional EGF-R [EGF-R (-/-)]. The present study aims to clarify which erbB ligands are important for islet development. Pancreatic expression of EGF, TGF-alpha, heparin-binding EGF, betacellulin (BTC), and NRG-4 was detected as early as <em>embryonic</em> d <em>1</em>3 (E<em>1</em>3). Effects of these ligands were studied in E<em>1</em>2.5 pancreatic explant cultures grown for 5 d ex vivo. None of the <em>growth</em> <em>factors</em> affected the ratio of endocrine to exocrine cells. However, significant effects within the endocrine cell populations were induced by EGF, BTC, and NRG-4. beta-Cell development was augmented by BTC, whereas the development of somatostatin-expressing delta-cells was stimulated by NRG-4. Both ligands decreased the numbers of glucagon-containing alpha-cells. The effect of BTC was abolished in the EGF-R (-/-) mice. A soluble erbB-4 binding fusion protein totally inhibited the effects of NRG-4 but not of BTC. Neutralization of endogenous NRG-4 activity in the model system effectively inhibited delta-cell development, indicating that this erbB4-ligand is an essential <em>factor</em> for delineation of the somatostatin-producing delta-cells. Our results suggest that ligands of the EGF-R/erbB-<em>1</em> and erbB-4 receptors regulate the lineage determination of islet cells during pancreatic development. BTC, acting through EGF-R/erbB-<em>1</em>, is important for the <em>differentiation</em> of beta-cells. This could be applied in the targeted <em>differentiation</em> of stem cells into insulin-producing cells.

The formation of new blood vessels proceeds by both vasculogenesis and angiogenesis. The development of models, which fully recapitulate spatio-temporal events involved during these processes, are crucial to fully understand their mechanisms of regulation. In vitro <em>differentiation</em> of murine <em>embryonic</em> stem (ES) cells has been shown to be a useful tool to investigate <em>factors</em> and genes potentially involved in vasculogenesis (Hirashima et al, <em>1</em>999; Risau et al, <em>1</em>988; Vittet et al, <em>1</em>996; Wang et al, <em>1</em>992; Wartenberg et al, <em>1</em>998). We asked here whether this model system can also recapitulate angiogenesis, which may offer new means to study mechanisms involved in this process. ES-derived embryoid bodies (EBs) obtained after <em>1</em><em>1</em> days of <em>differentiation</em>, in which a primitive vascular network had formed, were then subcultured into a type I collagen matrix. In the presence of angiogenic <em>growth</em> <em>factors</em>, EBs rapidly developed branching pseudopods. Whole mount immunostainings with a PECAM antibody revealed that more than 75% EBs displayed, within a few days, a large number of endothelial out<em>growth</em>s that can give tube-like structures with concomitant <em>differentiation</em> of alpha-smooth muscle actin positive cells, thus evoking sprouting angiogenesis. High expression levels of flk<em>1</em> (VEGFR2), flt<em>1</em> (VEGFR<em>1</em>), tie-<em>1</em>, and tie-2 are also found, indicating that budding endothelial cells displayed an angiogenic phenotype. The endothelial sprouting response was specifically induced by angiogenic <em>factors</em> with a major contribution of vascular endothelial <em>growth</em> <em>factor</em> (VEGF). Known angiostatic agents, such as platelet <em>factor</em> 4 (PF4), angiostatin, and endostatin inhibited the formation of endothelial sprouts induced by angiogenic <em>factors</em>. Moreover, consistent with the in vivo phenotype, VE-cadherin deficient EBs failed to develop angiogenesis in this model. ES cell <em>differentiation</em> can then recapitulate, in addition to vasculogenesis, the early stages of sprouting angiogenesis. This model system, in which genetic modifications can be easily introduced, may be of particular interest to investigate unsolved questions and molecular mechanisms involved in blood vessel formation.

Organotypic cocultures of keratinocytes and fibroblasts generate a normal epidermis irrespective of the species and tissue origin of fibroblasts. The use of mouse fibroblasts and human keratinocytes facilitates the identification of the origin of compounds involved in epidermal tissue reconstitution and <em>growth</em> regulation. Moreover, the functional significance for the keratinocyte phenotype of genetically modified fibroblasts from transgenic or knockout mice, even those exhibiting an <em>embryonic</em> lethal phenotype, can be studied in such heterologous in vitro tissue equivalents. Here we communicate results of such studies revealing the antagonistic function of mouse fibroblasts defective in the AP-<em>1</em> constituents c-Jun and JunB, respectively, on human keratinocyte <em>growth</em> and <em>differentiation</em>. Furthermore, the hematopoietic <em>growth</em> <em>factor</em> granulocyte macrophage-colony stimulating <em>factor</em> has been identified as a novel regulator of keratinocyte <em>growth</em> and <em>differentiation</em>. As will be reported in detail elsewhere both granulocyte macrophage-colony stimulating <em>factor</em> and keratinocyte <em>growth</em> <em>factor</em> have been identified as major mediators of fibroblast-keratinocyte interactions and their expression is induced via AP-<em>1</em> by interleukin-<em>1</em> released by the epithelial cells. Thus, these heterologous cocultures provide a novel promising tool for elucidating molecular mechanisms of epithelial-mesenchymal interactions and their consequences on epithelial cell proliferation and <em>differentiation</em>.

We have developed a serum-free medium, designated ESF7, in which leukemia inhibitory <em>factor</em> (LIF) clearly stimulated murine <em>embryonic</em> stem (ES) cell proliferation accompanied by increased expression of nanog and Rex-<em>1</em> and decreased FGF-5 expression. These effects were dependent on the concentration of LIF. The ES cells maintained in ESF7 medium for more than 2 yr retained an undifferentiated phenotype, as manifested by the expression of the transcription <em>factor</em> Oct-3/4, the stem cell marker SSEA-<em>1</em>, and alkaline phosphatase. Withdrawal of LIF from ESF7 medium resulted in ES cell apoptosis. Addition of serum to ESF7 medium promoted ES cell <em>differentiation</em>. Addition of BMP4 promoted ES cell <em>differentiation</em> into simple epithelial-like cells. In contrast, FGF-2 promoted ES cell <em>differentiation</em> into neuronal and glial-like cells. Under serum-free culture conditions, LIF was sufficient to stimulate cell proliferation, it inhibited cell <em>differentiation</em>, and it maintained self-renewal of ES cells. Because this simple serum-free adherent monoculture system supports the long-term propagation of pluripotent ES cells in vitro, it will allow the elucidation of ES cell responses to <em>growth</em> <em>factors</em> under defined conditions.

Cell therapy using stem cells is awaited by stroke patients with impaired movement and cognitive functions, although intravenous alteplase-administration ameliorated outcomes of patients receiving the therapy within 3 h of onset. In this study, we explored the therapeutic effects of neural progenitor cells (NPC) upon middle cerebral artery occlusion (MCAO) model of rats with exploration of the <em>differences</em> between adult and <em>embryonic</em> NPCs in therapeutic effects. GFP-labeled adult or <em>embryonic</em> NPCs were transplanted for transient MCAO model of rats at <em>1</em>h after reperfusion. Rats were examined behaviorally using limb placement test, rotarod test and cylinder test with neuroradiological assessment using magnetic resonance imaging (MRI). Consequently after euthanasia, rats were immunohistochemically investigated to explore graft survival and immune reaction. MRI of rats receiving NPCs revealed significant reduction of infarct volumes, compared to vehicle-treated rats with corresponding behavioral amelioration. The transplanted cells were surviving in rats receiving NPCs, although the number of <em>embryonic</em> NPCs was significantly higher than that of adult NPCs. Iba-<em>1</em>-positive inflammatory cells of rats receiving adult NPCs were prominent, compared to those receiving <em>embryonic</em> NPCs, which might be a rationale for the <em>differences</em> between rats receiving adult and <em>embryonic</em> NPCs in the number of surviving NPCs. On the contraries, adult NPCs surely demonstrated therapeutic effects with a few surviving cells, thus indicating that the therapeutic effects might be due to trophic/<em>growth</em> <em>factor</em>-secretion from transplanted NPCs, rather than replacement of damaged host neurons. Therapeutic effects of NPCs for MCAO model of rats were clarified in this study. Transplantation of NPCs will be a hopeful strategy for stroke patients, although further studies are required for the patient safety and underlying mechanisms.

Coronary vessel formation is a special case in the context of <em>embryonic</em> vascular development. A major part of the coronary cellular precursors (endothelial, smooth muscle, and fibroblastic cells) derive from the proepicardium and the epicardium in what can be regarded as a late event of angioblastic and smooth muscle cell <em>differentiation</em>. Thus, coronary morphogenesis is dependent on the epithelial-mesenchymal transformation of the proepicardium and the epicardium. In this study, we present several novel observations about the process of coronary vasculogenesis in avian embryos, namely: (<em>1</em>) The proepicardium displays a high vasculogenic potential, both in vivo (as shown by heterotopic transplants) and in vitro, which is modulated by vascular endothelial <em>growth</em> <em>factor</em> (VEGF) and basic fibroblast <em>growth</em> <em>factor</em> signals; (2) Proepicardial and epicardial cells co-express receptors for platelet-derived <em>growth</em> <em>factor</em>-BB and VEGF; (3) Coronary angioblasts (found all through the epicardial, subepicardial, and compact myocardial layers) express the Wilms' tumor associated transcription <em>factor</em> and the retinoic acid-synthesizing enzyme retinaldehyde-dehydrogenase-2, two markers of the coelomic epithelium involved in coronary endothelium development. All these results contribute to the development of our knowledge on the vascular potential of proepicardial/epicardial cells, the existent interrelationships between the differentiating coronary cell lineages, and the molecular mechanisms involved in the regulation of coronary morphogenesis.

pim-<em>1</em> and pim-3 encode serine/threonine kinases involved in the regulation of cell proliferation and apoptosis in response to cytokine stimulation. We analyzed the regulation of pim-<em>1</em> and pim-3 by the leukemia inhibitory <em>factor</em> (LIF)/gp<em>1</em>30/signal transducer and activator of transcription-3 (STAT3) pathway and the role of Pim-<em>1</em> and Pim-3 kinases in mouse <em>embryonic</em> stem (ES) cell self-renewal. Making use of ES cells expressing a granulocyte colony-stimulating <em>factor</em>:gp<em>1</em>30 chimeric receptor and a hormone-dependent signal transducer and activator of transcription-3 estrogen receptor (STAT3-ER(T2)), we showed that expression of pim-<em>1</em> and pim-3 was upregulated by LIF/gp<em>1</em>30-dependent signaling and the STAT3 transcription <em>factor</em>. ES cells overexpressing pim-<em>1</em> and pim-3 had a greater capacity to self-renew and displayed a greater resistance to LIF starvation based on a clonal assay. In contrast, knockdown of pim-<em>1</em> and pim-3 increased the rate of spontaneous <em>differentiation</em> in a self-renewal assay. Knockdown of pim-<em>1</em> and pim-3 was also detrimental to the <em>growth</em> of undifferentiated ES cell colonies and increased the rate of apoptosis. These findings provide a novel role of Pim-<em>1</em> and Pim-3 kinases in the control of self-renewal of ES cells. Disclosure of potential conflicts of interest is found at the end of this article.

OBJECTIVE

Exocrine acinar cells in the pancreas are highly differentiated cells that retain a remarkable degree of plasticity. After isolation and an initial phase of dedifferentiation in vitro, rodent acinar cells can convert to endocrine beta-cells when cultured in the presence of appropriate factors. The mechanisms regulating this phenotypic conversion are largely unknown.

METHODS

Using rat acinar cell cultures, we studied the role of Notch signaling in a model of acinar-to-beta-cell conversion.

RESULTS

We report a novel lectin-based cell labeling method to demonstrate the acinar origin of newly formed insulin-expressing beta-cells. This method allows for specific tracing of the acinar cells. We demonstrate that growth factor-induced conversion of adult acinar cells to beta-cells is negatively regulated by Notch1 signaling. Activated Notch1 signaling prevents the reexpression of the proendocrine transcription factor Neurogenin-3, the key regulator of endocrine development in the embryonic pancreas. Interfering with Notch1 signaling allows modulating the acinar cell susceptibility to the differentiation-inducing factors. Its inhibition significantly improves beta-cell neoformation with approximately 30% of acinar cells that convert to beta-cells. The newly formed beta-cells mature when transplanted ectopically and are capable of restoring normal blood glycemia in diabetic recipients.

CONCLUSIONS

We report for the first time an efficient way to reprogram one third of the acinar cells to beta-cells by adult cell type conversion. This could find application in cell replacement therapy of type 1 diabetes, provided that it can be translated from rodent to human models.

To examine the long-term effects of stress experienced early in gestation on the programming of offspring feeding behaviors and energy balance, pregnant mice were exposed to stress during early pregnancy (days <em>1</em>-7) and adult offspring examined on chow and high fat diets for long-term outcomes. Placental <em>1</em><em>1</em> beta-hydroxysteroid dehydrogenase type 2 (<em>1</em><em>1</em> beta-HSD2) and insulin-like <em>growth</em> <em>factor</em> 2 (IGF-2) expression was measured to determine the possible sex-specific contribution of prenatal stress (PNS) on fetal programming of embryo <em>growth</em> and development during early pregnancy. PNS mice showed a basal hyperphagia when on chow diet. Prenatal treatment <em>differences</em> were ameliorated when adult mice were on a high fat diet. Interestingly, PNS male mice also had significantly reduced body weights compared to control males on both chow and high fat diets. Body composition analyses revealed reduced body fat and increased lean mass in PNS mice on the high fat diet, but no <em>differences</em> were detected in plasma leptin or insulin-like <em>growth</em> <em>factor</em> <em>1</em> (IGF-<em>1</em>) levels. Mechanistic examination of gene expression in <em>embryonic</em> day <em>1</em>2 placentas found that early PNS was associated with increased IGF-2 expression and sex-dependent effects of stress on <em>1</em><em>1</em> beta-HSD2, supporting specific aspects of early pregnancy. These studies suggest that the long-term effects of stress during pregnancy on programming of feeding behavior and energy homeostasis begin much earlier in development than previously thought.