The cardiac conduction system is a network of cells responsible for the rhythmic and coordinated excitation of the heart. Components of the murine conduction system, including the peripheral Purkinje fibers, are morphologically indistinguishable from surrounding cardiomyocytes, and a paucity of molecular markers exists to identify these cells. The murine conduction system develops in close association with the endocardium. Using the recently identified CCS-lacZ line of reporter mice, in which lacZ expression delineates the <em>embryonic</em> and fully mature conduction system, we tested the ability of several endocardial-derived paracrine <em>factors</em> to convert contractile cardiomyocytes into conduction-system cells as measured by ectopic reporter gene expression in the heart. In this report we show that neuregulin-<em>1</em>, a <em>growth</em> and <em>differentiation</em> <em>factor</em> essential for ventricular trabeculation, is sufficient to induce ectopic expression of the lacZ conduction marker. This inductive effect of neuregulin-<em>1</em> was restricted to a window of sensitivity between 8.5 and <em>1</em>0.5 days postcoitum. Using the whole mouse embryo culture system, neuregulin-<em>1</em> was shown to regulate lacZ expression within the <em>embryonic</em> heart, whereas its expression in other tissues remained unaffected. We describe the electrical activation pattern of the 9.5-days postcoitum <em>embryonic</em> mouse heart and show that treatment with neuregulin-<em>1</em> results in electrophysiological changes in the activation pattern consistent with a recruitment of cells to the conduction system. This study supports the hypothesis that endocardial-derived neuregulins may be the major endogenous ligands responsible for inducing murine <em>embryonic</em> cardiomyocytes to differentiate into cells of the conduction system.

Genetic and molecular evidence indicates that visceral endoderm, an extra<em>embryonic</em> cell lineage, is required for gastrulation, early anterior neural patterning, cell death and specification of posterior mesodermal cell fates. We show that variant Hepatocyte Nuclear <em>Factor</em> <em>1</em> (vHNF<em>1</em>), a homeodomain-containing transcription <em>factor</em> first expressed in the primitive endoderm, is required for the specification of visceral endoderm. vHnf<em>1</em>-deficient mouse embryos develop normally to the blastocyst stage, start implantation, but die soon afterwards, with abnormal or absent extra<em>embryonic</em> region, poorly organised ectoderm and no discernible visceral or parietal endoderm. However, immunostaining analysis of E5.5 nullizygous mutant embryos revealed the presence of parietal endoderm-like cells lying on an abnormal basal membrane. Homozygous mutant blastocyst out<em>growth</em>s or differentiated <em>embryonic</em> stem cells do not express early or late visceral endoderm markers. In addition, in vHnf<em>1</em> null embryoid bodies there is no activation of the transcription <em>factors</em> HNF-4alpha<em>1</em>, HNF<em>1</em>alpha and HNF-3gamma. Aggregation of vHnf<em>1</em>-deficient <em>embryonic</em> stem cells with wild-type tetraploid embryos, which contribute exclusively to extra<em>embryonic</em> tissues, rescues periimplantation lethality and allows development to progress to early organogenesis. Our results place vHNF<em>1</em> in a preeminent position in the regulatory network that specifies the visceral endoderm and highlight the importance of this cell lineage for proper <em>growth</em> and <em>differentiation</em> of primitive ectoderm in pregastrulating embryos.

BACKGROUND

Recent studies have suggested that both embryonic stem cells and adult bone marrow stem cells can participate in the regeneration and repair of diseased adult organs, including the lungs. However, the extent of airway epithelial remodeling with adult marrow stem cells is low, and there are no available in vivo data with embryonic stem cells. Human umbilical cord blood contains both hematopoietic and nonhematopoietic stem cells, which have been used clinically as an alternative to bone marrow transplantation for hematologic malignancies and other diseases.

OBJECTIVE

We hypothesized that human umbilical cord blood stem cells might be an effective alternative to adult bone marrow and embryonic stem cells for regeneration and repair of injured airway epithelium.

METHODS

Human cord blood was obtained from normal deliveries at the University of Vermont. Cultured plastic adherent cells were characterized as mesenchymal stem cells (MSCs) by flow cytometry and differentiation assays. Cord blood-derived MSCs (CB-MSCs) were cultured in specialized airway growth media or with specific growth factors, including keratinocyte growth factor and retinoic acid. mRNA and protein expression were analyzed with PCR and immunofluorescent staining. CB-MSCs were systematically administered to immunotolerant, nonobese diabetic/severe combined immunodeficiency (NOD-SCID) mice. Lungs were analyzed for presence of human cells.

RESULTS

When cultured in specialized airway growth media or with specific growth factors, CB-MSCs differentially expressed Clara cell secretory protein (CCSP), cystic fibrosis transmembrane conductance regulator (CFTR), surfactant protein C, and thyroid transcription factor-1 mRNA, and CCSP and CFTR protein. Furthermore, CB-MSCs were easily transduced with recombinant lentiviral vectors to express human CFTR. After systemic administration to immunotolerant, NOD-SCID, mice, rare cells were found in the airway epithelium that had acquired cytokeratin and human CFTR expression.

CONCLUSIONS

CB-MSCs appear to be comparable to MSCs obtained from adult bone marrow in ability to express phenotypic markers of airway epithelium and to participate in airway remodeling in vivo.

Human <em>embryonic</em> stem cells (hESC) are pluripotent stem cells. A major challenge in the field of hESC is the establishment of specific <em>differentiation</em> protocols that drives hESC down a particular lineage fate. So far, attempts to generate T cells from hESC in vitro were unsuccessful. In this study, we show that T cells can be generated in vitro from hESC-derived hematopoietic precursor cells present in hematopoietic zones (HZs). These zones are morphologically similar to blood islands during <em>embryonic</em> development, and are formed when hESC are cultured on OP9 stromal cells. Upon subsequent transfer of these HZs on OP9 cells expressing high levels of Delta-like <em>1</em> and in the presence of <em>growth</em> <em>factors</em>, cells expand and differentiate to T cells. Furthermore, we show that T cells derive exclusively from a CD34(high)CD43(low) population, further substantiating the notion that hESC-derived CD34(high)CD43(low) cells are formed in HZs and are the only population containing multipotent hematopoietic precursor cells. <em>Differentiation</em> to T cells sequentially passes through the physiological intermediates: CD34(+)CD7(+) T/NK committed, CD7(+)CD4(+)CD8(-) immature single positive, CD4(+)CD8(+) double positive, and finally CD3(+)CD<em>1</em>(-)CD27(+) mature T cell stages. TCRalphabeta(+) and TCRgammadelta(+) T cells are generated. Mature T cells are polyclonal, proliferate, and secrete cytokines in response to mitogens. This protocol for the de novo generation of T cells from hESC could be clinically and scientifically relevant.

Understanding <em>embryonic</em> stem cell (ESC) regulation is important for realizing how best to control their <em>growth</em> and <em>differentiation</em> ex vivo for potential therapeutic benefit. Stromal cell-derived <em>factor</em>-<em>1</em> (SDF-<em>1</em>/CXCL<em>1</em>2) and its receptor, CXCR4, have been implicated as important regulators of a number of fetal and adult cell functions, including survival/antiapoptosis and migration/homing of hematopoietic stem and progenitor cells. We hypothesized that the SDF-<em>1</em>/CXCL<em>1</em>2-CXCR4 axis would also be important for regulation of murine ESC functions. ESCs secreted low levels of SDF-<em>1</em>/CXCL<em>1</em>2 and expressed low levels of CXCR4; however, both increased with <em>differentiation</em> of ESCs. Endogenously produced/released SDF-<em>1</em>/CXCL<em>1</em>2 enhanced survival/antiapoptosis of ESCs in the presence of leukemia inhibitory <em>factor</em> but absence of serum, and survival/antiapoptosis was further enhanced by exogenous administration of SDF-<em>1</em>/CXCL<em>1</em>2. Furthermore, SDF-<em>1</em>/CXCL<em>1</em>2 induced chemotaxis of ESCs, and chemotaxis could be enhanced by diprotin A inhibition of CD26/dipeptidylpeptidase IV. Endogenous and exogenous SDF-<em>1</em>/CXCL<em>1</em>2 enhanced embryoid body production of primitive and definitive erythroid, granulocyte-macrophage, and multipotential progenitors. SDF-<em>1</em>/CXCL<em>1</em>2 did not noticeably affect production of hemangioblasts. These results demonstrate functional activities of SDF-<em>1</em>/CXCL<em>1</em>2 on survival, chemotaxis, and hematopoietic <em>differentiation</em> of murine ESCs that may be relevant for their ex vivo manipulation.

Blood vessel formation requires the integrated regulation of endothelial cell proliferation and branching morphogenesis, but how this coordinated regulation is achieved is not well understood. Flt-<em>1</em> (vascular endothelial <em>growth</em> <em>factor</em> [VEGF] receptor <em>1</em>) is a high affinity VEGF-A receptor whose loss leads to vessel over<em>growth</em> and dysmorphogenesis. We examined the ability of Flt-<em>1</em> isoform transgenes to rescue the vascular development of <em>embryonic</em> stem cell-derived flt-<em>1</em>-/- mutant vessels. Endothelial proliferation was equivalently rescued by both soluble (sFlt-<em>1</em>) and membrane-tethered (mFlt-<em>1</em>) isoforms, but only sFlt-<em>1</em> rescued vessel branching. Flk-<em>1</em> Tyr-<em>1</em><em>1</em>73 phosphorylation was increased in flt-<em>1</em>-/- mutant vessels and partially rescued by the Flt-<em>1</em> isoform transgenes. sFlt-<em>1</em>-rescued vessels exhibited more heterogeneous levels of pFlk than did mFlt-<em>1</em>-rescued vessels, and reporter gene expression from the flt-<em>1</em> locus was also heterogeneous in developing vessels. Our data support a model whereby sFlt-<em>1</em> protein is more efficient than mFlt-<em>1</em> at amplifying initial expression <em>differences</em>, and these amplified <em>differences</em> set up local discontinuities in VEGF-A ligand availability that are important for proper vessel branching.

Genome endoreduplication during mammalian development is a rare event for which the mechanism is unknown. It first appears when fibroblast <em>growth</em> <em>factor</em> 4 (FGF4) deprivation induces <em>differentiation</em> of trophoblast stem (TS) cells into the nonproliferating trophoblast giant (TG) cells required for embryo implantation. Here we show that RO3306 inhibition of cyclin-dependent protein kinase <em>1</em> (CDK<em>1</em>), the enzyme required to enter mitosis, induced <em>differentiation</em> of TS cells into TG cells. In contrast, RO3306 induced abortive endoreduplication and apoptosis in <em>embryonic</em> stem cells, revealing that inactivation of CDK<em>1</em> triggers endoreduplication only in cells programmed to differentiate into polyploid cells. Similarly, FGF4 deprivation resulted in CDK<em>1</em> inhibition by overexpressing two CDK-specific inhibitors, p57/KIP2 and p2<em>1</em>/CIP<em>1</em>. TS cell mutants revealed that p57 was required to trigger endoreduplication by inhibiting CDK<em>1</em>, while p2<em>1</em> suppressed expression of the checkpoint protein kinase CHK<em>1</em>, thereby preventing induction of apoptosis. Furthermore, Cdk2(-/-) TS cells revealed that CDK2 is required for endoreduplication when CDK<em>1</em> is inhibited. Expression of p57 in TG cells was restricted to G-phase nuclei to allow CDK activation of S phase. Thus, endoreduplication in TS cells is triggered by p57 inhibition of CDK<em>1</em> with concomitant suppression of the DNA damage response by p2<em>1</em>.

The epidermal <em>growth</em> <em>factor</em> (EGF) receptor (or ErbB<em>1</em>) and the related ErbB4 are transmembrane receptor protein tyrosine kinases which bind extracellular ligands of the EGF family. ErbB2 and ErbB3 are "co-receptors" structurally related to ErbB<em>1</em>/ErbB4, but ErbB2 is an "orphan" receptor and ErbB3 lacks tyrosine kinase activity. However, both are important in transmembrane signalling. All ErbB receptors/ligands are intimately involved in the regulation of cell <em>growth</em>, <em>differentiation</em> and survival, and their dysregulation contributes to some human malignancies. After extracellular ligand binding, receptor dimerisation and transautophosphorylation of intracellular C-terminal tyrosine residues, they bind signalling proteins which recognise specific tyrosine-phosphorylated motifs. This leads to activation of multiple signalling pathways, notably the extracellular signal-regulated kinase <em>1</em>/2 (ERK<em>1</em>/2) cascade and the phosphoinositide 3-kinase (PI3K)/protein kinase B [PKB/(Akt)] pathway. In heart, targeted deletion of ErbB2, ErbB3, ErbB4 and some ErbB receptor extracellular ligands leads to <em>embryonic</em> lethality resulting from cardiovascular defects. ErbB receptor ligands improve cardiac myocyte viability and are hypertrophic, partly because of activation of ERK<em>1</em>/2 and/or PI3K/PKB(Akt). Furthermore, ErbB transactivation by Gq protein-coupled receptor (GqPCR) signalling may mediate the hypertrophic effects of GqPCR agonists. The utility of anthracyclines in cancer chemotherapy can be limited by their cardiotoxic side effects and these may be counteracted by ErbB receptor ligands. ErbB2 is the target of anti-cancer monoclonal antibody trastuzumab (Herceptin), and its myocardial downregulation may account for the occasional cardiotoxicity of this therapy. Here, we review the basic biochemistry of ErbB receptors/ligands, and emphasise their particular roles in the myocardium.

<em>Growing</em> stem cells are subjected to mechanical forces, which may initiate <em>differentiation</em> programs. Mechanical strain stimulated cardiovascular <em>differentiation</em> of mouse <em>embryonic</em> stem (ES) cells as evaluated by quantification of contracting cardiac foci and capillary areas, respectively. Mechanical strain rapidly elevated intracellular reactive oxygen species (ROS). After 24 h up-regulation of NADPH oxidase subunits p22-phox, p47-phox, p67-phox, and Nox-4 as well as Nox-<em>1</em> and Nox-4 mRNA was observed. In parallel, mechanical strain increased hypoxia-inducible <em>factor</em>-<em>1</em>alpha (HIF-<em>1</em>alpha) and vascular endothelial <em>growth</em> <em>factor</em> (VEGF) mRNA and protein as well as MEF2C and GATA-4 mRNA, which are involved in cardiovascular development. Furthermore, phosphorylation of extracellular-regulated kinase <em>1</em>,2 (ERK<em>1</em>,2), p38, and c-jun N-terminal kinase (c-Jun NH2-terminal kinase (JNK)) was observed. Stimulation of cardiovascular commitment, HIF-<em>1</em>alpha, VEGF, and MEF2C expression as well as MAPK activation were abolished by free radical scavengers, whereas GATA-4 expression was increased. Cardiomyogenesis was inhibited by the p38 inhibitor SB203580, the ERK<em>1</em>,2 inhibitor UO<em>1</em>26, and the JNK inhibitor SP600<em>1</em>25. Vasculogenesis/angiogenesis was blunted following inhibition of ERK<em>1</em>,2 and JNK, whereas p38 inhibition was ineffective. Our data outline a role of ROS as mechanotransducing molecules in mechanical strain-stimulated cardiovascular <em>differentiation</em> of ES cells, and point toward a microenvironment of elevated ROS required for signaling cascades initiating cardiovascular <em>differentiation</em> programs.

Mesenchymal stem cells (MSCs) are a very important adult stem cell population with a multitude of potential applications in regenerative medicine. The thorough characterization of the bone marrow MSC (BM-MSC) population derived from the BALB/c species was essential, considering the significance of the murine model amongst animal models. In the present study, we examined the effect of gender, age, and in vitro culture on the basic properties (proliferation, <em>differentiation</em>, and immunosuppressive potential) of BM-MSCs. We found a decline in the progenitor frequencies from the BM of adult mice, lower MSC frequencies in all female donors, and an increase in the BM-MSC proliferation rate upon in vitro propagation. We also examined BM-MSCs for the expression of the 3 major <em>embryonic</em> stem cell transcription <em>factors</em>, Oct3/4, Sox-2, and Nanog, as well as 2 mRNA binding proteins, coding region determinant binding protein/insulin-like <em>growth</em> <em>factor</em> 2 mRNA binding protein <em>1</em> (Crd-bp/Imp<em>1</em>) and Deleted in azoospermia-like (Dazl), which are expressed in primitive stem cells, umbilical cord blood-hematopoietic stem cells and amniotic fluid stem cells, respectively. Further, it has been reported that these 2 genes are critical for <em>embryonic</em> development. In this study, therefore, we report, for the first time, the expression of Crd-bp/Imp<em>1</em> and Dazl in BM-MSCs. Dazl, Oct3/4, and Sox2 were detected in relatively low levels in contrast to Crd-bp/Imp<em>1</em>, its major target c-Myc, as well as Nanog, which were expressed redundantly, irrespective of sex, donor age, or in vitro passaging. These findings could further support the extrinsic theory of aging of the MSC population and the potential implication of <em>embryonic</em> genes in adult stem cell physiology.

Drosophila Notch and the related Caenorhabditis elegans proteins lin-<em>1</em>2 and glp-<em>1</em> function as mediators of local cell-cell interactions required for cell-fate decisions during invertebrate development. To investigate the possibility that similar proteins play determinative roles during mammalian development, we isolated cDNA clones encoding rat Notch. The deduced amino acid sequence of this protein contains 36 epidermal <em>growth</em> <em>factor</em> (EGF)-like repeats, and is remarkably similar in both its extracellular and cytoplasmic domains to the sequence of Xenopus Xotch and Drosophila Notch. In the developing central nervous system, in situ hybridisation analyses revealed that Notch transcripts were dramatically restricted to the ventricular proliferative zones of <em>embryonic</em> neuroepithelia. Notch was also strongly expressed during development of non-neural tissues, such as hair follicles and tooth buds, whose correct <em>differentiation</em> requires epithelial-mesenchymal interactions. These data support the hypothesis that Notch plays an essential role in mammalian development and pattern formation that closely parallels its role in the development of invertebrates.

Lineage analysis studies in the avian embryo have identified two types of smooth muscle cells (SMCs) in the tunica media of large elastic arteries; one that originates within the cardiac neural crest and is ectoderm in origin (Ect) and another that arises from local mesenchyme of mesodermal origin (Mes). To determine if <em>differences</em> in primary <em>embryonic</em> lineage can give rise to SMCs with stable <em>differences</em> in <em>growth</em> and <em>differentiation</em> properties, we isolated Ect and Mes SMCs from the Day <em>1</em>4 chick embryo aorta. We report that despite different primary <em>embryonic</em> origins, Ect and Mes SMCs express nearly identical levels of seven SMC <em>differentiation</em> markers in vitro, consistent with their common smooth muscle developmental fates in vivo. By contrast, Ect SMCs displayed a greater capacity for <em>growth</em> in serum-free medium than Mes SMCs, but only under conditions permitting short-range cell-cell interactions. Most of the peptide <em>growth</em> <em>factors</em> tested that might account for serum-independent <em>growth</em> (PDGF-AA, PDGF-BB, basic FGF, EGF, or activin) stimulated DNA synthesis to similar extents in Ect and Mes SMCs. However, we found dramatic, lineage-dependent <em>differences</em> in SMC responses to transforming <em>growth</em> <em>factor</em>-beta (TGF-beta). Exposure to TGF-beta <em>1</em> (0.4 to 400 pmole/liter) consistently increased DNA synthesis in Ect SMCs, whereas in paired cultures of Mes SMCs, TGF-beta <em>1</em> was <em>growth</em> inhibitory. In SMC cultures transfected with p3TP-lux, a luciferase reporter controlled by the TGF-beta <em>1</em>-response elements of the human PAI-<em>1</em> promoter, TGF-beta <em>1</em> (<em>1</em>20 pM) produced <em>1</em>2 +/- 2-fold increases in luciferase activity in Ect SMCs and only 3 +/- <em>1</em>.5-fold increases in Mes SMCs. Analysis of TGF-beta receptor phenotypes by Northern blot, radioligand binding, and crosslinking assays showed that Ect and Mes SMCs expressed similar levels of types I, II, and III TGF-beta receptors. However, using a polyclonal antibody specific for the chick type II TGF-beta receptor subunit, we demonstrate that Mes SMCs produce a fully glycosylated form of this protein while Ect SMCs elaborate only an unglycosylated type II TGF-beta receptor. These results show that Ect and Mes SMCs exhibit lineage-dependent <em>differences</em> in <em>growth</em> and receptor-mediated transcriptional responses to at least one important class of SMC morphogens and <em>growth</em> modifiers, e.g., the TGF-betas. Our findings suggest that different SMC populations within a common vessel wall may respond in lineage-dependent ways to signals that direct formation of the tunica media in the embryo and to <em>factors</em> involved in the progression of vascular disease later in life.

Homodimeric bone morphogenetic protein-2 (BMP-2) is a member of the transforming <em>growth</em> <em>factor</em> beta (TGF-beta) superfamily that induces bone formation and regeneration, and determines important steps during early stages of <em>embryonic</em> development in vertebrates and non-vertebrates. BMP-2 can interact with two types of receptor chains, as well as with proteins of the extracellular matrix and several regulatory proteins. We report here the crystal structure of human BMP-2 determined by molecular replacement and refined to an R-value of 24.2 % at 2.7 A resolution. A common scaffold of BMP-2, BMP-7 and the TGF-betas, i.e. the cystine-knot motif and two finger-like double-stranded beta-sheets, can be superimposed with r. m.s. deviations of around <em>1</em> A. In contrast to the TGF-betas, the structure of BMP-2 shows <em>differences</em> in the flexibility of the N terminus and the orientation of the central alpha-helix as well as two external loops at the fingertips with respect to the scaffold. This is also known from the BMP-7 model. Small secondary structure elements in the loop regions of BMP-2 and BMP-7 seem to be specific for the respective BMP-subgroup. Two identical helix-finger clefts and two distinct cavities located around the central 2-fold axis of the dimer show characteristic shapes, polarity and surface charges. The possible function of these specific features in the interaction of BMP-2 with its binding partners is discussed.

Tissue engineering and cell therapy approaches aim to take advantage of the repopulating ability and plasticity of multipotent stem cells to regenerate lost or diseased tissue. Recently, stage-specific <em>embryonic</em> kidney progenitor tissue was used to regenerate nephrons. Through fluorescence-activated cell sorting, microarray analysis, in vitro <em>differentiation</em> assays, mixed lymphocyte reaction, and a model of ischemic kidney injury, this study sought to identify and characterize multipotent organ stem/progenitor cells in the adult kidney. Herein is reported the existence of nontubular cells that express stem cell antigen-<em>1</em> (Sca-<em>1</em>). This population of small cells includes a CD45-negative fraction that lacks hematopoietic stem cell and lineage markers and resides in the renal interstitial space. In addition, these cells are enriched for beta<em>1</em>-integrin, are cytokeratin negative, and show minimal expression of surface markers that typically are found on bone marrow-derived mesenchymal stem cells. Global gene profiling reveals enrichment for many genes downstream of developmental signaling molecules and self-renewal pathways, such as TGF-beta/bone morphogenic protein, Wnt, or fibroblast <em>growth</em> <em>factor</em>, as well as for those that are involved in specification of mesodermal lineages (myocyte enhancer <em>factor</em> 2A, YY<em>1</em>-associated <em>factor</em> 2, and filamin-beta). In vitro, they are plastic adherent and slowly proliferating and result in inhibition of alloreactive CD8(+) T cells, indicative of an immune-privileged behavior. Furthermore, clonal-derived lines can be differentiated into myogenic, osteogenic, adipogenic, and neural lineages. Finally, when injected directly into the renal parenchyma, shortly after ischemic/reperfusion injury, renal Sca-<em>1</em>(+)Lin(-) cells, derived from ROSA26 reporter mice, adopt a tubular phenotype and potentially could contribute to kidney repair. These data define a unique phenotype for adult kidney-derived cells, which have potential as stem cells and may contribute to the regeneration of injured kidneys.

NADPH oxidase (Nox4) produces reactive oxygen species (ROS) that are important for vascular smooth muscle cell (SMC) behavior, but the potential impact of Nox4 in stem cell <em>differentiation</em> is unknown. When mouse <em>embryonic</em> stem (ES) cells were plated on collagen IV-coated dishes/flasks, a panel of SMC-specific genes was significantly and consistently upregulated. Nox4 expression was markedly correlated with such a gene induction as confirmed by real-time PCR, immunofluorescence, and Western blot analysis. Overexpression of Nox4 specifically resulted in increased SMC marker production, whereas knockdown of Nox4 induced a decrease. Furthermore, SMC-specific transcription <em>factors</em>, including serum response <em>factor</em> (SRF) and myocardin were activated by Nox4 gene expression. Moreover, Nox4 was demonstrated to drive SMC <em>differentiation</em> through generation of H(2)O(2). Confocal microscopy analysis indicates that SRF was translocated into the nucleus during SMC <em>differentiation</em> in which SRF was phosphorylated. Additionally, autosecreted transforming <em>growth</em> <em>factor</em> (TGF)-beta(<em>1</em>) activated Nox4 and promoted SMC <em>differentiation</em>. Interestingly, cell lines generated from stem cells by Nox4 transfection and G4<em>1</em>8 selection displayed a characteristic of mature SMCs, including expression of SMC markers and cells with contractile function. Thus we demonstrate for the first time that Nox4 is crucial for SMC <em>differentiation</em> from ES cells, and enforced Nox4 expression can maintain <em>differentiation</em> status and functional features of stem cell-derived SMCs, highlighting its impact on vessel formation in vivo and vascular tissue engineering in the future.

Hepatocyte <em>growth</em> <em>factor</em>/scatter <em>factor</em> (HGF/SF) is the mesenchymal ligand of the epithelial tyrosine kinase receptor c-Met. In vitro, HGF/SF has morphogenic properties, e.g., induces kidney epithelial cells to form branching ducts in collagen gels. Mutation of the HGF/SF gene in mice results in <em>embryonic</em> lethality due to severe liver and placenta defects. Here, we have evaluated the morphogenic activity of HGF/SF with a large variety of epithelial cells grown in three-dimensional collagen matrices. We found that HGF/SF induces SW <em>1</em>222 colon carcinoma cells to form crypt-like structures. In these organoids, cells exhibit apical/basolateral polarity and build a well-developed brush border towards the lumen. Capan 2 pancreas carcinoma cells, upon addition of HGF/SF, develop large hollow spheroids lined with a tight layer of polarized cells. Collagen inside the cysts is digested and the cells show features of pancreatic ducts. HGF/SF induces EpH4 mammary epithelial cells to form long branches with end-buds that resemble developing mammary ducts. pRNS-<em>1</em>-<em>1</em> prostate epithelial cells in the presence of HGF/SF develop long ducts with distal branching as found in the prostate. Finally, HGF/SF simulates alveolar <em>differentiation</em> in LX-<em>1</em> lung carcinoma cells. Expression of transfected HGF/SF cDNA in LX-<em>1</em> lung carcinoma and EpH4 mammary epithelial cells induce morphogenesis in an autocrine manner. In the cell lines tested, HGF/SF activated the Met receptor by phosphorylation of tyrosine residues. These data show that HGF/SF induces intrinsic, tissue-specific morphogenic activities in a wide variety of epithelial cells. Apparently, HGF/SF triggers respective endogenous programs and is thus an inductive, not an instructive, mesenchymal effector for epithelial morphogenesis.

During the invasive phase of implantation, trophoblasts and maternal decidual stromal cells secrete products that regulate trophoblast <em>differentiation</em> and migration into the maternal endometrium. Paracrine interactions between the extravillous trophoblast and the maternal decidua are important for successful <em>embryonic</em> implantation, including establishing the placental vasculature, anchoring the placenta to the uterine wall, and promoting the immunoacceptance of the fetal allograph. To our knowledge, global crosstalk between the trophoblast and the decidua has not been elucidated to date, and the present study used a functional genomics approach to investigate these paracrine interactions. Human endometrial stromal cells were decidualized with progesterone and further treated with conditioned media from human trophoblasts (TCM) or, as a control, with control conditioned media (CCM) from nondecidualized stromal cells for 0, 3, and <em>1</em>2 h. Total RNA was isolated and processed for analysis on whole-genome, high-density oligonucleotide arrays containing 54,600 genes. We found that <em>1</em>374 genes were significantly upregulated and that 3443 genes were significantly downregulated after <em>1</em>2 h of coincubation of stromal cells with TCM, compared to CCM. Among the most upregulated genes were the chemokines CXCL<em>1</em> (GRO<em>1</em>) and IL8,CXCR4, and other genes involved in the immune response (CCL8 [SCYA8], pentraxin 3 (PTX3), IL6, and interferon-regulated and -related genes) as well as TNFAIP6 (tumor necrosis <em>factor</em> alpha-induced protein 6) and metalloproteinases (MMP<em>1</em>, MMP<em>1</em>0, and MMP<em>1</em>4). Among the downregulated genes were <em>growth</em> <em>factors</em>, e.g., IGF<em>1</em>, FGF<em>1</em>, TGFB<em>1</em>, and angiopoietin-<em>1</em>, and genes involved in Wnt signaling (WNT4 and FZD). Real-time RT-PCR and ELISAs, as well as immunohistochemical analysis of human placental bed specimens, confirmed these data for representative genes of both up- and downregulated groups. The data demonstrate a significant induction of proinflammatory cytokines and chemokines, as well as angiogenic/static <em>factors</em> in decidualized endometrial stromal cells in response to trophoblast-secreted products. The data suggest that the trophoblast acts to alter the local immune environment of the decidua to facilitate the process of implantation and ensure an enriched cytokine/chemokine environment while limiting the mitotic activity of the stromal cells during the invasive phase of implantation.

Wnt-<em>1</em>-induced secreted protein <em>1</em> (WISP-<em>1</em>) is a member of the CCN (connective tissue <em>growth</em> <em>factor</em>, Cyr6<em>1</em>, NOV) family of <em>growth</em> <em>factors</em>. Experimental evidence suggests that CCN family members are involved in skeletogenesis and bone healing. To investigate the role of WISP-<em>1</em> in osteogenic processes, we characterized its tissue and cellular expression and evaluated its activity in osteoblastic and chondrocytic cell culture models. During <em>embryonic</em> development, WISP-<em>1</em> expression was restricted to osteoblasts and to osteoblastic progenitor cells of the perichondral mesenchyme. In vitro, we showed that WISP-<em>1</em> expression in differentiating osteoblasts promotes BMP-2-induced osteoblastic <em>differentiation</em>. Using in situ and cell binding analysis, we demonstrated WISP-<em>1</em> interaction with perichondral mesenchyme and undifferentiated chondrocytes. We evaluated the effect of WISP-<em>1</em> on chondrocytes by generating stably transfected mouse chondrocytic cell lines. In these cells, WISP-<em>1</em> increased proliferation and saturation density but repressed chondrocytic <em>differentiation</em>. Because of the similarity between skeletogenesis and bone healing, we also analyzed WISP-<em>1</em> spatiotemporal expression in a fracture repair model. We found that WISP-<em>1</em> expression recapitulates the pattern observed during skeletal development. Our data demonstrate that WISP-<em>1</em> is an osteogenic potentiating <em>factor</em> promoting mesenchymal cell proliferation and osteoblastic <em>differentiation</em> while repressing chondrocytic <em>differentiation</em>. Therefore, we propose that WISP-<em>1</em> plays an important regulatory role during bone development and fracture repair.

After birth the proliferation of cardiac cells declines, and further <em>growth</em> of the heart occurs by hypertrophic cell <em>growth</em>. In the present study the cell proliferation capacity of mouse <em>embryonic</em> stem (ES) cells versus neonatal cardiomyocytes and the effects of reactive oxygen species (ROS) on cardiomyogenesis and cardiac cell proliferation of ES cells was investigated. Low levels of hydrogen peroxide stimulated cardiomyogenesis of ES cells and induced proliferation of cardiomyocytes derived from ES cells and neonatal mice, as investigated by nuclear translocation of cyclin D<em>1</em>, downregulation of p27(Kip<em>1</em>), phosphorylation of retinoblastoma (Rb), increase of Ki-67 expression and incorporation of BrdU. The observed effects were blunted by the free radical scavengers vitamin E and 2-mercaptoglycin (NMPG). In ES cells ROS induced expression of the cardiac-specific genes encoding alpha-actin, beta-MHC, MLC2a, MLC2v and ANP as well as the transcription <em>factors</em> GATA-4, Nkx-2.5, MEF2C, DTEF-<em>1</em> and the <em>growth</em> <em>factor</em> BMP-<em>1</em>0. During <em>differentiation</em> ES cells expressed the NADPH oxidase isoforms Nox-<em>1</em>, Nox-2 and Nox-4. Treatment of cardiac cells with ROS increased Nox-<em>1</em>, Nox-4, p22-phox, p47-phox and p67-phox proteins as well as Nox-<em>1</em> and Nox-4 mRNA, indicating feed-forward regulation of ROS generation. Inhibition of NADPH oxidase with diphenylen iodonium chloride (DPI) and apocynin abolished ROS-induced cardiomyogenesis of ES cells. Our data suggest that proliferation of neonatal and ES-cell-derived cardiac cells involves ROS-mediated signalling cascades and point towards an involvement of NADPH oxidase in cardiovascular <em>differentiation</em> of ES cells.

Research on the cell fate determination of <em>embryonic</em> stem cells is of enormous interest given the therapeutic potential in regenerative cell therapy. Human <em>embryonic</em> stem cells (hESCs) have the ability to renew themselves and differentiate into all three germ layers. The main focus of this study was to examine <em>factors</em> affecting derivation and further proliferation of multipotent neuroepithelial (NEP) cells from hESCs. hESCs cultured in serum-deprived defined medium developed distinct tube structures and could be isolated either by dissociation or adherently. Dissociated cells survived to form colonies of cells characterized as NEP when conditioned medium from human hepatocellular carcinoma HepG2 cell line (MEDII) was added. However, cells isolated adherently developed an enriched population of NEP cells independent of MEDII medium. Further characterization suggested that they were NEP cells because they had a similar phenotype profile to in vivo NEP cells and expression SOX<em>1</em>, SOX2, and SOX3 genes. They were positive for Nestin, a neural intermediate filament protein, and Musashi-<em>1</em>, a neural RNA-binding protein, but few cells expressed further <em>differentiation</em> markers, such as PSNCAM, A2B5, MAPII, GFAP, or O4, or other lineage markers, such as muscle actin, alpha fetoprotein, or the pluripotent marker Oct4. Further <em>differentiation</em> of these putative NEP cells gave rise to a mixed population of progenitors that included A2B5-positive and PSNCAM-positive cells and postmitotic neurons and astrocytes. To proliferate and culture these derived NEP cells, ideal conditions were obtained using neurobasal medium supplemented with B27 and basic fibroblast <em>growth</em> <em>factor</em> in 5% oxygen. NEP cells were continuously propagated for longer than 6 months without losing their multipotent cell characteristics and maintained a stable chromosome number.

Sphingolipids and their metabolites, ceramide, sphingosine and sphingosine-<em>1</em>-phosphate, are involved in a variety of cellular processes including <em>differentiation</em>, cellular senescence, apoptosis and proliferation. Ceramide is the main second messenger, and is produced by sphingomyelinase-induced hydrolysis of sphingomyelin and by de novo synthesis. Many stimuli, e. g. <em>growth</em> <em>factors</em>, cytokines, G protein-coupled receptor agonists and stress (UV irradiation) increase cellular ceramide levels. Sphingomyelin in the plasma membrane is located primarily in the outer (extracellular) leaflet of the bilayer, whilst sphingomyelinases are found at the inner (cytosolic) face and within lysosomes/endosomes. Such cellular compartmentalisation restricts the site of ceramide production and subsequent interaction with target proteins. Glycosphingolipids and sphingomyelin together with cholesterol are major components of specialised membrane microdomains known as lipid rafts, which are involved in receptor aggregation and immune responses. Many signalling molecules, for example Src family tyrosine kinases and glycosylinositolphosphate-anchored proteins, are associated with rafts, and disruption of these domains affects cellular responses such as apoptosis. Sphingosine and sphingosine-<em>1</em>-phosphate derived from ceramide are also signalling molecules. In particular, sphingosine-<em>1</em>-phosphate is involved in proliferation, <em>differentiation</em> and apoptosis. Sphingosine-<em>1</em>-phosphate can act both extracellularly through endothelial-differentiating gene (EDG) family G protein-coupled receptors and intracellularly through direct interactions with target proteins. The importance of sphingolipid signalling in cardiovascular development has been reinforced by recent reports implicating EDG receptors in the regulation of <em>embryonic</em> cardiac and vascular morphogenesis.

Cellular activities that lead to organogenesis are mediated by epithelial-mesenchymal interactions, which ultimately result from local activation of complex gene networks. Fibroblast <em>growth</em> <em>factor</em> (FGF) signaling is an essential component of the regulatory network present in the <em>embryonic</em> lung, controlling proliferation, <em>differentiation</em> and pattern formation. However, little is known about how FGFs interact with other signaling molecules in these processes. By using cell and organ culture systems, we provide evidence that FGFs, Sonic hedgehog (Shh), bone morphogenetic protein 4 (BMP-4), and TGFbeta-<em>1</em> form a regulatory circuit that is likely relevant for lung development in vivo. Our data show that FGF-<em>1</em>0 and FGF-7, important for patterning and <em>growth</em> of the lung bud, are differentially regulated by FGF-<em>1</em>, -2 and Shh. In addition, we show that FGFs regulate expression of Shh, BMP-4 and other FGF family members. Our data support a model in which Shh, TGFbeta-<em>1</em> and BMP-4 counteract the bud promoting effects of FGF-<em>1</em>0, and where FGF levels are maintained throughout lung development by other FGFs and Shh.

NGFI-A (also known as EGR-<em>1</em>, zif/268, and Krox-24) is a zinc finger transcription <em>factor</em> induced in many cell types by a variety of <em>growth</em> and <em>differentiation</em> stimuli. To determine if NGFI-A plays a requisite role in these processes, we used homologous recombination to mutate both alleles of NGFI-A in <em>embryonic</em> stem (ES) cells and examined its effect on <em>growth</em> and <em>differentiation</em>. We find that ES cells lacking NGFI-A exhibit similar <em>growth</em> rates and serum-induced gene expression profiles compared to wild-type parental cells. They are capable of differentiating into neurons, cardiac myocytes, chondrocytes, and squamous epithelium. Chimeric mice were generated from targeted ES cells, and their progeny were crossed to produce homozygous mutant mice. <em>Growth</em> and histological analyses of mice lacking NGFI-A confirm the finding in ES cells that NGFI-A is not required for many of the processes associated with its expression and suggest that the function of NGFI-A is either more subtle in vivo or masked by redundant expression provided by other gene family members such as NGFI-C, Krox-20, or EGR3.

OBJECTIVE

We demonstrated previously that mouse embryonic stem (ES) cell-derived vascular endothelial growth factor receptor-2 (VEGF-R2)-positive cells can differentiate into both vascular endothelial cells and mural cells. This time, we investigated kinetics of differentiation of human ES cells to vascular cells and examined their potential as a source for vascular regeneration.

RESULTS

Unlike mouse ES cells, undifferentiated human ES cells already expressed VEGF-R2, but after differentiation, a VEGF-R2-positive but tumor rejection antigen 1-60 (TRA1-60)-negative population emerged. These VEGF-R2-positive but tumor rejection antigen 1-60-negative cells were also positive for platelet-derived growth factor receptor alpha and beta chains and could be effectively differentiated into both VE-cadherin+ endothelial cell and alpha-smooth muscle actin+ mural cell. VE-cadherin+ cells, which were also CD34+ and VEGF-R2+ and thought to be endothelial cells in the early differentiation stage, could be expanded while maintaining their maturity. Their transplantation to the hindlimb ischemia model of immunodeficient mice contributed to the construction of new blood vessels and improved blood flow.

CONCLUSIONS

We could identify the differentiation process from human ES cells to vascular cell components and demonstrate that expansion and transplantation of vascular cells at the appropriate differentiation stage may constitute a novel strategy for vascular regenerative medicine.