Cardiomyocytes from common experimental animals rapidly exit the cell cycle upon isolation, impeding studies of basic cell biology and applications such as myocardial repair. Here we examined proliferation of cardiomyocytes derived from human and mouse <em>embryonic</em> stem (ES) cells. While mouse ES cell-derived cardiomyocytes showed little proliferation, human cardiomyocytes were highly proliferative under serum-free conditions (<em>1</em>5-25% BrdU+/sarcomeric actin+). The cells exhibited only a small serum dose-response, and proliferation gradually slowed with increasing <em>differentiation</em> of the cells. Neither cell density nor different matrix attachment <em>factors</em> affected cardiomyocyte proliferation. Blockade of phosphatidylinositol 3-kinase (PI 3-kinase) and Akt significantly reduced cardiomyocyte proliferation, whereas MEK inhibition had no effect. Antibody blocking of the insulin-like <em>growth</em> <em>factor</em>-<em>1</em> (IGF-<em>1</em>) receptor significantly inhibited cardiomyocyte proliferation, while addition of IGF-<em>1</em> or IGF-2 stimulated cardiomyocyte proliferation in a dose-dependent manner. Thus, cardiomyocytes derived from human ES cells proliferate extensively in vitro, and their proliferation appears to be mediated primarily via the PI 3-kinase/Akt signaling pathway, using the IGF-<em>1</em> receptor as one upstream activator. This system should permit identification of regulatory pathways for human cardiomyocyte proliferation and may facilitate expansion of cardiomyocytes from human ES cells for therapeutic purposes.

Many applications of human <em>embryonic</em> stem cells (hESCs) will require fully defined <em>growth</em> and <em>differentiation</em> conditions including media devoid of fetal calf serum. To identify <em>factors</em> that control lineage <em>differentiation</em> we have analyzed a serum-free (SF) medium conditioned by the cell line END2, which efficiently induces hESCs to form cardiomyocytes. Firstly, we noted that insulin, a commonly used medium supplement, acted as a potent inhibitor of cardiomyogenesis in multiple hESC lines and was rapidly cleared by medium conditioning. In the presence of insulin or IGF-<em>1</em>, which also suppressed cardiomyocyte <em>differentiation</em>, the PI3/Akt pathway was activated in undifferentiated hESC, suggesting that insulin/IGF-<em>1</em> effects were mediated by this signaling cascade. Time course analysis and quantitative RT-PCR revealed impaired expression of endoderm and mesoderm markers in the presence of insulin, particularly if added during early stages of hESC <em>differentiation</em>. Relatively high levels of the neural ectoderm marker Sox<em>1</em> were expressed under these conditions. Secondly, comparative gene expression showed that two key enzymes in the prostaglandin I2 (PGI2) synthesis pathway were highly up-regulated in END2 cells compared with a related, but non-cardiogenic, cell line. Biochemical analysis confirmed 6-<em>1</em>0-fold higher PGI2 levels in END2 cell-conditioned medium (END2-CM) vs. controls. Optimized concentrations of PGI2 in a fully synthetic, insulin-free medium resulted in a cardiogenic activity equivalent to END2-CM. Addition of the p38 mitogen-activated protein kinase-inhibitor SB203580, which we have shown previously to enhance hESC cardiomyogenesis, to these insulin-free and serum-free conditions resulted in a cardiomyocyte content of>><em>1</em>0% in differentiated cultures without any preselection. This study represents a significant step toward developing scalable production for cardiomyocytes from hESC using clinically compliant reagents compatible with Good Manufacturing Practice.

Pancreatic acini and islets are believed to differentiate from common ductal precursors through a process requiring various <em>growth</em> <em>factors</em>. Epidermal <em>growth</em> <em>factor</em> receptor (EGF-R) is expressed throughout the developing pancreas. We have analyzed here the pancreatic phenotype of EGF-R deficient (-/-) mice, which generally die from epithelial immaturity within the first postnatal week. The pancreata appeared macroscopically normal. The most striking feature of the EGF-R (-/-) islets was that instead of forming circular clusters, the islet cells were mainly located in streak-like structures directly associated with pancreatic ducts. Based on BrdU-labelling, proliferation of the neonatal EGF-R (-/-) beta-cells was significantly reduced (2.6+/-0.4 versus 5.8+/-0.9%, P<0.0<em>1</em>) and the <em>difference</em> persisted even at 7-<em>1</em><em>1</em> days of age. Analysis of <em>embryonic</em> pancreata revealed impaired branching morphogenesis and delayed islet cell <em>differentiation</em> in the EGF-R (-/-) mice. Islet development was analyzed further in organ cultures of E<em>1</em>2.5 pancreata. The proportion of insulin-positive cells was significantly lower in the EGF-R (-/-) explants (27+/-6 versus 48+/-8%, P<0.0<em>1</em>), indicating delayed <em>differentiation</em> of the beta cells. Branching of the epithelium into ducts was also impaired. Matrix metalloproteinase (MMP-2 and MMP-9) activity was reduced 20% in EGF-R (-/-) late-gestation pancreata, as measured by gelatinase assays. Furthermore, the levels of secreted plasminogen activator inhibitor-<em>1</em> (PAI-<em>1</em>) were markedly higher, while no apparent <em>differences</em> were seen in the levels of active uPA and tPa between EGF-R (-/-) and wild-type pancreata. Our findings suggest that the perturbation of EGF-R-mediated signalling can lead to a generalized proliferation defect of the pancreatic epithelia associated with a delay in beta cell development and disturbed migration of the developing islet cells as they differentiate from their precursors. Upregulated PAI-<em>1</em> production and decreased gelatinolytic activity correlated to this migration defect. An intact EGF-R pathway appears to be a prerequisite for normal pancreatic development.

Impaired Ag-presenting function in dendritic cells (DCs) due to abnormal <em>differentiation</em> is an important mechanism of tumor escape from immune control. A major role for vascular endothelial <em>growth</em> <em>factor</em> (VEGF) and its receptors, VEGFR<em>1</em>/Flt-<em>1</em> and VEGFR2/KDR/Flk-<em>1</em>, has been documented in hemopoietic development. To study the roles of each of these receptors in DC <em>differentiation</em>, we used an in vitro system of myeloid DC <em>differentiation</em> from murine <em>embryonic</em> stem cells. Exposure of wild-type, VEGFR<em>1</em>(-/-), or VEGFR2(-/-) <em>embryonic</em> stem cells to exogenous VEGF or the VEGFR<em>1</em>-specific ligand, placental <em>growth</em> <em>factor</em>, revealed distinct roles of VEGF receptors. VEGFR<em>1</em> is the primary mediator of the VEGF inhibition of DC maturation, whereas VEGFR2 tyrosine kinase signaling is essential for early hemopoietic <em>differentiation</em>, but only marginally affects final DC maturation. SU54<em>1</em>6, a VEGF receptor tyrosine kinase inhibitor, only partially rescued the mature DC phenotype in the presence of VEGF, suggesting the involvement of both tyrosine kinase-dependent and independent inhibitory mechanisms. VEGFR<em>1</em> signaling was sufficient for blocking NF-kappaB activation in bone marrow hemopoietic progenitor cells. VEGF and placental <em>growth</em> <em>factor</em> affect the early stages of myeloid/DC <em>differentiation</em>. The data suggest that therapeutic strategies attempting to reverse the immunosuppressive effects of VEGF in cancer patients might be more effective if they specifically targeted VEGFR<em>1</em>.

Vascular endothelial <em>growth</em> <em>factor</em> (VEGF) is a candidate regulator of blood vessel <em>growth</em> during <em>embryonic</em> development and in tumors. To evaluate the role of VEGF receptor-<em>1</em>/flt-<em>1</em> (VEGFR<em>1</em>/flt-<em>1</em>) in the development of the vascular system, we have characterized the murine homolog of the human flt-<em>1</em> gene and have analyzed its expression pattern during mouse embryogenesis. Receptor binding studies using transfected COS cells revealed that the murine flt-<em>1</em> gene encodes a high affinity receptor for VEGF. The apparent Kd for VEGF binding, as determined by Scatchard analysis, was <em>1</em><em>1</em>4 pM, demonstrating that VEGFR<em>1</em>/flt-<em>1</em> has a higher affinity to VEGF than VEGF receptor-2/flk-<em>1</em> (VEGFR2/flk-<em>1</em>). By in situ hybridization, VEGFR<em>1</em>/flt-<em>1</em> was detected in the yolk sac mesoderm already at the early stages of vascular development, while the receptor ligand was expressed in the entire endoderm of 7.5-day mouse embryos. A comparison with VEGFR2/flk-<em>1</em> showed that the two receptors shared a common expression domain in the yolk sac mesoderm, but were expressed at different sites in the ectoplacental cone. The differential expression of the two VEGF receptors persisted in the developing placenta, where VEGFR<em>1</em>/flt-<em>1</em> mRNA was detected in the spongiotrophoblast layer, whereas VEGFR2/flk-<em>1</em> transcripts were present in the labyrinthine layer which is the site of VEGF expression. In the embryo proper, VEGFR<em>1</em>/flt-<em>1</em> mRNA was specifically localized in blood vessels and capillaries of the developing organs, closely resembling the pattern of VEGFR2/flk-<em>1</em> transcript distribution. In the developing brain, the expression of VEGF receptors in the perineural capillary plexus and in capillary sprouts which have invaded the neuro-ectoderm correlated with endothelial cell proliferation and brain angiogenesis. The data are consistent with the hypothesis that VEGF and its receptors have an important function both in the <em>differentiation</em> of the endothelial lineage and in the neovascularization of developing organs, and act in a paracrine fashion.

Cells in the musculoskeletal system can respond to mechanical stimuli, supporting tissue homeostasis and remodeling. Recent studies have suggested that mechanical stimulation also influences the <em>differentiation</em> of MSCs, whereas the effect on <em>embryonic</em> cells is still largely unknown. In this study, we evaluated the influence of dynamic mechanical compression on chondrogenesis of bone marrow-derived MSCs and <em>embryonic</em> stem cell-derived (human embryoid body-derived [hEBd]) cells encapsulated in hydrogels and cultured with or without transforming <em>growth</em> <em>factor</em> beta-<em>1</em> (TGF-beta<em>1</em>). Cells were cultured in hydrogels for up to 3 weeks and exposed daily to compression for <em>1</em>, 2, 2.5, and 4 hours in a bioreactor. When MSCs were cultured, mechanical stimulation quantitatively increased gene expression of cartilage-related markers, Sox-9, type II collagen, and aggrecan independently from the presence of TGF-beta<em>1</em>. Extracellular matrix secretion into the hydrogels was also enhanced. When hEBd cells were cultured without TGF-beta<em>1</em>, mechanical compression inhibited their <em>differentiation</em> as determined by significant downregulation of cartilage-specific genes. However, after initiation of chondrogenic <em>differentiation</em> by administration of TGF-beta<em>1</em>, the hEBd cells quantitatively increased expression of cartilage-specific genes when exposed to mechanical compression, similar to the bone marrow-derived MSCs. Therefore, when appropriately directed into the chondrogenic lineage, mechanical stimulation is beneficial for further <em>differentiation</em> of stem cell tissue engineered constructs.

The density of native (preexisting) collaterals and their capacity to enlarge into large conduit arteries in ischemia (arteriogenesis) are major determinants of the severity of tissue injury in occlusive disease. Mechanisms directing arteriogenesis remain unclear. Moreover, nothing is known about how native collaterals form in healthy tissue. Evidence suggests vascular endothelial <em>growth</em> <em>factor</em> (VEGF), which is important in <em>embryonic</em> vascular patterning and ischemic angiogenesis, may contribute to native collateral formation and arteriogenesis. Therefore, we examined mice heterozygous for VEGF receptor-<em>1</em> (VEGFR-<em>1</em>(+/-)), VEGF receptor-2 (VEGFR-2(+/-)), and overexpressing (VEGF(hi/+)) and underexpressing VEGF-A (VEGF(lo/+)). Recovery from hindlimb ischemia was followed for 2<em>1</em> days after femoral artery ligation. All statements below are P<0.05. Compared to wild-type mice, VEGFR-2(+/-) showed similar: ischemic scores, recovery of hindlimb perfusion, pericollateral leukocytes, collateral enlargement, and angiogenesis. In contrast, VEGFR-<em>1</em>(+/-) showed impaired: perfusion recovery, pericollateral leukocytes, collateral enlargement, worse ischemic scores, and comparable angiogenesis. Compared to wild-type mice, VEGF(lo/+) had 2-fold lower perfusion immediately after ligation (suggesting fewer native collaterals which was confirmed by angiography) and blunted recovery of perfusion. VEGF(hi/+) mice had 3-fold greater perfusion immediately after ligation, more native collaterals, and improved recovery of perfusion. These <em>differences</em> were confirmed in the cerebral pial cortical circulation where, compared to VEGF(hi/+) mice, VEGF(lo/+) formed fewer collaterals during the perinatal period when adult density was established, and had 2-fold larger infarctions after middle cerebral artery ligation. Our findings indicate VEGF and VEGFR-<em>1</em> are determinants of arteriogenesis. Moreover, we describe the first signaling molecule, VEGF-A, that specifies formation of native collaterals in healthy tissues.

Members of the bone morphogenetic protein (BMP) family have been implicated in multiple aspects of neural development in both the CNS and peripheral nervous system. BMP ligands and receptors, as well as the BMP antagonist noggin, are expressed in the developing cerebral cortex, making the BMPs likely candidates for regulating cortical development. To define the role of these <em>factors</em> in the developing cerebral cortex, we examined the effects of BMP2 and BMP4 on cortical cells in vitro. Cells were cultured from <em>embryonic</em> day <em>1</em>3 (E<em>1</em>3) and E<em>1</em>6 rat cerebral cortex in the absence or presence of different concentrations of fibroblast <em>growth</em> <em>factor</em> 2, a known regulator of cortical cell proliferation and <em>differentiation</em>. At E<em>1</em>3, the BMPs promoted cell death and inhibited proliferation of cortical ventricular zone cells, resulting in the generation of fewer neurons and no glia. At E<em>1</em>6, the effects of the BMPs were more complex. Concentrations of BMP2 in the range of <em>1</em>-<em>1</em>0 ng/ml promoted neuronal and astroglial <em>differentiation</em> and inhibited oligodendroglial <em>differentiation</em>, whereas <em>1</em>00 ng/ml BMP2 promoted cell death and inhibited proliferation. Addition of the BMP antagonist noggin promoted oligodendrogliogenesis in vitro, demonstrating that endogenous BMP signaling influences the <em>differentiation</em> of cortical cells in vitro. The distribution of BMP2 and noggin within the developing cortex suggests that local concentrations of ligands and antagonists define gradients of BMP signaling during corticogenesis. Together, these results support the hypothesis that the BMPs and their antagonist noggin co-regulate cortical cell fate and morphogenesis.

The cardiovascular system develops early in embryogenesis from cells of mesodermal origin. To study the molecular and cellular processes underlying this transition, we have isolated mesodermal cells from murine embryos at E7.5 with characteristic properties of endothelial progenitors by using a combination of stromal cell layers and <em>growth</em> conditions. The isolated <em>embryonic</em> cells displayed unlimited stem-cell-like <em>growth</em> potential and a stable phenotype in culture. RNA analysis revealed that the <em>embryonic</em> cells express the endothelial-specific genes tie-2 and thrombomodulin (TM) as well as the early mesodermal marker fgf-3. The GSL I-B4 isolectin, a marker of early endothelial cells, specifically binds to the isolated cells. The in vitro <em>differentiation</em> with retinoic acid and cAMP led to a 5- to <em>1</em>0-fold induction of flk-<em>1</em>, von Willebrand <em>Factor</em> (vWF), TM, GATA-4 and GATA-6. Electron microscopy revealed that in vitro <em>differentiation</em> is associated with increased amounts of rER and Golgi, and a dramatic increase in secretory vesicles packed with vWF. When cultured in Matrigel, the <em>embryonic</em> cells assume the characteristic endothelial cobblestone morphology and form tubes. Injection into chicken embryos showed incorporation of the <em>embryonic</em> cells in the endocardium and the brain vasculature. The expression of TM, tie-2, GATA-4 and GATA-6 suggests that the isolated <em>embryonic</em> endothelial cell progenitors are derived from the proximal lateral mesoderm where the pre-endocardial tubes form. The properties of the endothelial cell progenitors described here provide a novel approach to analyze mediators, signaling pathways and transcriptional control in early vascular development.

OBJECTIVE

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta family and play an important role in the regulation of embryonic vasculogenesis but their role in postnatal angiogenesis remains to be clarified. In this study we investigated a possible role of BMP-2 in the promotion of tumor angiogenesis.

METHODS

We studied the effect of BMP-2 on human dermal microvascular endothelial cells (HDMECs) and examined a possible angiogenic activity of BMP-2 with the mouse sponge assay. The effect of BMP-2 overexpression on tumor vascularization was also analyzed in xenografts of human BMP-2 transfected MCF-7 breast cancer cells (MCF-7/BMP2) in mice.

RESULTS

BMP receptor activation selectively induced the phosphorylation of p38 mitogen-activated protein kinase (MAPK) in contrast to the ERK1/2 MAP kinases. In keeping with this finding, BMP-2 had no significant effect on endothelial cell proliferation but promoted HDMEC tube formation in the matrigel assay. The transcription factor inhibitor of differentiation 1 (Id1), which is known to play an important role in neovascularization of tumors, was confirmed as a BMP target in HDMECs. Immunohistochemical analysis of sponge sections revealed that BMP-2 induced vascularization and showed an additive enhancement of angiogenesis with VEGF. In the murine breast cancer xenograft model, human MCF-7 cells with stable overexpression of BMP-2 developed vascularized tumors while empty vector control MCF-7 cells failed to form tumors.

CONCLUSIONS

We conclude that activation of the BMP pathway by BMP-2 can promote vascularization and might be involved in tumor angiogenesis possibly by stimulating the Id1 and p38 MAPK pathway.

We present evidence for unique localization and specific biological activities for transforming <em>growth</em> <em>factor</em>-beta s (TGF-beta s) 2 and 3, as compared to TGF-beta <em>1</em>, in the nervous system of the <em>1</em>2-<em>1</em>8 day mouse embryo. Each TGF-beta isoform was localized immunohistochemically by specific antibodies raised to peptides corresponding to unique sequences in the respective TGF-beta proteins. Staining for TGF-beta <em>1</em> was principally in the meninges, while TGF-beta s 2 and 3 co-localized in neuronal perikarya and axons, as well as in radial glial cells. In the central nervous system, staining was most prominent in zones where neuronal <em>differentiation</em> occurs and less intense in zones of active proliferation, while in the peripheral nervous system, many nerve fibers as well as their cell bodies were strongly immunoreactive for TGF-beta s 2 and 3. Functionally, we have also found that in the presence of an extract of chick eye tissue, TGF-beta s 2 and 3 inhibit survival of cultured <em>embryonic</em> chick ciliary ganglionic neurons in a dose-dependent fashion; TGF-beta <em>1</em> shows no inhibitory effects. Our data suggest that TGF-beta s 2 and 3 may play a role in regulation of neuronal migration and <em>differentiation</em>, as well as in glial cell proliferation and <em>differentiation</em>.

Mesothelial Fibroblast <em>Growth</em> <em>Factor</em> 9 (Fgf9) has been demonstrated by inactivation studies in mouse to be critical for the proliferation of the mesenchyme. We now show that Fgf9 is also expressed at significant levels in the distal epithelium from the mid-pseudoglandular stages. Using mesenchymal-free lung endoderm culture, we show that FGF9 triggers the proliferation of the distal epithelium leading to the formation of a cyst-like structure. On <em>embryonic</em> Fgfr2b-/- lungs, FGF9 induces proliferation of the mesenchyme but fails to trigger a similar effect on the epithelium, therefore involving the FGFR2b receptor in the proliferative response of the epithelium to FGF9. While FGF9 inhibits the <em>differentiation</em> of the mesenchyme, the epithelium appears to differentiate normally. At the molecular level, FGF9 up-regulates Fgf<em>1</em>0 expression in the mesenchyme likely via increased expression of Tbx4 and 5 and controls the transcription of Hedgehog targets Ptc and Gli-<em>1</em> in a Hedgehog-independent manner. We also show that FGF9 inhibits the activation of the canonical Wnt pathway in the epithelium by increasing Dkk<em>1</em> expression, a canonical Wnt antagonist. Our work shows for the first time that FGF9 acts on the epithelium involving FGFR2b to control its proliferation but not its <em>differentiation</em> and contributes to the regulation of canonical Wnt signaling in the epithelium.

OBJECTIVE

The role of stem cells in regenerative medicine is evolving rapidly. Here, we describe the application, for kidney regeneration, of a novel non-genetically modified stem cell, derived from human amniotic fluid. We show that these pluripotent cells can develop and differentiate into de novo kidney structures during organogenesis in vitro.

METHODS

Human amniotic fluid-derived stem cells (hAFSCs) were isolated from human male amniotic fluid obtained between 12 and 18 weeks gestation. Green fluorescent protein and Lac-Z-transfected hAFSCs were microinjected into murine embryonic kidneys (12.5-18 days gestation) and were maintained in a special co-culture system in vitro for 10 days. Techniques of live microscopy, histology, chromogenic in situ hybridization and reverse transcriptase polymerase chain reaction were used to characterize the hAFSCs during their integration and differentiation in concert with the growing organ.

RESULTS

Green fluorescent protein and Lac-Z-transfected hAFSCs demonstrated long-term viability in organ culture. Histological analysis of injected kidneys revealed that hAFSCs were capable of contributing to the development of primordial kidney structures including renal vesicle, C- and S-shaped bodies. Reverse transcriptase polymerase chain reaction confirmed expression of early kidney markers for: zona occludens-1, glial-derived neurotrophic factor and claudin.

CONCLUSIONS

Human amniotic fluid-derived stem cells may represent a potentially limitless source of ethically neutral, unmodified pluripotential cells for kidney regeneration.

Human <em>embryonic</em> stem cells (hESCs) have great potential for use in research and regenerative medicine, but very little is known about the <em>factors</em> that maintain these cells in the pluripotent state. We investigated the role of three major mitogenic agents present in serum--sphingosine-<em>1</em>-phosphate (S<em>1</em>P), lysophosphatidic acid (LPA), and platelet-derived <em>growth</em> <em>factor</em> (PDGF)--in maintaining hESCs. We show here that although LPA does not affect hESC <em>growth</em> or <em>differentiation</em>, coincubation of S<em>1</em>P and PDGF in a serum-free culture medium successfully maintains hESCs in an undifferentiated state. Our studies indicate that signaling pathways activated by tyrosine kinase receptors act synergistically with those downstream from lysophospholipid receptors to maintain hESCs in the undifferentiated state. This study is the first demonstration of a role for lysophospholipid receptor signaling in the maintenance of stem cell pluri-potentiality.

Mitogen-activated protein (MAP) kinase cascades play a central role in mediating extracellular stimuli-induced intracellular signaling during cell activation. The fourth and least studied mammalian MAP kinase pathway, big MAP kinase <em>1</em> (BMK<em>1</em>), also known as extracellular signal regulated kinase 5 (ERK5), is activated in response to <em>growth</em> <em>factors</em> and stress. Activation of this signaling pathway has been implicated not only in physiological functions such as cell survival, proliferation and <em>differentiation</em> but also in pathological processes such as carcinogenesis, cardiac hypertrophy and atherosclerosis. In recent years a series of gene-targeted mice lacking components within the BMK<em>1</em> cascade have been generated, which have enabled us to investigate the role of the BMK<em>1</em> pathway within different tissues. Analyses of these knockout mice have led to major discoveries in the role of BMK<em>1</em> signaling in angiogenesis and in cardiac development. Moreover, studies using conditional BMK<em>1</em> knockout mice, which circumvent the early <em>embryonic</em> lethality of BMK<em>1</em> knockouts, have unveiled the importance of BMK<em>1</em> in endothelial survival and maintenance of vascular integrity during adulthood. Here we summarize current understanding of the function of BMK<em>1</em>, as well as include new data generated from a series of tissue-specific BMK<em>1</em> knockout mice in an attempt to dissect the role of the BMK<em>1</em> pathway in various cell types in animals.

OBJECTIVE

Human embryonic stem cells (hESCs) offer a sustainable source of endothelial cells for therapeutic vascularization and tissue engineering, but current techniques for generating these cells remain inefficient. We endeavored to induce and isolate functional endothelial cells from differentiating hESCs.

RESULTS

To enhance endothelial cell differentiation above a baseline of approximately 2% in embryoid body (EB) spontaneous differentiation, 3 alternate culture conditions were compared. Vascular endothelial growth factor (VEGF) treatment of EBs showed the best induction, with markedly increased expression of endothelial cell proteins CD31, VE-Cadherin, and von Willebrand Factor, but not the hematopoietic cell marker CD45. CD31 expression peaked around days 10 to 14. Continuous VEGF treatment resulted in a 4- to 5-fold enrichment of CD31(+) cells but did not increase endothelial proliferation rates, suggesting a primary effect on differentiation. CD31(+) cells purified from differentiating EBs upregulated ICAM-1 and VCAM-1 in response to TNFalpha, confirming their ability to function as endothelial cells. These cells also expressed multiple endothelial genes and formed lumenized vessels when seeded onto porous poly(2-hydroxyethyl methacrylate) scaffolds and implanted in vivo subcutaneously in athymic rats. Collagen gel constructs containing hESC-derived endothelial cells and implanted into infarcted nude rat hearts formed robust networks of patent vessels filled with host blood cells.

CONCLUSIONS

VEGF induces functional endothelial cells from hESCs independent of endothelial cell proliferation. This enrichment method increases endothelial cell yield, enabling applications for revascularization as well as basic studies of human endothelial biology. We demonstrate the ability of hESC-derived endothelial cells to facilitate vascularization of tissue-engineered implants.

Stem cells expressing c-kit have been identified in the adult epicardium. In mice, after myocardial infarction, these cells proliferate, migrate to the injury site and differentiate toward myocardial and vascular phenotype. We hypothesized that, acutely after myocardial infarction, pericardial sac integrity and pericardial fluid (PF) may play a role on epicardial cell gene expression, proliferation and <em>differentiation</em>. Microarray analysis indicated that, in the presence of an intact pericardial sac, myocardial infarction modulated 246 genes in epicardial cells most of which were related to cell proliferation, cytoskeletal organization, wound repair and signal transduction. Interestingly, WT<em>1</em>, Tbx<em>1</em>8 and RALDH2, notably involved in epicardial <em>embryonic</em> development, were markedly up-regulated. Importantly, coexpression of stem cell antigen c-kit and WT<em>1</em> and/or Tbx<em>1</em>8 was detected by immunohistochemistry in the mouse epicardium during embryogenesis as well as in adult mouse infarcted heart. Injection of human pericardial fluid from patients with acute myocardial ischemia (PFMI) in the pericardial cavity of non-infarcted mouse hearts, enhanced, epicardial cell proliferation and WT<em>1</em> expression. Further, PFMI supplementation to hypoxic cultured human epicardial c-kit(+) cells increased WT<em>1</em> and Tbx<em>1</em>8 mRNA expression. Finally, insulin-like <em>growth</em> <em>factor</em> <em>1</em>, hepatocyte <em>growth</em> <em>factor</em> and high mobility group box <em>1</em> protein, previously involved in cardiac c-kit(+) cell proliferation and <em>differentiation</em>, were increased in PFMI compared to the pericardial fluid of non ischemic patients. In conclusion, myocardial infarction reactivates an <em>embryonic</em> program in epicardial c-kit(+) cells; soluble <em>factors</em> released in the pericardial fluids following myocardial necrosis may play a role in this process.

Recent neuroimaging and postmortem studies have reported abnormalities in white matter of schizophrenic brains, suggesting the involvement of oligodendrocytes in the etiopathology of schizophrenia. This view is being supported by gene microarray studies showing the downregulation of genes related to oligodendrocyte function and myelination in schizophrenic brain compared to control subjects. However, there is currently little information available on the response of oligodendrocytes to antipsychotic drugs (APDs), which could be invaluable for corroborating the oligodendrocyte hypothesis. In this study we found: (<em>1</em>) quetiapine (QUE, an atypical APD) treatment in conjunction with addition of <em>growth</em> <em>factors</em> increased the proliferation of neural progenitors isolated from the cerebral cortex of <em>embryonic</em> rats; (2) QUE directed the <em>differentiation</em> of neural progenitors to oligodendrocyte lineage through extracellular signal-related kinases; (3) addition of QUE increased the synthesis of myelin basic protein and facilitated myelination in rat <em>embryonic</em> cortical aggregate cultures; (4) chronic administration of QUE to C57BL/6 mice prevented cortical demyelination and concomitant spatial working memory impairment induced by cuprizone, a neurotoxin. These findings suggest a new neural mechanism of antipsychotic action of QUE, and help to establish a role for oligodendrocytes in the etiopathology and treatment of schizophrenia.

<em>Growth</em> of functional arteries is essential for the restoration of blood flow to ischemic organs. Notch signaling regulates arterial <em>differentiation</em> upstream of ephrin-B2 during <em>embryonic</em> development, but its role during postnatal arteriogenesis is unknown. Here, we identify the Notch ligand Delta-like <em>1</em> (Dll<em>1</em>) as an essential regulator of postnatal arteriogenesis. Dll<em>1</em> expression was specifically detected in arterial endothelial cells, but not in venous endothelial cells or capillaries. During ischemia-induced arteriogenesis endothelial Dll<em>1</em> expression was strongly induced, Notch signaling activated and ephrin-B2 upregulated, whereas perivascular cells expressed proangiogenic vascular endothelial <em>growth</em> <em>factor</em>, and the ephrin-B2 activator EphB4. In heterozygous Dll<em>1</em> mutant mice endothelial Notch activation and ephrin-B2 induction after hindlimb ischemia were absent, arterial collateral <em>growth</em> was abrogated and recovery of blood flow was severely impaired, but perivascular vascular endothelial <em>growth</em> <em>factor</em> and EphB4 expression was unaltered. In vitro, angiogenic <em>growth</em> <em>factors</em> synergistically activated Notch signaling by induction of Dll<em>1</em>, which was necessary and sufficient to regulate ephrin-B2 expression and to induce ephrin-B2 and EphB4-dependent branching morphogenesis in human arterial EC. Thus, Dll<em>1</em>-mediated Notch activation regulates ephrin-B2 expression and postnatal arteriogenesis.

To facilitate the exploitation of <em>embryonic</em> stem cells (ESCs) and ESC-derived cells, scale-up of cell production and optimization of culture conditions are necessary. Conventional ESC culture methods are impractical for large-scale cell production and lack robust microenvironmental control. We developed two stirred-suspension culture systems for the propagation of undifferentiated ESCs--microcarrier and aggregate cultures--and compared them with tissue-culture flask and Petri dish controls. ESCs cultured on glass microcarriers had population doubling times (approximately <em>1</em>4-<em>1</em>7 hours) comparable to tissue-culture flask controls. ESC <em>growth</em> could be elicited in shear-controlled stirred-suspension culture, with population doubling times ranging between 24 and 39 hours at <em>1</em>00 rpm impeller speed. Upon removal of leukemia inhibitory <em>factor</em>, the size-controlled ESC aggregates developed into embryoid bodies (EBs) capable of multilineage <em>differentiation</em>. A comprehensive analysis of ESC developmental potential, including flow cytometry for Oct-4, SSEA-<em>1</em>, and E-cadherinprotein expression, reverse transcription-polymerase chain reaction for Flk-<em>1</em>, HNF3-beta, MHC, and Sox-<em>1</em> gene expression, and EB <em>differentiation</em> analysis, demonstrated that the suspension-cultured ESCs retained the developmental potential of the starting cell population. Analysis of E-cadherin-/- and E-cadherin+/- cells using both systems provided insight into the mechanisms behind the role of cell aggregation control, which is fundamental to these observations. These cell-culture tools should prove useful for both the production of ESCs and ESC-derived cells and for investigations into adhesion, survival, and <em>differentiation</em> phenomena during ESC propagation and <em>differentiation</em>.

The Jun proteins Jun, JunB and JunD are core members of activator protein-<em>1</em> (AP-<em>1</em>), a dimeric transcription <em>factor</em> complex consisting of homo- and heterodimers of the Jun, Fos, activating transcription <em>factor</em> (ATF) and musculoaponeurotic fibrosarcoma (Maf) families. <em>Growth</em> <em>factors</em>, hormones and a variety of environmental stresses activate mitogen activated protein kinase (MAPK) cascades that enhance Jun/AP-<em>1</em> activity, e.g. through phosphorylation thereby regulating cell proliferation, <em>differentiation</em>, transformation and/or apoptosis. <em>Embryonic</em> lethality of various AP-<em>1</em> knock-outs, e.g. for Jun, JunB, Fra-<em>1</em> and Fra-2 largely prevented functional studies in vivo. Therefore, conditional knock-out strategies, in particular for the epidermis, have become an important model to study the regulation and function of AP-<em>1</em> subunits in physiological and pathological processes in vivo. Jun is regarded as a positive regulator of keratinocyte proliferation/<em>differentiation</em> during development and in skin cancer through its direct transcriptional effect on epidermal <em>growth</em> <em>factor</em> receptor (EGFR) expression. In contrast, JunB can antagonize proliferation of keratinocytes and hematopoietic stem cells. Furthermore, it has been demonstrated in patient's samples and an inducible mouse model that down-regulation of JunB/AP-<em>1</em> in keratinocytes is one initiating event in the aetiology of psoriasis which is characterized by increased cell proliferation and deregulated cytokine expression.

The superfamily of transforming <em>growth</em> <em>factors</em>-beta (TGF-beta) comprises an expanding list of multifunctional proteins serving as regulators of cell proliferation and <em>differentiation</em>. Prominent members of this family include the TGF-beta s <em>1</em>-5, activins, bone morphogenetic proteins and a recently discovered glial cell line-derived neurotrophic <em>factor</em> (GDNF). In the present study we demonstrate and compare the survival promoting and neuroprotective effects of TGF-beta <em>1</em>, -2 and -3, activin A and GDNF for midbrain dopaminergic neurons in vitro. All proteins increase the survival of tyrosine hydroxylase-immunoreactive dopaminergic neurons isolated from the <em>embryonic</em> day (E) <em>1</em>4 rat mesencephalon floor to varying extents (TGF-beta s 2.5-fold, activin A and GDNF <em>1</em>.6-fold). TGF-beta s, activin A and GDNF did not augment numbers of very rarely observed astroglial cells visualized by using antibodies to glial fibrillary acidic protein and had no effect on cell proliferation monitored by incorporation of BrdU. TGF-beta <em>1</em> and activin A protected dopaminergic neurons against N-methyl-4-phenylpiridinium ion toxicity. Reverse transcription-polymerase chain reaction (RT-PCR) analysis indicated that TGF-beta 2 mRNA, but not GDNF mRNA, is expressed in the E<em>1</em>4 rat midbrain floor and in mesencephalic cultures. We conclude that TGF-beta s <em>1</em>-3, activin A and GDNF share a neurotrophic capacity for developing dopaminergic neurons, which is not mediated by astroglial cells and not accompanied by an increase in cell proliferation.

Fetal antigen <em>1</em> (FA<em>1</em>) is a circulating EGF multidomain glycoprotein. FA<em>1</em> and its membrane-associated precursor is defined by the mRNAs referred to as delta-like (dlk), preadipocyte <em>factor</em> <em>1</em> (pref-<em>1</em>) or zona glomerulosa-specific <em>factor</em> (ZOG). Using a polyclonal antibody recognising both forms, the localisation of FA<em>1</em>/dlk was analysed in <em>embryonic</em> and fetal tissues between week 5 to 25 of gestation and related to germinal origin and development. FA<em>1</em> was observed in endodermally derived hepatocytes, glandular cells of the pancreas anlage, and in respiratory epithelial cells. FA<em>1</em> was also present in mesodermally derived cells of the renal proximal tubules, adrenal cortex, Leydig and Hilus cells of the testes and ovaries, fetal chondroblasts, and skeletal myotubes. Ectodermally derived neuro- and adenohypophysial cells, cells in the floor of the 3rd ventricle and plexus choroideus were also FA<em>1</em> positive. The number of cells expressing FA<em>1</em> decreased during fetal development where the expression became restricted to specific functional cells. Epidermis, gut epithelium, gall bladder, blood cells, spleen, thyroid gland, salivary glands, and smooth muscle cells were FA<em>1</em> negative. Analysis of extra-<em>embryonic</em> tissues from normal and pathological pregnancies revealed FA<em>1</em> in stromal cells surrounding the blood islands of the yolk sac as well as in placental fibroblasts where the expression was most pronounced in diploid, androgenic complete hydatidiform moles. However, as measured by ELISA, the circulating maternal FA<em>1</em> levels in complete moles were not different from normal pregnancies. The results presented suggest that FA<em>1</em> is a <em>growth</em> and/or <em>differentiation</em> <em>factor</em> extensively expressed in immature cells and down-regulated during fetal development. FA<em>1</em> down-regulation was associated with a shift in the subcellular localisation indicating differential post-translational/post-transcriptional modifications during fetal development. FA<em>1</em> may be a new marker of cellular subtypes with a regenerative potential and of specific cells with endocrine or neuroendocrine functions.

Understanding the tissue interactions that induce pancreatic progenitor cells from the <em>embryonic</em> endoderm provides insights into congenital malformations, tissue repair, and differentiating stem cells to a pancreatic fate. The specification of pancreatic progenitors within the dorsal endodermal epithelium has been thought to involve two phases of mesodermal interactions; first with the lateral plate mesoderm and notochord and then with aortic endothelial cells. Afterwards, branching morphogenesis of the pancreatic bud is induced by Isl-<em>1</em>-positive dorsal mesenchyme cells, whose <em>growth</em> is stimulated by <em>factors</em> in the circulation. Using mouse genetic models and embryo tissue explants, we show that the aortic endothelium and dorsal mesenchyme each possess an additional role in pancreatic induction, prior to the branching morphogenesis step. Specifically, we find that aortic endothelial cells promote the survival of nearby, Isl-<em>1</em>-positive dorsal mesenchyme, independently of <em>factors</em> from the circulation. Furthermore, we find that FGF<em>1</em>0 signaling from the mesenchyme cells maintains Ptf<em>1</em>a expression in the dorsal pancreatic bud and appears genetically redundant with a role for the transcription <em>factor</em> gene HNF6 in promoting the induction of Pdx-<em>1</em>-positive dorsal endoderm. Together, these studies reveal a relay pathway from aortic endothelium to dorsal mesenchyme and then to the endoderm, along with functions of the dorsal mesenchyme that promote the initial <em>differentiation</em> of the dorsal pancreatic endoderm, prior to organ morphogenesis.