Constitutive ablation of the Yin Yang <em>1</em> (YY<em>1</em>) transcription <em>factor</em> in mice results in peri-implantation lethality. In this study, we used homologous recombination to generate knockout mice carrying yy<em>1</em> alleles expressing various amounts of YY<em>1</em>. Phenotypic analysis of yy<em>1</em> mutant embryos expressing approximately 75%, approximately 50%, and approximately 25% of the normal complement of YY<em>1</em> identified a dosage-dependent requirement for YY<em>1</em> during late embryogenesis. Indeed, reduction of YY<em>1</em> levels impairs <em>embryonic</em> <em>growth</em> and viability in a dose-dependent manner. Analysis of the corresponding mouse <em>embryonic</em> fibroblast cells also revealed a tight correlation between YY<em>1</em> dosage and cell proliferation, with a complete ablation of YY<em>1</em> inducing cytokinesis failure and cell cycle arrest. Consistently, RNA interference-mediated inhibition of YY<em>1</em> in HeLa cells prevents cytokinesis, causes proliferative arrest, and increases cellular sensitivity to various apoptotic agents. Genome-wide expression profiling identified a plethora of YY<em>1</em> target genes that have been implicated in cell <em>growth</em>, proliferation, cytokinesis, apoptosis, development, and <em>differentiation</em>, suggesting that YY<em>1</em> coordinates multiple essential biological processes through a complex transcriptional network. These data not only shed new light on the molecular basis for YY<em>1</em> developmental roles and cellular functions, but also provide insight into the general mechanisms controlling eukaryotic cell proliferation, apoptosis, and <em>differentiation</em>.

Hypoxia-inducible <em>factor</em>-<em>1</em> (HIF-<em>1</em>) regulates the transcription of genes whose products play critical roles in energy metabolism, erythropoiesis, angiogenesis, and cell survival. Limited information is available concerning its function in mammalian hematopoiesis. Previous studies have demonstrated that homozygosity for a targeted null mutation in the Hif<em>1</em>alpha gene, which encodes the hypoxia-responsive alpha subunit of HIF-<em>1</em>, causes cardiac, vascular, and neural malformations resulting in lethality by <em>embryonic</em> day <em>1</em>0.5 (E<em>1</em>0.5). This study revealed reduced myeloid multilineage and committed erythroid progenitors in HIF-<em>1</em>alpha-deficient embryos, as well as decreased hemoglobin content in erythroid colonies from HIF-<em>1</em>alpha-deficient yolk sacs at E9.5. Dysregulation of erythropoietin (Epo) signaling was evident from a significant decrease in mRNA levels of Epo receptor (EpoR) in Hif<em>1</em>alpha-/- yolk sac as well as Epo and EpoR mRNA in Hif<em>1</em>alpha-/- embryos. The erythropoietic defects in HIF-<em>1</em>alpha-deficient erythroid colonies could not be corrected by cytokines, such as vascular endothelial <em>growth</em> <em>factor</em> and Epo, but were ameliorated by Fe-SIH, a compound delivering iron into cells independently of iron transport proteins. Consistent with profound defects in iron homeostasis, Hif<em>1</em>alpha-/- yolk sac and/or embryos demonstrated aberrant mRNA levels of hepcidin, Fpn<em>1</em>, Irp<em>1</em>, and frascati. We conclude that dysregulated expression of genes encoding Epo, EpoR, and iron regulatory proteins contributes to defective erythropoiesis in Hif<em>1</em>alpha-/- yolk sacs. These results identify a novel role for HIF-<em>1</em> in the regulation of iron homeostasis and reveal unexpected regulatory <em>differences</em> in Epo/EpoR signaling in yolk sac and <em>embryonic</em> erythropoiesis.

The thiazolidinedione (TZD) class of peroxisome proliferator-activated receptor (PPAR) gamma ligands, known for their ability to induce adipocyte <em>differentiation</em> and increase insulin sensitivity, also exhibits anticancer properties. Currently, TZDs are being tested in clinical trials for treatment of human cancers expressing high levels of PPARgamma because it is assumed that activation of PPARgamma mediates their anticancer activity. Using PPARgamma(-/-) and PPARgamma(+/+) mouse <em>embryonic</em> stem cells, we report here that inhibition of cell proliferation and tumor <em>growth</em> by TZDs is independent of PPARgamma. Our studies demonstrate that these compounds block G(<em>1</em>)-S transition by inhibiting translation initiation. Inhibition of translation initiation is the consequence of partial depletion of intracellular calcium stores and the resulting activation of protein kinase R that phosphorylates the alpha subunit of eukaryotic initiation <em>factor</em> 2 (eIF2), thus rendering eIF2 inactive. PPARgamma-independent inhibition of translation initiation most likely accounts for the anticancer properties of thiazolidinediones.

Inhibins and activins are dimeric <em>growth</em> <em>factors</em> of the transforming <em>growth</em> <em>factor</em>-beta superfamily, a class of peptides that can regulate the <em>growth</em> and <em>differentiation</em> of a variety of cell types. Recently, activins have been implicated in early vertebrate development through their ability to evoke, in Xenopus embryo explants, both morphological and molecular changes characteristic of mesoderm induction. To understand these processes further, we have used homologous recombination in <em>embryonic</em> stem cells to create mouse strains carrying mutations in the gene encoding the activin/inhibin beta B subunit. These mice are expected to be deficient in activin B (beta B:beta B), activin AB (beta A:beta B), and inhibin B (alpha:beta B). Viable mutant animals were generated, indicating that the beta B subunit is not essential for mesoderm formation in the mouse. Mutant animals suffered, however, from distinct developmental and reproductive defects. An apparent failure of eyelid fusion during late <em>embryonic</em> development led to eye lesions in mutant animals. Whereas beta B-deficient males bred normally, mutant females manifested a profoundly impaired reproductive ability, characterized by perinatal lethality of their offspring. The phenotype of mutant mice suggests that activin beta B (<em>1</em>) plays a role in late fetal development and (2) is critical for female fecundity. In addition, we have found that expression of the related beta A subunit of activin is highly upregulated in ovaries of mutant females. Altered regulation of beta A activin in beta B-deficient mice may contribute to the mutant phenotype.

We investigated the potential of mouse <em>embryonic</em> stem (ES) cells to differentiate into hepatocytes in vitro. Differentiating ES cells expressed endodermal-specific genes, such as alpha-fetoprotein, transthyretin, alpha <em>1</em>-anti-trypsin and albumin, when cultured without additional <em>growth</em> <em>factors</em> and late differential markers of hepatic development, such as tyrosine aminotransferase (TAT) and glucose-6-phosphatase (G6P), when cultured in the presence of <em>growth</em> <em>factors</em> critical for late <em>embryonic</em> liver development. Further, induction of TAT and G6P expression was induced regardless of expression of the functional SEK<em>1</em> gene, which is thought to provide a survival signal for hepatocytes during an early stage of liver morphogenesis. The data indicate that the in vitro ES <em>differentiation</em> system has a potential to generate mature hepatocytes. The system has also been found useful in analyzing the role of <em>growth</em> <em>factors</em> and intracellular signaling molecules in hepatic development.

The receptor tyrosine kinase FLK<em>1</em> and the transcription <em>factor</em> SCL play crucial roles in the establishment of hematopoietic and endothelial cell lineages in mice. We have previously used an in vitro <em>differentiation</em> model of <em>embryonic</em> stem (ES) cells and demonstrated that hematopoietic and endothelial cells develop via sequentially generated FLK<em>1</em>(+) and SCL(+) cells. To gain a better understanding of cellular and molecular events leading to hematopoietic specification, we examined <em>factors</em> necessary for FLK<em>1</em>(+) and SCL(+) cell induction in serum-free conditions. We demonstrate that bone morphogenetic protein (BMP) 4 was required for the generation of FLK<em>1</em>(+) and SCL(+) cells, and that vascular endothelial <em>growth</em> <em>factor</em> (VEGF) was necessary for the expansion and <em>differentiation</em> of SCL-expressing hematopoietic progenitors. Consistently, Flk<em>1</em>-deficient ES cells responded to BMP4 and generated TER<em>1</em><em>1</em>9(+) and CD3<em>1</em>(+) cells, but they failed to expand in response to VEGF. The Smad<em>1</em>/5 and map kinase pathways were activated by BMP4 and VEGF, respectively. The overexpression of SMAD6 in ES cells resulted in a reduction of FLK<em>1</em>(+) cells. In addition, a MAP kinase kinase <em>1</em> specific inhibitor blocked the expansion of SCL(+) cells in response to VEGF. Finally, VEGF mediated expansion of hematopoietic and endothelial cell progenitors was inhibited by TGFbeta<em>1</em>, but was augmented by activin A. Our studies suggest that hematopoietic and endothelial commitment from the mesoderm occurs via BMP4-mediated signals and that expansion and/or <em>differentiation</em> of such progenitors is achieved by an interplay of VEGF, TGFbeta<em>1</em> and activin A signaling.

The <em>differentiation</em> of stem cells into smooth muscle cells (SMCs) plays an important role in vascular development and remodeling. In addition, stem cells represent a potential source of SMCs for regenerative medicine applications such as constructing vascular grafts. Previous studies have suggested that various biochemical <em>factors</em>, including transforming <em>growth</em> <em>factor</em>-beta (TGF-beta) and the Notch pathway, may play important roles in vascular <em>differentiation</em>. However, the interactions of these two signaling pathways in the <em>differentiation</em> of bone marrow mesenchymal stem cells (MSCs) have not been clearly defined. In this study, we profiled the gene expression in MSCs in response to TGF-beta, and showed that TGF-beta induced Notch ligand Jagged <em>1</em> (JAG<em>1</em>) and SMC markers, including smooth muscle alpha-actin (ACTA2), calponin <em>1</em> (CNN<em>1</em>), and myocardin (MYOCD), which were dependent on the activation of SMAD3 and Rho kinase. In addition, knocking down JAG<em>1</em> expression partially blocked ACTA2 and CNN<em>1</em> expression and completely blocked MYOCD expression, suggesting that JAG<em>1</em> plays an important role in TGF-beta-induced expression of SMC markers. On the other hand, the activation of Notch signaling induced the expression of SMC markers in MSCs and human <em>embryonic</em> stem cells (hESCs). Notch activation in hESCs also resulted in an increase of neural markers and a decrease of endothelial markers. These results suggest that Notch signaling mediates TGF-beta regulation of MSC <em>differentiation</em> and that Notch signaling induces the <em>differentiation</em> of MSCs and hESCs into SMCs, which represents a novel mechanism involved in stem cell <em>differentiation</em>.

BACKGROUND

MicroRNAs (miRNAs) are a newly discovered endogenous class of small, noncoding RNAs that play important posttranscriptional regulatory roles by targeting messenger RNAs for cleavage or translational repression. Human embryonic stem cells are known to express miRNAs that are often undetectable in adult organs, and a growing body of evidence has implicated miRNAs as important arbiters of heart development and disease.

RESULTS

To better understand the transition between the human embryonic and cardiac "miRNA-omes," we report here the first miRNA profiling study of cardiomyocytes derived from human embryonic stem cells. Analyzing 711 unique miRNAs, we have identified several interesting miRNAs, including miR-1, -133, and -208, that have been previously reported to be involved in cardiac development and disease and that show surprising patterns of expression across our samples. We also identified novel miRNAs, such as miR-499, that are strongly associated with cardiac differentiation and that share many predicted targets with miR-208. Overexpression of miR-499 and -1 resulted in upregulation of important cardiac myosin heavy-chain genes in embryoid bodies; miR-499 overexpression also caused upregulation of the cardiac transcription factor MEF2C.

CONCLUSIONS

Taken together, our data give significant insight into the regulatory networks that govern human embryonic stem cell differentiation and highlight the ability of miRNAs to perturb, and even control, the genes that are involved in cardiac specification of human embryonic stem cells.

We present the current form of a provisional DNA sequence-based regulatory gene network that explains in outline how endomesodermal specification in the sea urchin embryo is controlled. The model of the network is in a continuous process of revision and <em>growth</em> as new genes are added and new experimental results become available; see http://www.its.caltech.edu/~mirsky/endomeso.htm (End-mes Gene Network Update) for the latest version. The network contains over 40 genes at present, many newly uncovered in the course of this work, and most encoding DNA-binding transcriptional regulatory <em>factors</em>. The architecture of the network was approached initially by construction of a logic model that integrated the extensive experimental evidence now available on endomesoderm specification. The internal linkages between genes in the network have been determined functionally, by measurement of the effects of regulatory perturbations on the expression of all relevant genes in the network. Five kinds of perturbation have been applied: (<em>1</em>) use of morpholino antisense oligonucleotides targeted to many of the key regulatory genes in the network; (2) transformation of other regulatory <em>factors</em> into dominant repressors by construction of Engrailed repressor domain fusions; (3) ectopic expression of given regulatory <em>factors</em>, from genetic expression constructs and from injected mRNAs; (4) blockade of the beta-catenin/Tcf pathway by introduction of mRNA encoding the intracellular domain of cadherin; and (5) blockade of the Notch signaling pathway by introduction of mRNA encoding the extracellular domain of the Notch receptor. The network model predicts the cis-regulatory inputs that link each gene into the network. Therefore, its architecture is testable by cis-regulatory analysis. Strongylocentrotus purpuratus and Lytechinus variegatus genomic BAC recombinants that include a large number of the genes in the network have been sequenced and annotated. Tests of the cis-regulatory predictions of the model are greatly facilitated by interspecific computational sequence comparison, which affords a rapid identification of likely cis-regulatory elements in advance of experimental analysis. The network specifies genomically encoded regulatory processes between early cleavage and gastrula stages. These control the specification of the micromere lineage and of the initial veg(2) endomesodermal domain; the blastula-stage separation of the central veg(2) mesodermal domain (i.e., the secondary mesenchyme progenitor field) from the peripheral veg(2) endodermal domain; the stabilization of specification state within these domains; and activation of some downstream <em>differentiation</em> genes. Each of the temporal-spatial phases of specification is represented in a subelement of the network model, that treats regulatory events within the relevant <em>embryonic</em> nuclei at particular stages.

Human <em>embryonic</em> stem cells (hESCs) have enormous potential as a source of cells for cell replacement therapies and as a model for early human development. In this study we examined the differentiating potential of hESCs into hepatocytes in two- and three-dimensional (2D and 3D) culture systems. Embryoid bodies (EBs) were inserted into a collagen scaffold 3D culture system or cultured on collagen-coated dishes and stimulated with exogenous <em>growth</em> <em>factors</em> to induce hepatic histogenesis. Immunofluorescence analysis revealed the expression of albumin (ALB) and cytokeratin-<em>1</em>8 (CK-<em>1</em>8). The differentiated cells in 2D and 3D culture system displayed several characteristics of hepatocytes, including expression of transthyretin, alpha-<em>1</em>-antitrypsin, cytokeratin 8, <em>1</em>8, <em>1</em>9, tryptophan-2,3-dioxygenase, tyrosine aminotransferase, glucose-6-phosphatase (G6P), cytochrome P450 subunits 7a<em>1</em> and secretion of alpha-fetoprotein (AFP) and ALB and production of urea. In 3D culture, ALB and G6P were detected earlier and higher levels of urea and AFP were produced, when compared with 2D culture. Electron microscopy of differentiated hESCs showed hepatocyte-like ultrastructure, including glycogon granules, well-developed Golgi apparatuses, rough and smooth endoplasmic reticuli and intercellular canaliculi. The <em>differentiation</em> of hESCs into hepatocyte-like cells within 3D collagen scaffolds containing exogenous <em>growth</em> <em>factors</em>, gives rise to cells displaying morphological features, gene expression patterns and metabolic activities characteristic of hepatocytes and may provide a source of differentiated cells for treatment of liver diseases.

The murine Vgr-<em>1</em> (Vg-related) and BMP-2a (bone morphogenetic protein 2a) genes are members of the decapentaplegic subgroup of the transforming <em>growth</em> <em>factor</em>-beta (TGF beta) superfamily. Although genetic and biochemical studies suggest that the members of this subgroup play important roles in development, little is known about their function in mammals. Therefore, we investigated the expression of Vgr-<em>1</em> and BMP-2a RNAs in <em>embryonic</em>, newborn, and adult tissues by in situ hybridization. Vgr-<em>1</em> RNA is maternally encoded in ovarian oocytes but declines in fertilized eggs and is undectable by the two- to four-cell stage. Only low levels of transcripts are seen in blastocysts and early postimplantation stages. From mid-gestation on, Vgr-<em>1</em> RNA is expressed at high levels in developing skin, especially in the suprabasal cells of the proliferating epidermis but not in the dermis or hair follicles, both of which contain TGF beta <em>1</em> and/or TGF beta 2 RNAs. In contrast, BMP-2a transcripts are seen only in the hair follicles in the cells of the hair bulb cortex. Temporally and spatially distinct patterns of BMP-2a, Vgr-<em>1</em>, TGF beta <em>1</em>, and TGF beta 2 expression are also seen in different populations of mesenchymal cells in the developing skeletal system (cartilage and bone). Our results suggest that the coordinated expression of several members of the TGF beta superfamily is required to control the progression of specific cell types through their <em>differentiation</em> pathways.

Semaphorin 3A (Sema3A) is a secreted <em>factor</em> known to guide axon/dendrite <em>growth</em> and neuronal migration. We found that it also acts as a polarizing <em>factor</em> for axon/dendrite development in cultured hippocampal neurons. Exposure of the undifferentiated neurite to localized Sema3A suppressed its <em>differentiation</em> into axon and promoted dendrite formation, resulting in axon formation away from the Sema3A source, and bath application of Sema3A to polarized neurons promoted dendrite <em>growth</em> but suppressed axon <em>growth</em>. Fluorescence resonance energy transfer (FRET) imaging showed that Sema3A elevated the cGMP but reduced cAMP and protein kinase A (PKA) activity, and its axon suppression is attributed to the downregulation of PKA-dependent phosphorylation of axon determinants LKB<em>1</em> and GSK-3β. Downregulating Sema3A signaling in rat <em>embryonic</em> cortical progenitors via in utero electroporation of siRNAs against the Sema3A receptor neuropilin-<em>1</em> also resulted in polarization defects in vivo. Thus, Sema3A regulates the earliest step of neuronal morphogenesis by polarizing axon/dendrite formation.

Signaling through the type <em>1</em> IGF receptor (IGF<em>1</em>R) after interaction with IGF-I is crucial to the normal brain development. Manipulations of the mouse genome leading to changes in the expression of IGF-I or IGF<em>1</em>R significantly alters brain <em>growth</em>, such that IGF-I overexpression leads to brain over<em>growth</em>, whereas null mutations in either IGF-I or the IGF<em>1</em>R result in brain <em>growth</em> retardation. IGF-I signaling stimulates the proliferation, survival, and <em>differentiation</em> of each of the major neural lineages, neurons, oligodendrocytes, and astrocytes, as well as possibly influencing neural stem cells. During <em>embryonic</em> life, IGF-I stimulates neuron progenitor proliferation, whereas later it promotes neuron survival, neuritic out<em>growth</em>, and synaptogenesis. IGF-I also stimulates oligodendrocyte progenitor proliferation although inhibiting apoptosis in oligodendrocyte lineage cells and stimulating myelin production. These pleiotropic IGF-I activities indicate that other <em>factors</em> provide instructive signals for specific cellular events and that IGF-I acts to facilitate them. Studies of the few humans with IGF-I and/or IGF<em>1</em>R gene mutations indicate that IGF-I serves a similar role in man.

To date, three closely-related TGF beta genes have been found in the mouse; TGF beta <em>1</em>, TGF beta 2 and TGF beta 3. Previous experiments have indicated that TGF beta <em>1</em> and TGF beta 2 may play important roles during mouse embryogenesis. The present study now reports the distribution of transcripts of TGF beta 3 in comparison to the other two genes and reveals overlapping but distinct patterns of RNA expression. TGF beta 3 RNA is expressed in a diverse array of tissues including perichondrium, bone, intervertebral discs, mesenteries, pleura, heart, lung, palate, and amnion, as well as in central nervous system (CNS) structures such as the meninges, choroid plexus and the ol<em>factor</em>y bulbs. Furthermore, in several organ systems, TGF beta 3 transcripts are expressed during periods of active morphogenesis suggesting that the protein may be an important <em>factor</em> for the <em>growth</em> and <em>differentiation</em> of many <em>embryonic</em> tissues.

Transforming <em>growth</em> <em>factor</em>-beta (TGF-beta) stimulates collagen synthesis and accumulation, and aberrant TGF-beta signaling is implicated in pathological organ fibrosis. Regulation of type I procollagen gene (COL<em>1</em>A2) transcription by TGF-beta involves the canonical Smad signaling pathway as well as additional protein and lipid kinases, coactivators, and DNA-binding transcription <em>factors</em> that constitute alternate non-Smad pathways. By using Affymetrix microarrays to detect cellular genes whose expression is regulated by Smad3, we identified early <em>growth</em> response <em>factor</em>-<em>1</em> (EGR-<em>1</em>) as a novel Smad3-inducible gene. Previous studies implicated Egr-<em>1</em> in cell <em>growth</em>, <em>differentiation</em>, and survival. We found that TGF-beta induced rapid and transient accumulation of Egr-<em>1</em> protein and mRNA in human skin fibroblasts. In transient transfection assays, TGF-beta stimulated the activity of the Egr-<em>1</em> gene promoter, as well as that of a minimal Egr-<em>1</em>-responsive reporter construct. Furthermore, TGF-beta enhanced endogenous Egr-<em>1</em> interaction with a consensus Egr-<em>1</em>-binding site element and with GC-rich DNA sequences of the human COL<em>1</em>A2 promoter in vitro and in vivo. Forced expression of Egr-<em>1</em> by itself caused dose-dependent up-regulation of COL<em>1</em>A2 promoter activity and further enhanced the stimulation induced by TGF-beta. In contrast, the TGF-beta response was abrogated when the Egr-<em>1</em>-binding sites of the COL<em>1</em>A2 promoter were mutated or deleted. Furthermore, Egr-<em>1</em>-deficient <em>embryonic</em> mouse fibroblasts showed attenuated TGF-beta responses despite intact Smad activation, and forced expression of ectopic EGR-<em>1</em> in these cells could restore COL<em>1</em>A2 stimulation in a dose-dependent manner. Taken together, these findings identify Egr-<em>1</em> as a novel intracellular TGF-beta target that is necessary for maximal stimulation of collagen gene expression in fibroblasts. The results therefore implicate Egr-<em>1</em> in the profibrotic responses elicited by TGF-beta and suggest that Egr-<em>1</em> may play a new and important role in the pathogenesis of fibrosis.

Notch signaling has been shown to regulate various aspects of vascular development. However, a specific role of the ligand Delta-like <em>1</em> (DLL<em>1</em>) has not been shown thus far. Here, we demonstrate that during fetal development, DLL<em>1</em> is an essential Notch ligand in the vascular endothelium of large arteries to activate Notch<em>1</em> and maintain arterial identity. DLL<em>1</em> was detected in fetal arterial endothelial cells beginning at <em>embryonic</em> day <em>1</em>3.5. While DLL4-mediated activation has been shown to suppress vascular endothelial <em>growth</em> <em>factor</em> (VEGF) pathway components in <em>growing</em> capillary beds, DLL<em>1</em>-Notch signaling was required for VEGF receptor expression in fetal arteries. In the absence of DLL<em>1</em> function, VEGF receptor 2 (VEGFR2) and its coreceptor, neuropilin-<em>1</em> (NRP<em>1</em>), were down-regulatedin mutant arteries, which was followed by up-regulation of chicken ovalbumin upstream promoter-transcription <em>factor</em> II (COUP-TFII), a repressor of arterial <em>differentiation</em> and Nrp<em>1</em> expression in veins. Consistent with a positive modulation of the VEGF pathway by DLL<em>1</em>, the Nrp<em>1</em> promoter contains several recombinant signal-binding protein <em>1</em> for J kappa (RBPJkappa)-binding sites and was responsive to Notch activity in cell culture. Our results establish DLL<em>1</em> as a critical endothelial Notch ligand required for maintaining arterial identity during mouse fetal development and suggest context-dependent interrelations of the VEGFA and Notch signaling pathways.

Partially purified extracts from newborn calf articular cartilage were found to induce cartilage and bone when subcutaneously implanted in rats. This activity showed characteristics of bone morphogenetic proteins (BMPs). Degenerate oligonucleotide primer sets derived from the highly conserved carboxyl-terminal region of the BMP family were designed and used in reverse transcription-polymerase chain reactions with poly(A)+ RNA from articular cartilage as template to determine which BMPs are produced by chondrocytes. Two novel members of the transforming <em>growth</em> <em>factor</em>-beta (TGF-beta) superfamily were identified and designated cartilage-derived morphogenetic protein-<em>1</em> (CDMP-<em>1</em>) and -2 (CDMP-2). Their carboxyl-terminal TGF-beta domains are 82% identical, thus defining a novel subfamily most closely related to BMP-5, BMP-6, and osteogenic protein-<em>1</em>. Northern analyses showed that both genes are predominantly expressed in cartilaginous tissues. In situ hybridization and immunostaining of sections from human embryos showed that CDMP-<em>1</em> was predominantly found at the stage of precartilaginous mesenchymal condensation and throughout the cartilaginous cores of the developing long bones, whereas CDMP-2 expression was restricted to the hypertrophic chondrocytes of ossifying long bone centers. Neither gene was detectable in the axial skeleton during human <em>embryonic</em> development. The cartilage-specific localization pattern of these novel TGF-beta superfamily members, which contrasts with the more ubiquitous presence of other BMP family members, suggests a potential role for these proteins in chondrocyte <em>differentiation</em> and <em>growth</em> of long bones.

Early mammalian embryogenesis is controlled by mechanisms governing the balance between pluripotency and <em>differentiation</em>. The expression of early lineage-specific genes can vary significantly between species, with implications for developmental control and stem cell derivation. However, the mechanisms involved in patterning the human embryo are still unclear. We analyzed the appearance and localization of lineage-specific transcription <em>factors</em> in staged preimplantation human embryos from the zygote until the blastocyst. We observed that the pluripotency-associated transcription <em>factor</em> OCT4 was initially expressed in 8-cell embryos at 3 days post-fertilization (dpf), and restricted to the inner cell mass (ICM) in <em>1</em>28-256 cell blastocysts (6dpf), approximately 2 days later than the mouse. The trophectoderm (TE)-associated transcription <em>factor</em> CDX2 was upregulated in 5dpf blastocysts and initially coincident with OCT4, indicating a lag in CDX2 initiation in the TE lineage, relative to the mouse. Once established, the TE expressed intracellular and cell-surface proteins cytokeratin-7 (CK7) and fibroblast <em>growth</em> <em>factor</em> receptor-<em>1</em> (FGFR<em>1</em>), which are thought to be specific to post-implantation human trophoblast progenitor cells. The primitive endoderm (PE)-associated transcription <em>factor</em> SOX<em>1</em>7 was initially heterogeneously expressed in the ICM where it co-localized with a sub-set of OCT4 expressing cells at 4-5dpf. SOX<em>1</em>7 was progressively restricted to the PE adjacent to the blastocoel cavity together with the transcription <em>factor</em> GATA6 by 6dpf. We observed low levels of Laminin expression in the human PE, though this basement membrane component is thought to play an important role in mouse PE cell sorting, suggesting divergence in <em>differentiation</em> mechanisms between species. Additionally, while stem cell lines representing the three distinct cell types that comprise a mouse blastocyst have been established, the identity of cell types that emerge during early human <em>embryonic</em> stem cell derivation is unclear. We observed that derivation from plating intact human blastocysts resulted predominantly in the out<em>growth</em> of TE-like cells, which impairs human <em>embryonic</em> stem cell derivation. Altogether, our findings provide important insight into developmental patterning of preimplantation human embryos with potential consequences for stem cell derivation.

Human erythropoiesis is a complex multistep developmental process that begins at the level of pluripotent hematopoietic stem cells (HSCs) at bone marrow microenvironment (HSCs niche) and terminates with the production of erythrocytes (RBCs). This review covers the basic and contemporary aspects of erythropoiesis. These include the: (a) cell-lineage restricted pathways of <em>differentiation</em> originated from HSCs and going downward toward the blood cell development; (b) model systems employed to study erythropoiesis in culture (erythroleukemia cell lines and <em>embryonic</em> stem cells) and in vivo (knockout animals: avian, mice, zebrafish, and xenopus); (c) key regulators of erythropoiesis (iron, hypoxia, stress, and <em>growth</em> <em>factors</em>); (d) signaling pathways operating at hematopoietic stem cell niche for homeostatic regulation of self renewal (SCF/c-kit receptor, Wnt, Notch, and Hox) and for erythroid <em>differentiation</em> (HIF and EpoR). Furthermore, this review presents the mechanisms through which transcriptional <em>factors</em> (GATA-<em>1</em>, FOG-<em>1</em>, TAL-<em>1</em>/SCL/MO2/Ldb<em>1</em>/E2A, EKLF, Gfi-<em>1</em>b, and BCL<em>1</em><em>1</em>A) and miRNAs regulate gene pattern expression during erythroid <em>differentiation</em>. New insights regarding the transcriptional regulation of alpha- and beta-globin gene clusters were also presented. Emphasis was also given on (i) the developmental program of erythropoiesis, which consists of commitment to terminal erythroid maturation and hemoglobin production, (two closely coordinated events of erythropoieis) and (ii) the capacity of human <em>embryonic</em> and umbilical cord blood (UCB) stem cells to differentiate and produce RBCs in culture with highly selective media. These most recent developments will eventually permit customized red blood cell production needed for transfusion.

Reendothelialization involves endothelial progenitor cell (EPC) homing, proliferation, and <em>differentiation</em>, which may be influenced by fluid shear stress and local flow pattern. This study aims to elucidate the role of laminar flow on <em>embryonic</em> stem (ES) cell <em>differentiation</em> and the underlying mechanism. We demonstrated that laminar flow enhanced ES cell-derived progenitor cell proliferation and <em>differentiation</em> into endothelial cells (ECs). Laminar flow stabilized and activated histone deacetylase 3 (HDAC3) through the Flk-<em>1</em>-PI3K-Akt pathway, which in turn deacetylated p53, leading to p2<em>1</em> activation. A similar signal pathway was detected in vascular endothelial <em>growth</em> <em>factor</em>-induced EC <em>differentiation</em>. HDAC3 and p2<em>1</em> were detected in blood vessels during embryogenesis. Local transfer of ES cell-derived EPC incorporated into injured femoral artery and reduced neointima formation in a mouse model. These data suggest that shear stress is a key regulator for stem cell <em>differentiation</em> into EC, especially in EPC <em>differentiation</em>, which can be used for vascular repair, and that the Flk-<em>1</em>-PI3K-Akt-HDAC3-p53-p2<em>1</em> pathway is crucial in such a process.

Cardiotrophin-<em>1</em> (CT-<em>1</em>) is a newly isolated cytokine that was identified based on its ability to induce cardiac myocyte hypertrophy. It is a member of the family of cytokines that includes interleukins-6 and -<em>1</em><em>1</em>, leukemia inhibitory <em>factor</em> (LIF), ciliary neurotrophic <em>factor</em>, and oncostatin M. These cytokines induce a pleiotropic set of <em>growth</em> and <em>differentiation</em> activities via receptors that use a common signaling subunit, gp<em>1</em>30. In this work we determine the activity of CT-<em>1</em> in six in vitro biological assays and examine the composition of its cell surface receptor. We find that CT-<em>1</em> is inactive in stimulating the <em>growth</em> of the hybridoma cell line, B9 and inhibits the <em>growth</em> of the mouse myeloid leukemia cell line, M<em>1</em>. CT-<em>1</em> induces a phenotypic switch in rat sympathetic neurons and promotes the survival of rat dopaminergic and chick ciliary neurons. CT-<em>1</em> also inhibits the <em>differentiation</em> of mouse <em>embryonic</em> stem cells. CT-<em>1</em> and LIF cross-compete for binding to M<em>1</em> cells, Kd [CT-<em>1</em>] approximately 0.7 nM, and this binding is inhibited by an anti-gp<em>1</em>30 monoclonal antibody. Both ligands can be specifically cross-linked to a protein on M<em>1</em> cells with the mobility of the LIF receptor (approximately 200 kDa). In addition, CT-<em>1</em> binds directly to a purified, soluble form of the LIF receptor in solution (Kd approximately 2 nM). These data show that CT-<em>1</em> has a wide range of hematopoietic, neuronal, and developmental activities and that it can act via the LIF receptor and the gp<em>1</em>30 signaling subunit.

<em>Differentiation</em> of mouse <em>embryonic</em> stem (ES) cells via embryoid bodies was established as a suitable model to study development in vitro. Here, we show that <em>differentiation</em> of ES cells in vitro into chondrocytes can be modulated by members of the transforming <em>growth</em> <em>factor</em>-beta family (TGF-beta(<em>1</em>), BMP-2 and -4). ES cell <em>differentiation</em> into chondrocytes was characterized by the appearance of Alcian blue-stained areas and the expression of cartilage-associated genes and proteins. Different stages of cartilage <em>differentiation</em> could be distinguished according to the expression pattern of the transcription <em>factor</em> scleraxis, and the cartilage matrix protein collagen II. The number of Alcian-blue-stained areas decreased slightly after application of TGF-beta(<em>1</em>), whereas BMP-2 or -4 induced chondrogenic <em>differentiation</em>. The inducing effect of BMP-2 was found to be dependent on the time of application, consistent with its role to recruit precursor cells to the chondrogenic fate.

Prolonged propagation of human <em>embryonic</em> stem (hES) cells is currently achieved by coculture with primary mouse <em>embryonic</em> fibroblasts (MEFs) serving as feeder cells. Unlike mouse ES cells, adding <em>growth</em> <em>factors</em> such as leukemia inhibitory <em>factor</em> is insufficient to maintain undifferentiated hES cells without feeder cells. The presence of uncharacterized rodent cells or crude extracts imposes a risk to the clinical applications of hES cells. While others looked for a replacement of MEFs with human fetal cells, we attempted to use easily accessible postnatal human cells such as human marrow stromal cells (hMSCs). Culture-expanded hMSCs from multiple donors were used as feeder cells to support <em>growth</em> of the H<em>1</em> hES cell line under a serum-free culture condition. Human ES cell colonies cultured on irradiated hMSCs amplified>><em>1</em>00-fold during the 30-day continuous culture (in five passages). The longest continuous expansion of hES cells on hMSCs tested to date is <em>1</em>3 passages. The expanded hES cells displayed the unique morphology and molecular markers characteristic of undifferentiated hES cells as observed when they were cultured on MEFs. They expressed the transcription <em>factor</em> Oct-4, a membrane alkaline phosphatase, and the stage-specific <em>embryonic</em> antigen (SSEA)-4, but not the SSEA-<em>1</em> marker. Expanded hES cells on hMSCs retained unique <em>differentiation</em> potentials in culture and a normal diploid karyotype. The well-studied hMSCs (and this animal cell- and serum-free system) may provide a clinically and ethically feasible method to expand hES cells for novel cell therapies. In addition, this system may help to identify cytokines and adhesion molecules that are required for the self-renewal of hES cells.

We propose a new methodology to enhance the vascular <em>differentiation</em> of human <em>embryonic</em> stem cells (hESCs) by encapsulation in a bioactive hydrogel. hESCs were encapsulated in a dextran-based hydrogel with or without immobilized regulatory <em>factors</em>: a tethered RGD peptide and microencapsulated VEGF(<em>1</em>65). The fraction of cells expressing vascular endothelial <em>growth</em> <em>factor</em> (VEGF) receptor KDR/Flk-<em>1</em>, a vascular marker, increased up to 20-fold, as compared to spontaneously differentiated embryoid bodies (EBs). The percentage of encapsulated cells in hydrogels with regulatory <em>factors</em> expressing ectodermal markers including nestin or endodermal markers including alpha-fetoprotein decreased 2- or 3-fold, respectively, as compared to EBs. When the cells were removed from these networks and cultured in media conditions conducive for further vascular <em>differentiation</em>, the number of vascular cells was higher than the number obtained through EBs, using the same media conditions. Functionalized dextran-based hydrogels could thus enable derivation of vascular cells in large quantities, particularly endothelial cells, for potential application in tissue engineering and regenerative medicine.