Exciting studies involving the molecular regulation of lymphangiogenesis in lymphatic-associated disorders (e.g., wound healing, lymphedema and tumor metastasis) have focused renewed attention on the intrinsic relationship between lymphatic endothelial cells (LECs) and extracellular matrix (ECM) microenvironment. ECM molecules and remodeling events play a key role in regulating lymphangiogenesis, and the "functionality"-relating molecules, especially hyaluronan, integrins, reelin, IL-7, and matrix metalloproteinases, provide the most fundamental and critical prerequisite for LEC <em>growth</em>, migration, tube formation, and survival, although lymphangiogenesis is directly or/and indirectly controlled by VEGF-C/-D/VEGFR- 3- Prox-<em>1</em>-, Syk/SLP76-, podoplanin/Ang-2/Nrp-2-, FOXC2-, and other signaling pathways in <em>embryonic</em> and pathological processes. New knowledge regarding the <em>differentiation</em> of initial lymphatics should enable improvements in understanding of a variety of cytokines, chemokines, and other <em>factors</em>. The lymphatic colocalization with histochemical staining by using the novel molecular markers (e.g., LYVE-<em>1</em>), along with subsequent injection technique with ferritin or some tracer, will reveal functional and structural features of newly formed and preexisting lymphatics. <em>Growing</em> recognition of the multiple functions of ECM and LEC molecules for important physiological and pathological events may be helpful in identifying the crucial changes in tissues subjected to lymph circulation and ultimately in the search for rational therapeutic approaches to prevent lymphatic-associated disorders.

The rat optic nerve contains three types of macroglial cells: type <em>1</em> astrocytes first appear at <em>embryonic</em> day <em>1</em>6 (E<em>1</em>6), oligodendrocytes at birth (E2<em>1</em>), and type 2 astrocytes between postnatal days 7 and <em>1</em>0. The oligodendrocytes and type 2 astrocytes develop from a common, bipotential O-2A progenitor cell. We show here that although O-2A progenitor cells in E<em>1</em>7 optic nerve prematurely stop dividing and differentiate into oligodendrocytes within 2 days in culture, when cultured on a monolayer of type <em>1</em> astrocytes, they continue to proliferate; moreover, the first cells differentiate into oligodendrocytes after 4 days in vitro, which is equivalent to the time that oligodendrocytes first appear in vivo. Our findings suggest that the timing of oligodendrocyte <em>differentiation</em> depends on an intrinsic clock in the O-2A progenitor cell that counts cell divisions that are driven by a <em>growth</em> <em>factor</em> (or <em>factors</em>) produced by type <em>1</em> astrocytes.

Genetic studies in mice and humans have revealed a pivotal function for transforming <em>growth</em> <em>factor</em>-beta (TGF-β) in vascular development and maintenance of vascular homeostasis. Mice deficient for various TGF-β signaling components develop an <em>embryonic</em> lethality due to vascular defects. In patients, mutations in TGF-β receptors have been linked to vascular dysplasia like Hereditary Hemorrhagic Telangiectasia (HHT) and pulmonary arterial hypertension (PAH). Besides indirect effects by regulating the expression of angiogenic regulators, TGF-β also has potent direct effects on endothelial cell <em>growth</em> and migration, and we have proposed that TGF-β regulates the activation state of the endothelium via two opposing type I receptor/Smad pathways, activin receptor-like kinase (ALK)<em>1</em> and ALK5. TGF-β is also critical for the <em>differentiation</em> of mural precursors into pericytes and smooth muscle cells. Furthermore, defective paracrine TGF-β signaling between endothelial and neighboring mural cells may be responsible for a leaky vessel phenotype that is characteristic of HHT. In this review, we discuss our current understanding of the TGF-β signaling pathway and its regulation of endothelial and vascular smooth muscle cell function.

Human <em>embryonic</em> stem cells (hESC) can proliferate indefinitely while retaining the capacity to form derivatives of all three germ layers. We have reported previously that hESC differentiate into cardiomyocytes when cocultured with a visceral endoderm-like cell line (END-2). Insulin/insulin-like <em>growth</em> <em>factors</em> and their intracellular downstream target protein kinase Akt are known to protect many cell types from apoptosis and to promote proliferation, including hESC-derived cardiomyocytes. Here, we show that in the absence of insulin, a threefold increase in the number of beating areas was observed in hESC/END-2 coculture. In agreement, the addition of insulin strongly inhibited cardiac <em>differentiation</em>, as evidenced by a significant reduction in beating areas, as well as in alpha-actinin and beta-myosin heavy chain (beta-MHC)-expressing cells. Real-time reverse transcription-polymerase chain reaction and Western blot analysis showed that insulin inhibited cardiomyogenesis in the early phase of coculture by suppressing the expression of endoderm (Foxa2, GATA-6), mesoderm (brachyury T), and cardiac mesoderm (Nkx2.5, GATA-4). In contrast to previous reports, insulin was not sufficient to maintain hESC in an undifferentiated state, since expression of the pluripotency markers Oct3/4 and nanog declined independently of the presence of insulin during coculture. Instead, insulin promoted the expression of neuroectodermal markers. Since insulin triggered sustained phosphorylation of Akt in hESC, we analyzed the effect of an Akt inhibitor during coculture. Indeed, the inhibition of Akt or insulin-like <em>growth</em> <em>factor</em>-<em>1</em> receptor reversed the insulin-dependent effects. We conclude that in hESC/END-2 cocultures, insulin does not prevent <em>differentiation</em> but favors the neuroectodermal lineage at the expense of mesendodermal lineages.

Cells derived from the epicardium are required for coronary vessel development. Transforming <em>growth</em> <em>factor</em> beta (TGFbeta) induces loss of epithelial character and smooth muscle <em>differentiation</em> in chick epicardial cells. Here, we show that epicardial explants from <em>embryonic</em> day (E) <em>1</em><em>1</em>.5 mouse embryos incubated with TGFbeta<em>1</em> or TGFbeta2 lose epithelial character and undergo smooth muscle <em>differentiation</em>. To further study TGFbeta Signaling, we generated immortalized mouse epicardial cells. Cells from E<em>1</em>0.5, <em>1</em><em>1</em>.5, and <em>1</em>3.5 formed tightly packed epithelium and expressed the epicardial marker Wilm's tumor <em>1</em> (WT<em>1</em>). TGFbeta induced the loss of zonula occludens-<em>1</em> (ZO-<em>1</em>) and the appearance of SM22alpha and calponin consistent with smooth muscle <em>differentiation</em>. Inhibition of activin receptor-like kinase (ALK) 5 or p<em>1</em>60 rho kinase activity prevented the effects of TGFbeta while inhibition of p38 mitogen activated protein (MAP) kinase did not. These data demonstrate that TGFbeta induces epicardial cell <em>differentiation</em> and that immortalized epicardial cells provide a suitable model for <em>differentiation</em>.

<em>Embryonic</em> submandibular salivary gland (SMG) initiation and branching morphogenesis are dependent on cell-cell communications between and within epithelium and mesenchyme. Such communications are typically mediated in other organs (teeth, lung, lacrimal glands) by <em>growth</em> <em>factors</em> in such a way as to translate autocrine, juxtacrine and paracrine signals into specific gene responses regulating cell division and histodifferentiation. Using Wnt<em>1</em>-Cre/R26R transgenic mice, we demonstrate that <em>embryonic</em> SMG mesenchyme is derived exclusively from cranial neural crest. This origin contrasts to that known for tooth mesenchyme, previously shown to be derived from both neural crest and nonneural crest cells. Thus, although both SMGs and teeth are mandibular derivatives, we can expect overlap and <em>differences</em> in the details of their early inductive interactions. In addition, since <em>embryonic</em> SMG branching morphogenesis is analogous to that seen in other branching organs, we also expect similarities of expression regarding those molecules known to be ubiquitous regulators of morphogenesis. In this study, we performed an analysis of the distribution of specific fibroblast <em>growth</em> <em>factors</em> (FGFs), FGF receptors, bone morphogenetic proteins (BMPs) and Pax transcription <em>factors</em>, previously shown to be important for tooth development and/or branching morphogenesis, from the time of initiation of <em>embryonic</em> SMG development until early branching morphogenesis. In addition, we report abnormal SMG phenotypes in FgfR2- IIIc(+/Delta), BMP7(-/-) and Pax6(-/-) mice. Our results, in comparison with functional studies in other systems, suggest that FGF-2/FGFR-<em>1</em>, FGF-8/FGFR-2(IIIc) and FGF-<em>1</em>0/FGFR-2(IIIb) signaling have different paracrine and juxtacrine functions during SMG initial bud formation and branching. Finally, our observations of abnormal SMGs in BMP7(-/-) and Pax6(-/-) indicate that both BMP7 and Pax6 play important roles during <em>embryonic</em> SMG branching morphogenesis.

Vascular cells provide a neural stem/progenitor cell (NSPC) niche that regulates expansion and <em>differentiation</em> of NSPCs within the germinal zones of the <em>embryonic</em> and adult brain under both physiologic and pathologic conditions. Here, we examined the NSPC-endothelial cell (NSPC/EC) interaction under conditions of ischemia, both in vitro and after intracerebral transplantation. In culture, <em>embryonic</em> mouse NSPCs supported capillary morphogenesis and protected ECs from cell death induced by serum starvation or by transient oxygen and glucose deprivation (OGD). Neural stem/progenitor cells constitutively expressed hypoxia-inducible <em>factor</em> <em>1</em>alpha (HIF-<em>1</em>alpha) transcription <em>factor</em> and vascular endothelial <em>growth</em> <em>factor</em> (VEGF), both of which were increased approximately twofold after the exposure of NSPCs to OGD. The protective effects of NSPCs on ECs under conditions of serum starvation and hypoxia were blocked by pharmacological inhibitors of VEGF signaling, SU<em>1</em>498 and Flt-<em>1</em>-Fc. After intracerebral transplantation, NSPCs continued to express HIF-<em>1</em>alpha and VEGF, and promoted microvascular density after focal ischemia. These studies support a role for NSPCs in stabilization of vasculature during ischemia, mediated via HIF-<em>1</em>alpha-VEGF signaling pathways, and suggest therapeutic application of NSPCs to promote revascularization and repair after brain injury.

The availability of human cardiomyocytes derived from <em>embryonic</em> stem cells (ESCs) has generated -considerable excitement, as these cells are an excellent model system for studying myocardial development and may have eventual application in cell-based cardiac repair. Cardiomyocytes derived from the related induced pluripotent stem cells (iPSCs) have similar properties, but also offer the prospects of patient-specific disease modeling and cell therapies. Unfortunately, the methods by which cardiomyocytes have been historically generated from pluripotent stem cells are unreliable and typically result in preparations of low cardiac purity (typically (<em>1</em>% cardiomyocytes). We detail here the methods for a recently reported directed cardiac <em>differentiation</em> protocol, which involves the serial application of two <em>growth</em> <em>factors</em> known to be involved in early <em>embryonic</em> heart development, activin A, and bone morphogenetic protein-4 (BMP-4). This protocol reliably yields preparations of 30-60% cardiomyocytes, which can then be further enriched to >90% cardiomyocytes using straightforward physical methods.

The purpose of the present work is to examine some of the mechanisms responsible for the early architectonic <em>differentiation</em> of the central nervous system, as well as for the abnormal development which occurs in certain hereditary malformations. In order to approach these questions, the <em>embryonic</em> development of the cerebral cortex, the cerebellum, the inferior olivary complex and the facial nerve nucleus has been studied in normal and reeler mutant mice, using morphological methods. The adult reeler phenotype is characterized not only by extreme laminar abnormalities of cell positioning in the telencephalic and cerebellar cortices, but also by relatively less extreme, though distinct abnormal architectonics in non-cortical structures such as the inferior olive and the facial nerve nucleus. Study of the <em>embryonic</em> development of these structures reveals that neurons are generated at the normal time and migrate along normal pathways. Moreover, the processes of directional axonal <em>growth</em>, <em>differentiation</em> of class specific features of neurons and glia, and synaptogenesis appear similar in both genotypes and are probably not directly affected by the reeler mutation. However, in all instances, the early architectonic organization achieved by reeler cortical, Purkinje, olivary or facial neurons at the end of their migration is consistently less regular than in normal embryos. In addition, these anomalies become amplified during the later developmental period. This evidence for the early appearance of abnormalities in reeler embryos indicates that the disposition of neurons at maturity cannot be exclusively regarded as secondary to the maturation of cells, neurites and connections, but is contingent upon a specific mechanism. One may infer that the presence of a normal allele at the reeler locus is necessary for the normal completion of this histogenetic step, which consequently is submitted to genetic control. Although the <em>factor</em>(s) responsible for the stable configuration of the early architectonics is unknown, various hypotheses are considered. Several lines of evidence are presented which argue against a major role being played by diffusible <em>factors</em>, mesodermal components and afferent fiber systems. Two mechanisms are considered particularly worth evaluating: (<em>1</em>) a diminution of relative adhesivity between neurons and radial glial fibers at the end of migration, and (2) a stabilization of neuronal configuration by selective recognition-adhesion among postmigratory neurons. The reeler gene could, directly or indirectly, affect these cell-cell interactions.(ABSTRACT TRUNCATED AT 400 WORDS)

The emergence of hepatocyte based clinical and pharmaceutical technologies, has been limited by the absence of a stable hepatocyte cell source. <em>Embryonic</em> stem cells may represent a potential solution to this cell source limitation problem since they are highly proliferative, renewable, and pluripotent. Although many investigators have described techniques to effectively differentiate stem cells into a variety of mature cell lineages, their practicality is limited by: (<em>1</em>) low yields of fully differentiated cells, (2) absence of large scale processing considerations, and (3) ineffective downstream enrichment protocols. Thus, a <em>differentiation</em> platform that may be modified to induce and sustain differentiated cell function and scaled to increase differentiated cell yield would improve current stem cell <em>differentiation</em> strategies. Microencapsulation provides a vehicle for the discrete control of key cell culture parameters such as the diffusion of <em>growth</em> <em>factors</em>, metabolites, and wastes. In addition, both cell seeding density and bead composition may be manipulated. In order to assess the feasibility of directing stem cell <em>differentiation</em> via microenvironment regulation, we have developed a murine <em>embryonic</em> stem cell (ES) alginate poly-l-lysine microencapsulation hepatocyte <em>differentiation</em> system. Our results indicate that the alginate microenvironment maintains cell viability, is conducive to ES cell <em>differentiation</em>, and maintains differentiated cellular function. This system may ultimately assist in developing scalable stem cell <em>differentiation</em> strategies.

The MUC<em>1</em> protein is aberrantly expressed on an estimated 75% of all human solid tumor cancers. We recently reported that a transmembrane cleavage product, MUC<em>1</em>*, is the predominant form of the protein on cancer cells [<em>1</em>]. Further, our evidence indicated that MUC<em>1</em>* functions as a <em>growth</em> <em>factor</em> receptor on tumor cells, while the full-length protein appeared to have no <em>growth</em> promoting activity. Here, we report that MUC<em>1</em>* acts as a <em>growth</em> <em>factor</em> receptor on undifferentiated human <em>embryonic</em> stem cells (hESCs). Cleavage of the full-length ectodomain to form MUC<em>1</em>*, a membrane receptor, appears to make binding to its ligand, NM23, possible. Unexpectedly, we found that newly differentiated cells no longer express the cleaved form, MUC<em>1</em>*, or its ligand, NM23. Newly differentiated stem cells exclusively present full-length MUC<em>1</em>. Antibody-induced dimerization of the MUC<em>1</em>* receptor on hESCs stimulated cell <em>growth</em> to a far greater degree than currently used methods that require the addition of exogenous basic fibroblast <em>growth</em> <em>factor</em> (bFGF) as well as <em>factors</em> secreted by fibroblast "feeder cells". Further, MUC<em>1</em>* mediated <em>growth</em> was shown to be independent of <em>growth</em> stimulated by bFGF or the milieu of <em>factors</em> secreted by feeder cells. Stimulating the MUC<em>1</em>* receptor with either the cognate antibody or its ligand NM23 enabled hESC <em>growth</em> in a feeder cell-free system and produced pluripotent colonies that resisted spontaneous <em>differentiation</em>. These findings suggest that this primal <em>growth</em> mechanism could be utilized to propagate large numbers of pluripotent stem cells for therapeutic interventions.

BACKGROUND

The epicardium contributes to the majority of nonmyocardial cells in the adult heart. Recent studies have reported that the epicardium is derived from Nkx2.5-positive progenitors and can differentiate into cardiomyocytes. Not much is known about the relation between the myocardial and epicardial lineage during development, whereas insights into these embryonic mechanisms could facilitate the design of future regenerative strategies.

OBJECTIVE

Acquiring insight into the signaling pathways involved in the lineage separation leading to the differentiation of myocardial and (pro)epicardial cells at the inflow of the developing heart.

RESULTS

We made 3D reconstructions of Tbx18 gene expression patterns to give insight into the developing epicardium in relation to the developing myocardium. Next, using DiI tracing, we show that the (pro)epicardium separates from the same precursor pool as the inflow myocardium. In vitro, we show that this lineage separation is regulated by a crosstalk between bone morphogenetic protein (BMP) signaling and fibroblast growth factor (FGF) signaling. BMP signaling via Smad drives differentiation toward the myocardial lineage, which is inhibited by FGF signaling via mitogen-activated protein kinase kinase (Mek)1/2. Embryos exposed to recombinant FGF2 in vivo show enhanced epicardium formation, whereas a misbalance between FGF and BMP by Mek1/2 inhibition and BMP stimulation causes a developmental arrest of the epicardium and enhances myocardium formation at the inflow of the heart.

CONCLUSIONS

Our data show that FGF signaling via Mek1/2 is dominant over BMP signaling via Smad and is required to separate the epicardial lineage from precardiac mesoderm. Consequently, myocardial differentiation requires BMP signaling via Smad and inhibition of FGF signaling at the level of Mek1/2. These findings are of clinical interest for the development of regeneration-based therapies for heart disease.

The targeted disruption of the two nonallelic insulin genes in mouse was reported previously to result in intrauterine <em>growth</em> retardation, severe diabetes immediately after suckling, and death within 48 h of birth. We have further used these animals to investigate the morphology and cell biology of the endocrine pancreas in late gestation and at birth when insulin is absent throughout development. Pancreatic beta-cells were identified by detecting the activity of the LacZ gene inserted at the Ins2 locus. A significant increase in the mean area of the islets was found at <em>embryonic</em> d <em>1</em>8.5 (E<em>1</em>8.5) and in the newborn in Ins<em>1</em>-/-, Ins2-/- animals compared with Ins<em>1</em>-/-, Ins2+/- and wild-type controls, whereas the blood glucose levels were unaltered. The individual size of the beta-cells in the insulin-deficient fetuses was similar to controls, suggesting that the relative increase in islet size was due to an increase in cell number. Immunohistochemistry for proliferating cell nuclear antigen within the pancreatic ductal epithelium showed no <em>differences</em> in labeling index between insulin-deficient and control mice, and no change in the number of beta-cells associated with ducts, but the relative size distribution of the islets was altered so that fewer islets under 5,000 microm(2) and more islets greater than <em>1</em>0,000 microm(2) were present in Ins<em>1</em>-/-, Ins2-/- animals. This suggests that the greater mean islet size seen in insulin-deficient animals represented an enlargement of formed islets and was not associated with an increase in islet neogenesis. The proportional contribution of alpha- and beta-cells to the islets was not altered. This was supported by an increase in the number of cells containing immunoreactive proliferating cell nuclear antigen in both islet alpha- and beta-cells at E<em>1</em>8.5 in insulin-deficient mice, and a significantly lower incidence of apoptotic cells, as determined by molecular histochemistry using the terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end labeling reaction. The density of blood vessels within sections of whole pancreas, or within islets, was determined by immunohistochemistry for the endothelial cell marker CD3<em>1</em> and was found to be increased 2-fold in insulin-deficient mice compared with controls at E<em>1</em>8.5. However, no changes were found in the steady-state expression of mRNAs encoding vascular endothelial <em>growth</em> <em>factor</em>, its receptor Flk-<em>1</em>, IGF-I or -II, the IGF-I and insulin receptors, or insulin receptor substrates-<em>1</em> or -2 in pancreata from Ins<em>1</em>-/-, Ins2-/- mice compared with Ins<em>1</em>-/-, Ins2+/- controls. Thus, we conclude that the relative hyperplasia of the islets in late gestation in the insulin-deficient mice was due to an increased islet cell proliferation coupled with a reduced apoptosis, which may be related to an increased vascularization of the pancreas.

<em>Embryonic</em> stem (ES) cells differentiating in vitro reproduce many facets of early <em>embryonic</em> development, including the expression of developmentally regulated transcription <em>factors</em> and the <em>differentiation</em> of multipotent precursor cells. ES cells were evaluated for their ability to differentiate into pancreatic and islet lineage-restricted stages including pancreatic duodenal homeobox <em>1</em> (PDX<em>1</em>)-positive pancreatic precursor cells, early endocrine cell progenitors, and islet hormone-producing cells. Following <em>growth</em> and <em>differentiation</em> in nonselective medium containing serum, murine ES cells spontaneously differentiated into cells individually expressing each of the four major islet hormones: insulin, glucagon, somatostatin, and pancreatic polypeptide. PDX<em>1</em> immunostaining cells appeared first, before hormone-positive cells had emerged. Hormone-positive cells appeared within focal clusters of cells coexpressing PDX<em>1</em> and the nonclassical hormone markers peptide YY (YY) and islet amyloid polypeptide (IAPP) in combination with the definitive hormones, characteristic of endocrine cells appearing during early pancreaticogenesis. This system allows the investigation of many facets of islet development since it promotes the appearance of the complete range of islet phenotypes and reproduces important developmental stages of normal islet cyto<em>differentiation</em> in differentiating ES cell cultures.

We report the three-dimensional structure of osteogenic protein <em>1</em> (OP-<em>1</em>, also known as bone morphogenetic protein 7) to 2.8-A resolution. OP-<em>1</em> is a member of the transforming <em>growth</em> <em>factor</em> beta (TGF-beta) superfamily of proteins and is able to induce new bone formation in vivo. Members of this superfamily share sequence similarity in their C-terminal regions and are implicated in <em>embryonic</em> development and adult tissue repair. Our crystal structure makes possible the structural comparison between two members of the TGF-beta superfamily. We find that although there is limited sequence identity between OP-<em>1</em> and TGF-beta 2, they share a common polypeptide fold. These results establish a basis for proposing the OP-<em>1</em>/TGF-beta 2 fold as the primary structural motif for the TGF-beta superfamily as a whole. Detailed comparison of the OP-<em>1</em> and TGF-beta 2 structures has revealed striking <em>differences</em> that provide insights into how these <em>growth</em> <em>factors</em> interact with their receptors.

Vasculogenesis, the de novo formation of <em>embryonic</em> blood vessels from their angioblastic precursors in situ, is supposed to be under the control of polypeptide <em>growth</em> <em>factors</em> and their receptors. The receptor tyrosine kinase flk-<em>1</em> and its high-affinity ligand vascular endothelial <em>growth</em> <em>factor</em> (VEGF) represent an endothelial specific signal transduction system expressed during <em>embryonic</em> vascular <em>growth</em> in the mouse. We have cloned the quail homologs of VEGF and flk-<em>1</em> using PCR and have investigated their expression pattern in vivo. As shown by Northern analysis and reverse transcription PCR, VEGF and flk-<em>1</em> mRNA (3.9 and 5.8 kb, respectively) were already present in the unincubated blastodisc at low levels and were largely upregulated during gastrulation at <em>Embryonic</em> Day <em>1</em>. As detected by in situ hybridization, flk-<em>1</em> mRNA was initially present in the entire mesoderm of Day <em>1</em> embryos but from Day 2 on was restricted to endothelial cells. At Day 2 VEGF was ubiquitously expressed in the embryo proper and was mainly restricted to the vascularized part (area vasculosa) in the yolk sac. Later on VEGF expression was detected in all organs. In the kidney VEGF mRNA was mainly localized to the glomeruli. This pattern of expression is consistent with the pattern found during mouse embryogenesis. We have recently established an in vitro model of vasculogenesis in which hemangioblastic precursors are induced in cell cultures from the unincubated quail blastodisc by basic fibroblast <em>growth</em> <em>factor</em> (bFGF) and give rise to blood vessels in vitro. Taking advantage of this in vitro model we examined whether FGF and VEGF act in concert during vasculogenesis. We found that the flk-<em>1</em> receptor mRNA is dramatically upregulated within 24 hr upon the addition of FGF to quail blastodisc cell cultures. This inducibility in response to FGF is confined to the first 24 hr of culture. The early expression of the flk-<em>1</em> mRNA may characterize the <em>differentiation</em> of hemangioblastic precursors from pluripotent epiblast cells which in vivo is initiated during gastrulation. Thus, the time course and the pattern of expression during embryogenesis in different species suggest a major role for the VEGF/flk-<em>1</em> signal transduction system in vasculogenesis and angiogenesis.

The relationship between expression of the pituitary-specific transcription <em>factor</em>, GHF-<em>1</em>, and activation of the <em>growth</em> hormone and prolactin genes during mouse anterior pituitary development was investigated. While GHF-<em>1</em> transcripts were detected within 24 hr of the first observable events in anterior pituitary <em>differentiation</em>, no GHF-<em>1</em> protein could be detected until about 3 days later. The appearance of GHF-<em>1</em> protein showed good temporal and spatial correlation with activation of the <em>growth</em> hormone gene. Prolactin gene expression, on the other hand, was observed transiently during <em>embryonic</em> day <em>1</em>6 in two different populations of cells, of which the major one does not contain GHF-<em>1</em> or <em>growth</em> hormone. These results suggest that expression of GHF-<em>1</em> is controlled both transcriptionally and posttranscriptionally. The spatial and temporal correlation between the appearance of GHF-<em>1</em> protein and <em>growth</em> hormone gene activation suggests that GHF-<em>1</em> is responsible for this very last step in the specialization of somatotrophic cells.

OBJECTIVE

Cord blood banks provide fully human leukocyte antigen-typed cells, from which a set of standard induced pluripotent stem (iPS) cells for use in allogenic transplantation can be derived. Hence, the ability to generate iPS cells from cord blood cells has the potential to provide a suitable source for clinical transplantation. The aim of this work is to determine the reprogramming methods, culture conditions, and cell fractions that can be used to generate iPS cells from cord blood cells effectively.

METHODS

CD34(+), mononucleated, and derived adherent cells from cord blood were cultured in hematopoietic medium (X-vivo<em>1</em>0 containing 50 ng/mL interleukin-6, 50 ng/mL soluble interleukin-6 receptor, 50 ng/mL stem cell <em>factor</em>, <em>1</em>0 ng/mL thrombopoietin, and 20 ng/mL Flit3/4 ligand) 3 days prior to viral infection. Cells were then infected with retroviral constructs driving the expression of OCT3/4, SOX2, Krüppel-like <em>factor</em> 4, c-MYC, and enhanced green fluorescent protein together with or without the p53 knockdown lentiviral construct Shp53 pLKO.<em>1</em>-puro. Infected cells were then cultured for an additional 4 days in hematopoietic culture medium before being transferred onto mouse <em>embryonic</em> fibroblast (MEF) or SNL76/7 feeder cells in human <em>embryonic</em> stem cell medium (Dulbecco's modified Eagle medium/F-<em>1</em>2 containing 20% knockout serum replacement, 200 mM L-glutamine, <em>1</em>% non-essential amino acids (NEAA), 0.<em>1</em> mM 2-mercaptoethanol, and 4 ng/mL basic fibroblast <em>growth</em> <em>factor</em>). Subsequently, the number of <em>embryonic</em> stem cell-like colonies that emerged in the following 4 weeks was scored. Expression of a number of pluripotency makers were examined by immunochemistry and reverse transcriptase polymerase chain reaction. Finally, the <em>differentiation</em> potential of selected colonies was determined by teratoma formation in severe combined immunodeficient mice and in vitro culture.

RESULTS

Repression of p53 expression by the addition of a lentiviral p53 short-hairpin RNA expression vector increased the frequency of formation of iPS-like colonies from <em>1</em> (on average) to around <em>1</em>00 per 2 x <em>1</em>0(4) cells when infected cells were grown on SNL feeder cells.

CONCLUSIONS

iPS cells can be generated easily from CD34(+) cord blood cells through the addition of p53 inhibition to standard reprogramming conditions.

Human <em>embryonic</em> stem (hES) cells provide an important means to evaluate specific soluble and cell-bound stimuli that regulate development of specific cell lineages. Here, we examined specific cytokines and extracellular matrix (ECM) proteins that support <em>differentiation</em> of hES cells to hepatocytes. Tests of several different conditions determined that addition of fibroblast <em>growth</em> <em>factor</em> (FGF)-4 and hepatocyte <em>growth</em> <em>factor</em> in completely serum-free cultures of hES cell-derived embryoid bodies subsequently allowed to attach to type I collagen-coated dishes led to maximal <em>differentiation</em> into cells, not only with the morphologic and phenotypic characteristics of hepatocytes but also the functional characteristics. Expression of common hepatic transcription <em>factors</em> including HNF-3beta, HNF-<em>1</em>, and GATA-4 were all significantly induced under these conditions. Hepatocyte function was demonstrated by multiple complementary criteria: production of urea and albumin, phenobarbital-induced cytochrome P450 expression, and uptake of indocyanine green. These hES cell-derived hepatocytes will serve as a resource to understand normal human hepatocyte development and for applications such as cell replacement therapies and screening of pharmacologic drugs.

Neurogenesis persists in certain regions of the adult brain including the subgranular zone of the hippocampal dentate gyrus wherein its regulation is essential, particularly in relation to learning, stress and modulation of mood. Lysophosphatidic acid (LPA) is an extracellular signaling phospholipid with important neural regulatory properties mediated by specific G protein-coupled receptors, LPA(<em>1</em>-5). LPA(<em>1</em>) is highly expressed in the developing neurogenic ventricular zone wherein it is required for normal <em>embryonic</em> neurogenesis, and, by extension may play a role in adult neurogenesis as well. By means of the analyses of a variant of the original LPA(<em>1</em>)-null mutant mouse, termed the Malaga variant or "maLPA(<em>1</em>)-null," which has recently been reported to have defective neurogenesis within the <em>embryonic</em> cerebral cortex, we report here a role for LPA(<em>1</em>) in adult hippocampal neurogenesis. Proliferation, <em>differentiation</em> and survival of newly formed neurons are defective in the absence of LPA(<em>1</em>) under normal conditions and following exposure to enriched environment and voluntary exercise. Furthermore, analysis of trophic <em>factors</em> in maLPA(<em>1</em>)-null mice demonstrated alterations in brain-derived neurotrophic <em>factor</em> and insulin <em>growth</em> <em>factor</em> <em>1</em> levels after enrichment and exercise. Morphological analyses of doublecortin positive cells revealed the anomalous prevalence of bipolar cells in the subgranular zone, supporting the operation of LPA(<em>1</em>) signaling pathways in normal proliferation, maturation and <em>differentiation</em> of neuronal precursors.

The theory of bifurcating vascular systems predicts vessel diameters that are related to optimality criteria like minimization of pumping energy or of building material. However, mechanisms for producing the postulated optimality have not been described so far, and quantitative data on bifurcation diameters during development are scarce. We used an <em>embryonic</em> vascular bed that rapidly grows and adapts to changing hemodynamic conditions, the chicken chorioallantoic membrane (CAM), and correlated vascular cast and tissue section morphology with in vivo time-lapse video monitoring. The bifurcation exponent delta and associated parameters were quantitatively assessed in arterial and venous microvessels ranging in diameter from 30 to <em>1</em>00 microm. We observed emergence of optimality by means of intussusception, i.e., formation of transvascular tissue pillars. In addition to intussusceptive microvascular <em>growth</em> (IMG = expansion of capillary networks) and intussusceptive arborization (IAR = formation of feeding vessels from capillaries) the observed intussusception at bifurcations represents a third variant of nonsprouting angiogenesis. We call it intussusceptive branching remodeling (IBR). IBR occurred in vessels of considerable diameter by means of two alternative mechanisms: either through pillars arising close to a bifurcation, which increased in girth until they merged with the connective tissue in the bifurcation angle; or through pillars arising at some distance from the bifurcation point, which then expanded by formation of ingrowing tissue folds until they became connected to the tissue of the bifurcation angle. Morphologic evidence suggests that IBR is a wide-spread phenomenon, taking place also in lung, intestinal, kidney, eye, etc., vasculature. Irrespective of the mode followed, IBR led to a branching pattern close to the predicted optimum, delta = 3.0. Significant <em>differences</em> were observed between delta at arterial bifurcations (2.70 to 2.90) and delta at venous bifurcations (2.93 to 3.75). IBR, by means of eccentric pillar formation and fusion, was also involved in vascular pruning. Experimental changes in CAM hemodynamics (by locally increasing blood flow) induced onset of IBR within less than <em>1</em> hr. Our study provides morphologic and quantitative evidence that a similar cellular machinery is used for all three variants of vascular intussusception, IMG, IAR, and IBR. It thus provides a mechanism of efficiently generating complex blood transport systems from limited genetic information. Differential quantitative outcome of IBR in arteries and veins, and the experimental induction of IBR strongly suggest that hemodynamic <em>factors</em> can instruct <em>embryonic</em> vascular remodeling toward optimality.

<em>Embryonic</em> stem (ES) cells may differentiate along a hepatocyte lineage; however, currently there are no reports of culture conditions yielding high levels of hepatocyte-specific gene expression in these cells. We investigated culture conditions for differentiating ES cells into hepatocyte-like cells in vitro. Various combinations of culture media, <em>growth</em> and <em>differentiation</em> <em>factors</em>, and substratum precoatings were evaluated, and it was determined that a combination of Iscove's modified Dulbecco's medium with 20% fetal bovine serum, human insulin, dexamethasone. and collagen type I precoating was optimal for directing mouse ES cells along a hepatocyte lineage. Treatment of mouse ES cell with the optimal condition led to prealbumin gene expression 20% as high, and albumin synthesis 7% as high, as in mouse liver. The optimal culture condition also induced albumin gene expression in differentiated human ES cells <em>1</em>% as high as in normal human hepatocytes as shown by Western blot analysis, and cells were positive for human albumin by immunocytochemistry. In addition, our optimal condition led to high levels of albumin gene expression in primary mouse hepatocytes after 35 days of culture, levels <em>1</em>0-fold higher than with other hepatocyte <em>differentiation</em> media. In conclusion, our optimal condition directed both mouse and human ES cells along a hepatocyte lineage. This represents the initial step in establishing cell lines that can be employed in cell-based therapeutics in humans and for toxicology and pharmacology studies.

Pigment epithelium-derived <em>factor</em> (PEDF) is a member of the serine protease inhibitor (serpin) superfamily that has been shown previously to promote the survival and/or <em>differentiation</em> of rat cerebellar granule neurons and human retinoblastoma cells in vitro. However, in contrast to most serpins, PEDF has no inhibitory activity against any known proteases, and its described biological activities do not appear to require the serpin-reactive loop located toward the carboxy end of the polypeptide. Because another serpin, protease nexin-<em>1</em>, has been shown to promote the in vivo survival and <em>growth</em> of motor neurons, the authors investigated the potential neurotrophic effects of PEDF on spinal cord motor neurons in highly enriched cultures and in vivo after injury. Here, it is shown that native bovine and recombinant human PEDF promoted the survival and <em>differentiation</em> (neurite out<em>growth</em>) of <em>embryonic</em> chick spinal cord motor neurons in vitro in a dose-dependent manner. A truncated form of PEDF that lacks approximately 62% of the carboxy end of the polypeptide comprising the homologous serpin-reactive loop also exhibited neurotrophic activities similar to those of the full-length protein. Furthermore, the data here showed that PEDF was transported retrogradely and prevented the death and atrophy of spinal motor neurons in the developing neonatal mouse after axotomy. These results indicate that PEDF exerts trophic effects on motor neurons, and, together with previous reports, these findings suggest that this protein may be useful as a pharmacologic agent to promote the development and maintenance of motor neurons. J. Comp. Neurol. 4<em>1</em>2:506-5<em>1</em>4, <em>1</em>999. Published <em>1</em>999 Wiley-Liss, Inc.

Heparan sulfate (HS) glycosaminoglycans are essential modulators of fibroblast <em>growth</em> <em>factor</em> (FGF) activity and appear to act by coupling particular forms of FGF to appropriate FGF receptors. During neural development, one particular HS proteoglycan is able to rapidly switch its potentiating activity from FGF-2, as neural precursor cell proliferation occurs, to FGF-<em>1</em>, as neuronal <em>differentiation</em> occurs. Using various analytical techniques, including chemical and enzymatic cleavage, low pressure chromatography, and strong anion-exchange high performance liquid chromatography, we have analyzed the different HSs expressed during these crucial developmental stages. There are distinct alterations in patterns of 6-O-sulfation, total chain length, and the number of sulfated domains of the HS from the more mature <em>embryonic</em> brain. These changes correlate with a switch in the ability of the HS to potentiate the actions of FGF-<em>1</em> in triggering cell <em>differentiation</em>. It thus appears that each HS pool is designed to function in the modulation of an intricate interaction with a specific <em>growth</em> <em>factor</em> and its cognate receptor, and suggests tightly regulated expression of specific, bioactive disaccharide sequences. The data can be used to construct a simple model of controlled variations in HS chain structure which have functional consequences at a crucial stage of neuronal maturation.