Early development of mammalian embryos occurs in an environment of relative hypoxia. Nevertheless, human <em>embryonic</em> stem cells (hESC), which are derived from the inner cell mass of blastocyst, are routinely cultured under the same atmospheric conditions (2<em>1</em>% O(2)) as somatic cells. We hypothesized that O(2) levels modulate gene expression and <em>differentiation</em> potential of hESC, and thus, we performed gene profiling of hESC maintained under normoxic or hypoxic (<em>1</em>% or 5% O(2)) conditions. Our analysis revealed that hypoxia downregulates expression of pluripotency markers in hESC but increases significantly the expression of genes associated with angio- and vasculogenesis including vascular endothelial <em>growth</em> <em>factor</em> and angiopoitein-like proteins. Consequently, we were able to efficiently differentiate hESC to functional endothelial cells (EC) by varying O(2) levels; after 24 hours at 5% O(2), more than 50% of cells were CD34+. Transplantation of resulting endothelial-like cells improved both systolic function and fractional shortening in a rodent model of myocardial infarction. Moreover, analysis of the infarcted zone revealed that transplanted EC reduced the area of fibrous scar tissue by 50%. Thus, use of hypoxic conditions to specify the endothelial lineage suggests a novel strategy for cellular therapies aimed at repair of damaged vasculature in pathologies such as cerebral ischemia and myocardial infarction.

The isolation and characterization of <em>embryonic</em> and adult stem cells from higher-order mammalian species will enhance the understanding of the biology and therapeutic application of stem cells. The aim of this study was to purify rhesus mesenchymal stem cells (MSCs) from adult bone marrow and to characterize functionally their abilities to differentiate along diverse lineages. Adherent cells from adult rhesus macaque bone marrow were characterized for their <em>growth</em> characteristics, lineage <em>differentiation</em>, cell-surface antigen expression, telomere length, chromosome content, and transcription <em>factor</em> gene expression. Rhesus bone marrow MSCs (BMSCs) are very heterogeneous, composed of primarily long, thin cells and some smaller, round cells. The cells are capable of differentiating along osteogenic, chondrogenic, and adipogenic lineages in vitro. The cell morphology and multipotential <em>differentiation</em> capabilities are maintained throughout extended culture. They express CD59, CD90 (Thy-<em>1</em>), CD<em>1</em>05, and HLA-<em>1</em> and were negative for hematopoietic markers such as CD3, CD4, CD8, CD<em>1</em><em>1</em>b, CD<em>1</em>3, CD34, and platelet endothelial cell adhesion molecule-<em>1</em> (CD3<em>1</em>). BMSCs were also demonstrated to express the mRNA for important stem cell-related transcription <em>factors</em> such as Oct-4, Sox-2, Rex-<em>1</em>, and Nanog. Rhesus BMSCs have a normal chromosome content, and the shortening of telomeres is minimal during early passages. These data demonstrate that BMSCs isolated from rhesus macaques have a high degree of commonality with MSCs isolated from other species. Therefore, isolation of these cells provides an effective and convenient method for rapid expansion of pluripotent rhesus MSCs.

BACKGROUND

The density of native (preexisting) collaterals varies widely and is a significant determinant of variation in severity of stroke, myocardial infarction, and peripheral artery disease. However, little is known about mechanisms responsible for formation of the collateral circulation in healthy tissues.

OBJECTIVE

We previously found that variation in vascular endothelial growth factor (VEGF) expression causes differences in collateral density of newborn and adult mice. Herein, we sought to determine mechanisms of collaterogenesis in the embryo and the role of VEGF in this process.

RESULTS

Pial collaterals begin forming between embryonic day 13.5 and 14.5 as sprout-like extensions from arterioles of existing cerebral artery trees. Global VEGF-A overexpressing mice (Vegf(hi/+)) formed more, and Vegf(lo/+) formed fewer, collaterals during embryogenesis, in association with differences in vascular patterning. Conditional global reduction of Vegf or Flk1 only during collaterogenesis significantly reduced collateral formation, but now without affecting vascular patterning, and the effects remained in adulthood. Endothelial-specific Vegf reduction had no effect on collaterogenesis. Endothelial-specific reduction of a disintegrin-and-metalloprotease-domain-10 (Adam10) and inhibition of γ-secretase increased collateral formation, consistent with their roles in VEGF-induced Notch1 activation and suppression of prosprouting signals. Endothelial-specific knockdown of Adam17 reduced collateral formation, consistent with its roles in endothelial cell migration and embryonic vascular stabilization, but not in activation of ligand-bound Notch1. These effects also remained in adulthood.

CONCLUSIONS

Formation of pial collaterals occurs during a narrow developmental window via a sprouting angiogenesis-like mechanism, requires paracrine VEGF stimulation of fetal liver kinase 1-Notch signaling, and adult collateral number is dependent on embryonic collaterogenesis.

The molecular events of cardiac lineage specification and <em>differentiation</em> are largely unknown. Here we describe the involvement of a <em>growth</em> <em>factor</em> with an EGF-like domain, Cripto-<em>1</em> (Cr-<em>1</em>), in cardiac <em>differentiation</em>. During <em>embryonic</em> development, Cr-<em>1</em> is expressed in the mouse blastocyst, primitive streak, and later is restricted to the developing heart. To investigate the role of Cr-<em>1</em>, we have generated Cr-<em>1</em>-negative <em>embryonic</em> stem (ES) cell lines by homologous recombination. The resulting double "knockout" ES cells have selectively lost the ability to form beating cardiac myocytes, a process that can be rescued by reintroducing Cr-<em>1</em> gene back into the Cr(-/-) cells. Furthermore, the lack of functional Cr-<em>1</em> is correlated with absence of expression of cardiac-specific myosin light and heavy chain genes during <em>differentiation</em>. <em>Differentiation</em> into other cell types including skeletal muscle is not disrupted. These results suggest that Cr-<em>1</em> is essential for contractile cardiomyocyte formation in vitro.

This study represents a first step in investigating the possible involvement of transforming <em>growth</em> <em>factor</em>-beta (TGF-beta) in the regulation of <em>embryonic</em> chick limb cartilage <em>differentiation</em>. TGF-beta <em>1</em> and 2 (<em>1</em>-<em>1</em>0 ng/ml) elicit a striking increase in the accumulation of Alcian blue, pH <em>1</em>-positive cartilage matrix, and a corresponding twofold to threefold increase in the accumulation of 35S-sulfate- or 3H-glucosamine-labeled sulfated glycosaminoglycans (GAG) by high density micromass cultures prepared from the cells of whole stage 23/24 limb buds or the homogeneous population of chondrogenic precursor cells comprising the distal subridge mesenchyme of stage 25 wing buds. Moreover, TGF-beta causes a striking (threefold to sixfold) increase in the steady-state cytoplasmic levels of mRNAs for cartilage-characteristic type II collagen and the core protein of cartilage-specific proteoglycan. Only a brief (2 hr) exposure to TGF-beta at the initiation of culture is sufficient to stimulate chondrogenesis, indicating that the <em>growth</em> <em>factor</em> is acting at an early step in the process. Furthermore, TGF-beta promotes the formation of cartilage matrix and cartilage-specific gene expression in low density subconfluent spot cultures of limb mesenchymal cells, which are situations in which little, or no chondrogenic <em>differentiation</em> normally occurs. These results provide strong incentive for considering and further investigating the role of TGF-beta in the control of limb cartilage <em>differentiation</em>.

BACKGROUND

The human endometrium is unique in its capacity to remodel constantly throughout adult reproductive life. Although the processes of tissue damage and breakdown in the endometrium have been well studied, little is known of how endometrial regeneration is achieved after menstruation. Nodal, a member of the transforming growth factor-beta superfamily, regulates the processes of pattern formation and differentiation that occur during early embryo development.

METHODS

In this study, the expression of Nodal, Cripto (co-receptor) and Lefty A (antagonist) was examined by RT-PCR and immunohistochemistry across the menstrual cycle and in endometrial carcinomas.

RESULTS

Nodal and Cripto were found to be expressed at high levels in both stromal and epithelial cells during the proliferative phase of the menstrual cycle. Although immunoreactivity for both proteins in surface and glandular epithelium was maintained at relatively steady-state levels across the cycle, their expression was significantly decreased within the stromal compartment by the mid-secretory phase. Lefty expression, as has previously been reported, was primarily restricted to glandular epithelium and surrounding stroma during the late secretory and menstrual phases. In line with recent studies that have shown that Nodal pathway activity is upregulated in many human cancers, we found that Nodal and Cripto immunoreactivity increased dramatically in the transition from histologic Grade 1 to histologic Grades 2 and 3 endometrial carcinomas. Strikingly, Lefty expression was low or absent in all cancer tissues.

CONCLUSIONS

The expression of Nodal in normal and malignant endometrial cells that lack Lefty strongly supports an important role for this embryonic morphogen in the tissue remodelling events that occur across the menstrual cycle and in tumourogenesis.

The <em>differentiation</em> of precursor cells into neurons has been shown to be influenced by both the Notch signaling pathway and <em>growth</em> <em>factor</em> stimulation. In this study, the regulation of neuronal <em>differentiation</em> by these mechanisms was examined in the <em>embryonic</em> day <em>1</em>0 neuroepithelial precursor (NEP) population. By downregulating Notch<em>1</em> expression and by the addition of a Delta<em>1</em> fusion protein (Delta Fc), it was shown that signaling via the Notch pathway inhibited neuron <em>differentiation</em> in the NEP cells, in vitro. The expression of two of the Notch receptor homologs, Notch<em>1</em> and Notch3, and the ligand Delta<em>1</em> in these NEP cells was found to be influenced by a number of different <em>growth</em> <em>factors</em>, indicating a potential interaction between <em>growth</em> <em>factors</em> and Notch signaling. Interestingly, none of the <em>growth</em> <em>factors</em> examined promoted neuron <em>differentiation</em>; however, the fibroblast <em>growth</em> <em>factors</em> (FGFs) <em>1</em> and 2 potently inhibited <em>differentiation</em>. FGF<em>1</em> and FGF2 upregulated the expression of Notch and decreased expression of Delta<em>1</em> in the NEP cells. In addition, the inhibitory response of the cells to the FGFs could be overcome by downregulating Notch<em>1</em> expression and by disrupting Notch cleavage and signaling by the ablation of the Presenilin<em>1</em> gene. These results indicate that FGF<em>1</em> and FGF2 act via the Notch pathway, either directly or indirectly, to inhibit <em>differentiation</em>. Thus, signaling through the Notch receptor may be a common regulator of neuronal <em>differentiation</em> within the developing forebrain.

Implantation of an embryo induces rapid proliferation and <em>differentiation</em> of uterine stromal cells, forming a new structure, the decidua. One salient feature of decidua formation is a marked increase in maternal angiogenesis. Vascular endothelial <em>growth</em> <em>factor</em> (VEGF)-dependent pathways are active in the ovary, uterus, and embryo, and inactivation of VEGF function in any of these structures might prevent normal pregnancy development. We hypothesized that decidual angiogenesis is regulated by VEGF acting through specific VEGF receptors (VEGFRs). To test this hypothesis, we developed a murine pregnancy model in which systemic administration of a receptor-blocking antibody would act specifically on uterine angiogenesis and not on ovarian or <em>embryonic</em> angiogenesis. In our model, ovarian function was replaced with exogenous progesterone, and blocking antibodies were administered prior to <em>embryonic</em> expression of VEGFRs. After administration of a single dose of the anti-VEGFR-2 antibody during the peri-implantation period, no embryos were detected on <em>embryonic</em> d <em>1</em>0.5. The pregnancy was disrupted because of a significant reduction in decidual angiogenesis, which under physiological conditions peaks on <em>embryonic</em> d 5.5 and 6.5. Inactivation of VEGFR-3 reduced angiogenesis in the primary decidual zone, whereas administration of VEGFR-<em>1</em> blocking antibodies had no effect. Pregnancy was not disrupted after administration of anti-VEGFR-3 or anti-VEGFR-<em>1</em> antibodies. Thus, the VEGF/VEGFR-2 pathway plays a key role in the maintenance of early pregnancy through its regulation of peri-implantation angiogenesis in the uterine decidua. This newly formed decidual vasculature serves as the first exchange apparatus for the developing embryo until the placenta becomes functionally active.

A locally acting <em>growth</em> restraining feedback loop has been identified in the murine <em>embryonic</em> <em>growth</em> plate in which the level of parathyroid hormone-related peptide (PTHrP) expression regulates the pace of chondrocyte <em>differentiation</em>. To date, it is largely unknown whether this feedback loop also regulates the pace of chondrocyte <em>differentiation</em> in the <em>growth</em> plate after birth. We therefore characterized the spatio-temporal expression of Indian hedgehog (IHH), PTHrP, and their receptors in the postnatal <em>growth</em> plate from female and male rats of <em>1</em>, 4, 7, and <em>1</em>2 weeks of age. These stages are representative for early life and puberty in rats. Using semiquantitative reverse-transcription polymerase chain reaction (RT-PCR) on <em>growth</em> plate tissue, IHH and components of its receptor complex, patched (PTC) and smoothened (SMO), PTHrP and the type I PTH/PTHrP receptor messenger RNA (mRNA) were shown at all ages studied irrespective of gender. Using in situ hybridization, IHH, PTHrP, and PTH/PTHrP receptor mRNA were detected in prehypertrophic and hypertrophic chondrocytes in both sexes during development. In addition, especially in the younger age groups, faint expression of PTH/PTHrP receptor mRNA also was shown in stem cells and proliferative chondrocytes. Immunohistochemistry confirmed the observations made with in situ hybridization, by showing the presence of IHH, PTC, PTHrP, and PTH/PTHrP receptor protein in prehypertrophic and hypertrophic chondrocytes. In addition, staining for hedgehog, PTC, and PTHrP also was observed in <em>growth</em> plate stem cells. No <em>differences</em> in staining patterns were observed between the sexes. Furthermore, no mRNA or protein expression of the mentioned <em>factors</em> was detected in the perichondrium. Our data suggest that in contrast to the proposed feedback loop in the early <em>embryonic</em> <em>growth</em> plate, which requires the presence of the perichondrium, a feedback loop in the postnatal <em>growth</em> plate can be confined to the <em>growth</em> plate itself. In fact, two loops might exist: (<em>1</em>) a loop confined to the transition zone and early hypertrophic chondrocytes, which might in part be autocrine and (2) a loop involving the <em>growth</em> plate stem cells.

Postnatal skeletal muscle <em>growth</em> is classically attributed to fiber hypertrophy and myogenic <em>differentiation</em>, but these processes do not account for the size-independent increase of muscle mechanical performance that occurs during postnatal <em>growth</em>. There is also little knowledge about the precise time-course of contractile function or the underlying <em>factors</em> that affect it. The present study investigated morphological <em>factors</em> (muscle fiber size and myofibrillar packing), biochemical <em>factors</em> (myosin heavy chain isoform and desmin intermediate filament protein expression), and muscle architecture during postnatal development in mice. Physiological testing of the mouse tibialis anterior revealed that maximum isometric stress increased from 27+/-3 kPa at postnatal day <em>1</em> to <em>1</em>69+/-<em>1</em>0 kPa by postnatal day 28, roughly a sixfold increase. Morphological measurements revealed a robust increase in the size-independent packing of myofibrillar matrix material occurring with the functional improvement, with just 48.<em>1</em>+/-5.5% of the cross-sectional area filled with myofibrils at postnatal day <em>1</em> whereas 92.5+/-0.9% was filled by day 28. Expression of four myosin heavy chain isoforms (<em>embryonic</em>, neonatal, IIX and IIB), as well as desmin, correlated significantly with muscle mechanical function. Stepwise multiple regression showed that, of the variables measured, percentage content of neonatal myosin heavy chain was the best predictor of mechanical function during the postnatal time-course. These data provide the first specific structural basis for increases in muscle tension development during <em>growth</em>. Therefore, models of muscle <em>growth</em> must be modified to include an intrinsic quality enhancement component.

Platelet-derived <em>growth</em> <em>factor</em>-AA (PDGF-AA) has been used as a potent mitogen for the proliferation of oligodendrocyte progenitor cells (OPCs). Whether it plays a role in oligodendrocyte lineage <em>differentiation</em> of neural stem cells (NSCs) is unclear. Here we report that PDGF-AA is an instructional signal required for the <em>differentiation</em> of <em>embryonic</em> forebrain NSCs into O4-positive oligodendrocytes. Moreover, such PDGF-AA-induced oligodendrocyte <em>differentiation</em> appears to be mediated by the extracellular signal-regulated kinases <em>1</em> and 2 (Erk<em>1</em>/2) but not phosphatidylinositol-3 kinase (PI3K) pathway. Finally, PDGF-AA treatment resulted in a significant increase in the expression of the oligodendrocyte-specific transcriptional <em>factor</em> Olig2 in an Erk<em>1</em>/2-dependent mechanism at early stages of oligodendrogliogenesis. Together, our studies provide cellular and molecular evidence to suggest that PDGF-AA is a key molecule that regulates the <em>differentiation</em> of <em>embryonic</em> NSCs into oligodendrocytes. The action of PDGF-AA is mediated by the activation of Erk pathway which involves the downstream upregulation of transcriptional <em>factor</em> Olig2.

Islet transplantation has proven to be a successful strategy to restore normoglycemia in patients with type <em>1</em> diabetes (T<em>1</em>D). However, the dearth of cadaveric islets available for transplantation hampers the widespread application of this treatment option. Although human <em>embryonic</em> stem cells and induced pluripotent stem cells are capable of generating insulin-producing cells in vitro when provided with the appropriate inductive cues, the insulin-expressing cells that develop behave more like immature β-cells with minimal sensitivity to glucose stimulation. Here, we identify a set of signaling <em>factors</em> expressed in mouse <em>embryonic</em> mesenchyme during the time when foregut and pancreatic progenitors are specified and test their activities during in vitro <em>differentiation</em> of human <em>embryonic</em> stem cells. Several of the identified <em>factors</em> work in concert to expand the pancreatic progenitor pool. Interestingly, transforming <em>growth</em> <em>factor</em> (TGF)-β ligands, most potent in inducing pancreatic progenitors, display strong inhibitory effects on subsequent endocrine cell <em>differentiation</em>. Treatment with TGF-β ligands, followed by the addition of a TGF-β receptor antagonist, dramatically increased the number of insulin-producing cells in vitro, demonstrating the need for dynamic temporal regulation of TGF-β signaling during in vitro <em>differentiation</em>. These studies illustrate the need to precisely mimic the in vivo conditions to fully recapitulate pancreatic lineage specification in vitro.

Recent studies indicate that neonatal spermatogonial stem cells (SSCs) possess pluripotency. However, the mechanisms that regulate the pluripotent <em>differentiation</em> capacity of SSCs remain unclear. Here, we describe a new method to clonally derive pluripotent SSCs from neonatal mouse testis. By coculturing with testicular stromal cells, SSCs can be maintained and expanded in serum-free conditions. Unlike endogenous SSCs, these in vitro expanded SSCs showed strong alkaline phosphatase (AP) activity and displayed characteristics of <em>embryonic</em> stem cells and primordial germ cells, which were therefore designated as AP(+) germline stem cells (AP(+)GSCs). The pluripotency of AP(+)GSCs was confirmed by in vitro <em>differentiation</em> toward hepatic and neuronal lineages and formation of <em>embryonic</em> chimeras after injection into blastocysts. Further investigation revealed that insulin-like <em>growth</em> <em>factor</em>-<em>1</em> (IGF-<em>1</em>) secreted from Leydig cells was a key <em>factor</em> involved in maintaining the pluripotency of AP(+)GSCs. The blockage of IGF-<em>1</em> receptor phosphorylation and its downstream PI3K pathway by PPP or LY294002 dramatically reduced their AP activity and expression of pluripotent genes, such as Oct-4, Blimp<em>1</em>, and Nanog. In conclusion, the present study demonstrated that IGF-<em>1</em> secreted by testicular Leydig cells plays an important role in maintaining the pluripotency of SSCs in culture, which provides an insight into the molecular mechanism underlying germ cell pluripotency.

BACKGROUND

To gain insight into the role of satellite cells in skeletal muscle hypertrophy, the effect of radiation on small fiber formation, embryonic myosin heavy chain (embryonic MHC) production, and insulin-like growth factor I (IGF-I) production in overloaded adult rat soleus muscle was examined.

METHODS

Adult rat soleus muscle was overloaded by ablation of the synergistic gastrocnemius, plantaris, and flexor digitorum profundus muscles of the right hindlimb. Half of the rats were subjected to gamma irradiation of the right hindlimb prior to ablation in an attempt to prevent satellite cell proliferation.

RESULTS

Wet weight of the non-irradiated overloaded soleus muscle increased almost 40% compared to contralateral control muscle following 4 weeks of overload. Small fibers, which were rare in control muscle, accounted for 6.76 +/- 5.08% to 12.74 +/- 7.76% of the total fiber number of the non-irradiated soleus following 1 to 4 weeks of overload. Although usually absent in control muscle, IGF-I or embryonic MHC was immunolocalized in a small percentage (< 11%) of the mature fibers in the non-irradiated overloaded soleus. Irradiation prevented compensatory hypertrophy and nearly abolished small fiber formation in the overloaded soleus. However, irradiation did not diminish the percentage of mature fibers producing immunocytochemically detectable levels of embryonic MHC or IGF-I.

CONCLUSIONS

Irradiation may prevent hypertrophy by impairing activation, proliferation, and/or differentiation of satellite cells. Small fibers in overloaded muscle appear to be new fibers arising from satellite cells. IGF-I may have a role in muscle hypertrophy involving satellite cell activation, or perhaps a more direct role that requires additional factors.

Phenotypic heterogeneity has been observed among mesenchymal stem/stromal cell (MSC) populations, but specific genes associated with this variability have not been defined. To study this question, we analyzed two distinct isogenic MSC populations isolated from umbilical cord blood (UCB<em>1</em> and UCB2). The use of isogenic populations eliminated <em>differences</em> contributed by genetic background. We characterized these UCB MSCs for cell morphology, <em>growth</em> kinetics, immunophenotype, and potential for <em>differentiation</em>. UCB<em>1</em> displayed faster <em>growth</em> kinetics, higher population doublings, and increased adipogenic lineage <em>differentiation</em> compared to UCB2. However, osteogenic <em>differentiation</em> was stronger for the UCB2 population. To identify MSC-specific genes and developmental genes associated with observed phenotypic <em>differences</em>, we performed expression analysis using Affymetrix microarrays and compared them to bone marrow (BM) MSCs. We compared UCB<em>1</em>, UCB2, and BM and identified distinct gene expression patterns. Selected clusters were analyzed demonstrating that genes of multiple developmental pathways, such as transforming <em>growth</em> <em>factor</em>-beta (TGF-beta) and wnt genes, and markers of early <em>embryonic</em> stages and mesodermal <em>differentiation</em> displayed significant <em>differences</em> among the MSC populations. In undifferentiated UCB<em>1</em> cells, multiple genes were significantly up-regulated (p < 0.000<em>1</em>): peroxisome proliferation activated receptor gamma (PPARG), which correlated with adipogenic <em>differentiation</em> capacities, hepatocyte <em>growth</em> <em>factor</em> (HGF), and stromal-derived <em>factor</em> <em>1</em> (SDF<em>1</em>/CXCL<em>1</em>2), which could both potentially contribute to the higher <em>growth</em> kinetics observed in UCB<em>1</em> cells. Overall, the results confirmed the presence of two distinct isogenic UCB-derived cell populations, identified gene profiles useful to distinguish MSC types with different lineage <em>differentiation</em> potentials, and helped clarify the heterogeneity observed in these cells.

Little is known about the molecular mechanisms controlling primitive hematopoietic stem cells, especially during embryogenesis. Homeobox genes encode a family of transcription <em>factors</em> that have gained increasing attention as master regulators of developmental processes and recently have been implicated in the <em>differentiation</em> and proliferation of hematopoietic cells. Several Hox homeobox genes are now known to be differentially expressed in various subpopulations of human hematopoietic cells and one such gene, HOXB4, has recently been shown to positively determine the proliferative potential of primitive murine bone marrow cells, including cells with long-term repopulating ability. To determine if this gene might influence hematopoiesis at the earliest stages of development, <em>embryonic</em> stem (ES) cells were genetically modified by retroviral gene transfer to overexpress HOXB4 and the effect on their in vitro <em>differentiation</em> was examined. HOXB4 overexpression significantly increased the number of progenitors of mixed erythroid/myeloid colonies and definitive, but not primitive, erythroid colonies derived from embryoid bodies (EBs) at various stages after induction of <em>differentiation</em>. There appeared to be no significant effect on the generation of granulocytic or monocytic progenitors, nor on the efficiency of EB formation or <em>growth</em> rate. Analysis of mRNA from EBs derived from HOXB4-transduced ES cells on different days of primary <em>differentiation</em> showed a significant increase in adult beta-globin expression, with no detectable effect on GATA-<em>1</em> or <em>embryonic</em> globin (beta H-<em>1</em>). Thus, HOXB4 enhances the erythropoietic, and possibly more primitive, hematopoietic differentiative potential of ES cells. These results provide new evidence implicating Hox genes in the control of very early stages in the development of the hematopoietic system and highlight the utility of the ES model for gaining insights into the molecular genetic regulation of <em>differentiation</em> and proliferation events.

Although most imprinted genes show allelic <em>differences</em> in DNA methylation, it is not clear whether methylation regulates the expression of some or all imprinted genes in somatic cells. To examine the mechanisms of silencing of imprinted alleles, we generated novel uniparental mouse <em>embryonic</em> fibroblasts exclusively containing either the paternal or the maternal genome. These fibroblasts retain parent-of-origin allele-specific expression of <em>1</em>2 imprinted genes examined for more than 30 cell generations. We show that p57(Kip2) (cyclin-dependent kinase inhibitor protein 2) and Igf2 (insulin-like <em>growth</em> <em>factor</em> 2) are induced by inhibiting histone deacetylases; however, their activated state is reversed quickly by withdrawal of trichostatin A. In contrast, DNA demethylation results in the heritable expression of a subset of imprinted genes including H<em>1</em>9 (H<em>1</em>9 fetal liver mRNA), p57(Kip2), Peg3/Pw<em>1</em> (paternally expressed gene 3), and Zac<em>1</em> (zinc finger-binding protein regulating apoptosis and cell cycle arrest). Other imprinted genes such as Grb<em>1</em>0 (<em>growth</em> <em>factor</em> receptor-bound protein <em>1</em>0), Peg<em>1</em>/Mest (paternally expressed gene <em>1</em>/mesoderm-specific transcript), Sgce (epsilon-sarcoglycan), Snrpn (small nuclear ribonucleoprotein polypeptide N), and U2af<em>1</em> (U2 small nuclear ribonucleoprotein auxiliary <em>factor</em>), remain inactive, despite their exposure to inhibitors of histone deacetylases and DNA methylation. These results demonstrate that changes in DNA methylation but not histone acetylation create a heritable epigenetic state at some imprinted loci in somatic cells.

The unique pluripotential characteristic of human <em>embryonic</em> stem cells heralds their use in fields such as medicine, biotechnology, biopharmaceuticals, and developmental biology. However, the current availability of sufficient quantities of <em>embryonic</em> stem cells for such applications is limited, and generating sufficient numbers for downstream therapeutic applications is a key concern. In the absence of feeder layers or their conditioned media, human <em>embryonic</em> stem cells readily differentiate to form embryoid bodies, indicating that trophic <em>factors</em> secreted by the feeder layers are required for long-term proliferation and maintenance of pluripotency. Adding further complexity to the elucidation of the <em>factors</em> required for the maintenance of pluripotency is the variability of different fibroblast feeder layers (of mouse or human origin) to effectively support human <em>embryonic</em> stem cells. Currently, the deficiency of knowledge concerning the exact identity of <em>factors</em> within the pathways for self-renewal illustrates that a number of <em>factors</em> may be required to support pluripotent, undifferentiated <em>growth</em> of human <em>embryonic</em> stem cells. This study utilized a proteomic analysis (multidimensional chromatography coupled to tandem mass spectrometry) to isolate and identify proteins in the conditioned media of three mitotically inactivated fibroblast lines (human fetal, human neonatal, and mouse <em>embryonic</em> fibroblasts) used to support the undifferentiated <em>growth</em> of human <em>embryonic</em> stem cells. One-hundred seventy-five unique proteins were identified between the three cell lines using a </=<em>1</em>% false positive rate of identification. These proteins were organized into <em>1</em>7 categories. The <em>differentiation</em> and <em>growth</em> <em>factor</em> and extracellular matrix and remodeling categories contained proteins from many of the key pathways already implicated in the maintenance of human <em>embryonic</em> stem cell pluripotency including the Wnt, BMP/TGF-beta<em>1</em>, Activin/Inhibin, and insulin-like <em>growth</em> <em>factor</em>-<em>1</em> pathways. The conditioned media of fibroblast feeder layers is a complex system, and this study assists in narrowing potential candidates responsible for the support of undifferentiated human <em>embryonic</em> stem cells.

The role of Hedgehogs (Hh) in murine skeletal development was studied by overexpressing human Sonic Hedgehog (SHH) in chondrocytes of transgenic mice using the collagen II promoter/enhancer. Overexpression caused a lethal craniorachischisis with major alterations in long bones because of defects in chondrocyte differentiation.

BACKGROUND

Hedgehogs (Hhs) are a family of secreted polypeptides that play important roles in vertebrate development, controlling many critical steps of cell differentiation and patterning. Skeletal development is affected in many different ways by Hhs. Genetic defects and anomalies of Hhs signaling pathways cause severe abnormalities in the appendicular, axial, and cranial skeleton in man and other vertebrates.

METHODS

Genetic manipulation of mouse embryos was used to study in vivo the function of SHH in skeletal development. By DNA microinjection into pronuclei of fertilized oocytes, we have generated transgenic mice that express SHH specifically in chondrocytes using the cartilage-specific collagen II promoter/enhancer. Transgenic skeletal development was studied at different embryonic stages by histology. The expression pattern of specific chondrocyte molecules was studied by immunohistochemistry and in situ hybridization.

RESULTS

Transgenic mice died at birth with severe craniorachischisis and other skeletal defects in ribs, sternum, and long bones. Detailed analysis of long bones showed that chondrocyte differentiation was blocked at prehypertrophic stages, hindering endochondral ossification and trabecular bone formation, with specific defects in different limb segments. The growth plate was highly disorganized in the tibia and was completely absent in the femur and humerus, leading to skeletal elements entirely made of cartilage surrounded by a thin layer of bone. In this cartilage, chondrocytes maintained a columnar organization that was perpendicular to the bone longitudinal axis and directed toward its outer surface. The expression of SHH receptor, Patched-1 (Ptc1), was greatly increased in all cartilage, as well as the expression of parathyroid hormone-related protein (PTHrP) at the articular surface; while the expression of Indian Hedgehog (Ihh), another member of Hh family that controls the rate of chondrocyte maturation, was greatly reduced and restricted to the displaced chondrocyte columns. Transgenic mice also revealed the ability of SHH to upregulate the expression of Sox9, a major transcription factor implicated in chondrocyte-specific gene expression, in vivo and in vitro, acting through the proximal 6.8-kb-long Sox9 promoter.

CONCLUSIONS

Transgenic mice show that continuous expression of SHH in chondrocytes interferes with cell differentiation and growth plate organization and induces high levels and diffuse expression of Sox9 in cartilaginous bones.

Null-mutant (knockout) mice were obtained through disruption of the sixth exon of the endogenous transforming <em>growth</em> <em>factor</em>-beta <em>1</em> allele in murine <em>embryonic</em> stem cells via homologous recombination. Mice lacking transforming <em>growth</em> <em>factor</em>-beta <em>1</em> (mutants) were born grossly indistinguishable from wild-type littermates. With time, mutant mice exhibited a wasting phenotype that manifested itself in severe weight loss and dishevelled appearance (between <em>1</em>5 and 36 days of age). Examination of these moribund mice histologically revealed that transforming <em>growth</em> <em>factor</em>-beta <em>1</em>-deficient mice exhibit a moderate to severe, multifocal, organ-dependent, mixed inflammatory cell response adversely affecting the heart, stomach, diaphragm, liver, lung, salivary gland, and pancreas. Because of the known multifunctional nature of transforming <em>growth</em> <em>factor</em>-beta <em>1</em> on the control of <em>growth</em> and <em>differentiation</em> of many different cell types, it is important to determine the degree to which the inflammatory response interacts with or masks other deficiencies that are present. To this end, we examined the extent and nature of the inflammatory lesions in different ages of neonatal knockout mice (5, 7, <em>1</em>0, and <em>1</em>4 days of age) and older moribund mice >> <em>1</em>5 days of age) and compared them with the histology seen in wild-type normal animals. Mild inflammatory infiltrates were first observed in 5-day mutant mice in the heart, by day 7 in the lung, salivary gland, and pancreas, and by day <em>1</em>4 inflammatory lesions were found in almost all organs examined. Moderate to severe inflammation was not present until the mice were <em>1</em>0 to <em>1</em>4 days old. In the older animals, there was a slight increase in the severity of the inflammatory lesions as the mice aged.

We present evidence that the cholinergic <em>differentiation</em> <em>factor</em> (CDF), originally purified from cardiac and skeletal muscle cell-conditioned medium and found to be identical to leukemia inhibitory <em>factor</em> (LIF), promotes survival of <em>embryonic</em> day <em>1</em>4 rat motoneurons in vitro. These neurons were retrogradely labeled with the fluorescent tracer Dil and enriched on a density gradient or purified to homogeneity by fluorescence-activated cell sorting. Subnanomolar concentrations of CDF/LIF supported the survival of 85% of the motoneurons that would have died between days <em>1</em> and 4 of culture. The enhanced survival was accompanied by a 4-fold increase in choline acetyltransferase (ChAT) activity per culture. CDF/LIF also increased ChAT activity in dorsal spinal cord cultures, but had no detectable effect on ChAT levels in septal or striatal neuronal cultures. For comparison, other neurotrophic molecules were tested on motoneuron cultures. Ciliary neurotrophic <em>factor</em> had effects on motoneuron survival similar to those of CDF/LIF, whereas basic fibroblast <em>growth</em> <em>factor</em> was somewhat less effective. Nerve <em>growth</em> <em>factor</em> had no effect on the survival of rat motoneurons.

The urogenital sinus (UGS) is specified as prostate in mice around <em>embryonic</em> day <em>1</em>5.5 as indicated by expression of the transcription <em>factor</em> Nkx3.<em>1</em>. Shortly thereafter, <em>growth</em> of epithelial buds into the UGS mesenchyme initiates prostatic morphogenesis. A comparison of male and female UGSs in vivo demonstrated sexually dimorphic expression of branching morphogenesis regulatory genes coincident with epithelial budding including Bmp7, Gli<em>1</em>, Gli2, Fgf<em>1</em>0, Ptch<em>1</em>, and Shh. A comparison of UGSs grown with or without testosterone in serum-free organ cultures showed that some, but not all sexually dimorphic <em>differences</em> observed during prostate bud induction, were effectively modeled in vitro. Organ cultures were then used to investigate the role of fibroblast <em>growth</em> <em>factor</em> receptor (FGFR) signaling during prostatic induction. Blocking FGFR activation with PD<em>1</em>73074 showed that activation of extracellular signal-regulated kinase <em>1</em>/2 (ERK<em>1</em>/2) in the UGS is dependent on FGFR signaling. Furthermore, inhibiting either FGFR activation with PD<em>1</em>73074 or ERK<em>1</em>/2 activation with UO<em>1</em>26 blocked all morphogenesis, proliferation, and gene expression changes induced by androgens in the UGS. These data reveal a previously unknown role for ERK<em>1</em>/2 during prostate bud induction. They also show that signaling by FGFRs through ERK<em>1</em>/2 is required for androgen-induced budding morphogenesis, proliferation, and gene expression during prostate bud induction.

The delineation of regulatory networks involved in early endocrine pancreas specification will play a crucial role in directing the <em>differentiation</em> of <em>embryonic</em> stem cells toward the mature phenotype of beta cells for cell therapy of type <em>1</em> diabetes. The transcription <em>factor</em> Ngn3 is required for the specification of the endocrine lineage, but its direct targets and the scope of biological processes it regulates remain elusive. We show that stepwise <em>differentiation</em> of <em>embryonic</em> stem cells using successive in vivo patterning signals can lead to simultaneous induction of Ptf<em>1</em>a and Pdx<em>1</em> expression. In this cellular context, Ngn3 induction results in upregulation of its known direct target genes within <em>1</em>2 hours. Microarray gene expression profiling at distinct time points following Ngn3 induction suggested novel and diverse roles of Ngn3 in pancreas endocrine cell specification. Induction of Ngn3 expression results in regulation of the Wnt, integrin, Notch, and transforming <em>growth</em> <em>factor</em> beta signaling pathways and changes in biological processes affecting cell motility, adhesion, the cytoskeleton, the extracellular matrix, and gene expression. Furthermore, the combination of in vivo patterning signals and inducible Ngn3 expression enhances ESC <em>differentiation</em> toward the pancreas endocrine lineage. This is shown by strong upregulation of endocrine lineage terminal <em>differentiation</em> markers and strong expression of the hormones glucagon, somatostatin, and insulin. Importantly, all insulin(+) cells are also C-peptide(+), and glucose-dependent insulin release was <em>1</em>0-fold higher than basal levels. These data suggest that bona fide pancreas endocrine cells have been generated and that timely induction of Ngn3 expression can play a decisive role in directing ESC <em>differentiation</em> toward the endocrine lineage.

Coronary heart disease (CHD) is the leading cause of mortality in both developed and developing countries worldwide. Genome-wide association studies (GWAS) have now identified 46 independent susceptibility loci for CHD, however, the biological and disease-relevant mechanisms for these associations remain elusive. The large-scale meta-analysis of GWAS recently identified in Caucasians a CHD-associated locus at chromosome 6q23.2, a region containing the transcription <em>factor</em> TCF2<em>1</em> gene. TCF2<em>1</em> (Capsulin/Pod<em>1</em>/Epicardin) is a member of the basic-helix-loop-helix (bHLH) transcription <em>factor</em> family, and regulates cell fate decisions and <em>differentiation</em> in the developing coronary vasculature. Herein, we characterize a cis-regulatory mechanism by which the lead polymorphism rs<em>1</em>2<em>1</em>90287 disrupts an atypical activator protein <em>1</em> (AP-<em>1</em>) element, as demonstrated by allele-specific transcriptional regulation, transcription <em>factor</em> binding, and chromatin organization, leading to altered TCF2<em>1</em> expression. Further, this element is shown to mediate signaling through platelet-derived <em>growth</em> <em>factor</em> receptor beta (PDGFR-β) and Wilms tumor <em>1</em> (WT<em>1</em>) pathways. A second disease allele identified in East Asians also appears to disrupt an AP-<em>1</em>-like element. Thus, both disease-related <em>growth</em> <em>factor</em> and <em>embryonic</em> signaling pathways may regulate CHD risk through two independent alleles at TCF2<em>1</em>.