The extracellular signals which regulate the myogenic program are transduced to the nucleus by mitogen-activated protein kinases (MAPKs). We have investigated the role of two MAPKs, p38 and extracellular signal-regulated kinase (ERK), whose activities undergo significant changes during muscle <em>differentiation</em>. p38 is rapidly activated in myocytes induced to differentiate. This activation differs from those triggered by stress and cytokines, because it is not linked to Jun-N-terminal kinase stimulation and is maintained during the whole process of myotube formation. Moreover, p38 activation is independent of a parallel promyogenic pathway stimulated by insulin-like <em>growth</em> <em>factor</em> <em>1</em>. Inhibition of p38 prevents the <em>differentiation</em> program in myogenic cell lines and human primary myocytes. Conversely, deliberate activation of endogenous p38 stimulates muscle <em>differentiation</em> even in the presence of antimyogenic cues. Much evidence indicates that p38 is an activator of MyoD: (i) p38 kinase activity is required for the expression of MyoD-responsive genes, (ii) enforced induction of p38 stimulates the transcriptional activity of a Gal4-MyoD fusion protein and allows efficient activation of chromatin-integrated reporters by MyoD, and (iii) MyoD-dependent myogenic conversion is reduced in mouse <em>embryonic</em> fibroblasts derived from p38alpha(-/-) embryos. Activation of p38 also enhances the transcriptional activities of myocyte enhancer binding <em>factor</em> 2A (MEF2A) and MEF2C by direct phosphorylation. With MEF2C, selective phosphorylation of one residue (Thr293) is a tissue-specific activating signal in differentiating myocytes. Finally, ERK shows a biphasic activation profile, with peaks of activity in undifferentiated myoblasts and postmitotic myotubes. Importantly, activation of ERK is inhibitory toward myogenic transcription in myoblasts but contributes to the activation of myogenic transcription and regulates postmitotic responses (i.e., hypertrophic <em>growth</em>) in myotubes.

CYR6<em>1</em> (CCN<em>1</em>) is a member of the CCN family of secreted matricellular proteins that includes connective tissue <em>growth</em> <em>factor</em> (CCN2), NOV (CCN3), WISP-<em>1</em> (CCN4), WISP-2 (CCN5), and WISP-3 (CCN6). First identified as the product of a <em>growth</em> <em>factor</em>-inducible immediate-early gene, CYR6<em>1</em> is an extracellular matrix-associated angiogenic inducer that functions as a ligand of integrin receptors to promote cell adhesion, migration, and proliferation. Aberrant expression of Cyr6<em>1</em> is associated with breast cancer, wound healing, and vascular diseases such as atherosclerosis and restenosis. To understand the functions of CYR6<em>1</em> during development, we have disrupted the Cyr6<em>1</em> gene in mice. We show here that Cyr6<em>1</em>-null mice suffer <em>embryonic</em> death: approximately 30% succumbed to a failure in chorioallantoic fusion, and the reminder perished due to placental vascular insufficiency and compromised vessel integrity. These findings establish CYR6<em>1</em> as a novel and essential regulator of vascular development. CYR6<em>1</em> deficiency results in a specific defect in vessel bifurcation (nonsprouting angiogenesis) at the chorioallantoic junction, leading to an undervascularization of the placenta without affecting <em>differentiation</em> of the labyrinthine syncytiotrophoblasts. This unique phenotype is correlated with impaired Vegf-C expression in the allantoic mesoderm, suggesting that CYR6<em>1</em>-regulated expression of Vegf-C plays a role in vessel bifurcation. The genetic and molecular basis of vessel bifurcation is presently unknown, and these findings provide new insight into this aspect of angiogenesis.

Neuregulins (i.e. neuregulin-<em>1</em> (NRG<em>1</em>), also called neu <em>differentiation</em> <em>factor</em>, heregulin, glial <em>growth</em> <em>factor</em>, and acetylcholine receptor-inducing activity) are known to induce <em>growth</em> and <em>differentiation</em> of epithelial, glial, neuronal, and skeletal muscle cells. Unexpectedly, mice with loss of function mutations of NRG<em>1</em> or of either of two of their cognate receptors, ErbB2 and ErbB4, die during midembryogenesis due to the aborted development of myocardial trabeculae in ventricular muscle. To examine the role of NRG and their receptors in developing and postnatal myocardium, we studied the ability of a soluble NRG<em>1</em> (recombinant human glial <em>growth</em> <em>factor</em> 2) to promote proliferation, survival, and <em>growth</em> of isolated neonatal and adult rat cardiac myocytes. Both ErbB2 and ErbB4 receptors were found to be expressed by neonatal and adult ventricular myocytes and activated by rhGGF2. rhGGF2 (30 ng/ml) provoked an approximate 2-fold increase in <em>embryonic</em> cardiac myocyte proliferation. rhGGF2 also promoted survival and inhibited apoptosis of subconfluent, serum-deprived myocyte primary cultures and also induced hypertrophic <em>growth</em> in both neonatal and adult ventricular myocytes, which was accompanied by enhanced expression of prepro-atrial natriuretic <em>factor</em> and skeletal alpha-actin. Moreover, NRG<em>1</em> mRNA could be detected in coronary microvascular endothelial cell primary cultures prepared from adult rat ventricular muscle. NRG<em>1</em> expression in these cells was increased by endothelin-<em>1</em>, another locally acting cardiotropic peptide within the heart. The persistent expression of both a neuregulin and its cognate receptors in the postnatal and adult heart suggests a continuing role for neuregulins in the myocardial adaption to physiologic stress or injury.

Pluripotent stem cells self-renew indefinitely and possess characteristic protein-protein networks that remodel during <em>differentiation</em>. How this occurs is poorly understood. Using quantitative mass spectrometry, we analyzed the (phospho)proteome of human <em>embryonic</em> stem cells (hESCs) during <em>differentiation</em> induced by bone morphogenetic protein (BMP) and removal of hESC <em>growth</em> <em>factors</em>. Of 5222 proteins identified, <em>1</em>399 were phosphorylated on 3067 residues. Approximately 50% of these phosphosites were regulated within <em>1</em> hr of <em>differentiation</em> induction, revealing a complex interplay of phosphorylation networks spanning different signaling pathways and kinase activities. Among the phosphorylated proteins was the pluripotency-associated protein SOX2, which was SUMOylated as a result of phosphorylation. Using the data to predict kinase-substrate relationships, we reconstructed the hESC kinome; CDK<em>1</em>/2 emerged as central in controlling self-renewal and lineage specification. The findings provide new insights into how hESCs exit the pluripotent state and present the hESC (phospho)proteome resource as a complement to existing pluripotency network databases.

The proto-oncogene c-jun is the cellular homologue of v-jun, the transforming oncogene of the avian sarcoma virus <em>1</em>7 (ref. <em>1</em>). c-jun encodes one major component of the AP-<em>1</em> transcription <em>factor</em> complex and is expressed in many organs during mouse development and in the adult. Because of its rapid induction in cells following <em>growth</em> stimulation and the presence of AP-<em>1</em> binding sites in the promoter regions of many genes, the c-Jun protein is thought to have important functions in cell proliferation and <em>differentiation</em>. But <em>embryonic</em> stem (ES) cells lacking c-Jun are viable and have a normal in vitro <em>differentiation</em> capacity, although c-Jun appears to be important for <em>growth</em> of teratocarcinomas in vivo. To define the function of c-jun better, targeted ES cells were used to generate mice lacking c-Jun. Here we report that heterozygous mutant mice appear normal, but embryos lacking c-Jun die at mid- to late-gestation and exhibit impaired hepatogenesis, altered fetal liver erythropoiesis and generalized oedema. Interestingly, c-jun-/- ES cells can participate efficiently in the development of all somatic cells in chimaeric mice except liver cells, further suggesting an essential function of c-Jun in hepatogenesis.

R325-beta TK+, a herpes simplex virus <em>1</em> mutant carrying a 500-base-pair deletion in the alpha 22 gene and the wild-type (beta) thymidine kinase (TK) gene, was previously shown to grow efficiently in HEp-2 and Vero cell lines. We report that in rodent cell lines exemplified by the Rat-<em>1</em> line, plating efficiency was reduced and <em>growth</em> was multiplicity dependent. A similar multiplicity dependence for <em>growth</em> and lack of virus spread at low multiplicity was seen in resting, confluent human <em>embryonic</em> lung (HEL) cells. The shutoff of synthesis of beta proteins was delayed and the duration of synthesis of gamma proteins was extended in R325-beta TK+-infected HEL cells relative to cells infected with the wild-type parent, but no significant <em>differences</em> were seen in the total accumulation of viral DNA. To quantify the effect on late (gamma 2) gene expression, a recombinant carrying the deletion in the alpha 22 gene and a gamma 2-TK gene (R325-gamma 2 TK) was constructed and compared with a wild-type virus (R3<em>1</em><em>1</em>2) carrying a chimeric gamma 2-TK gene. In Vero cells, the gamma 2-TK gene of R325-gamma 2TK was expressed earlier than and at the same level as the gamma 2-TK gene of R3<em>1</em><em>1</em>2. In the confluent resting HEL cells, the expression of the gamma 2-TK gene of the alpha 22- virus was grossly reduced relative to that of the alpha 22+ virus. Electron microscopic studies indicated that the number of intranuclear capsids of R325-beta TK+ virus was reduced relative to that of the parent virus in resting confluent HEL cells, but the number of DNA-containing capsids was higher. Notwithstanding the grossly reduced neurovirulence on intracerebral inoculation in mice, R325-beta TK+ virus was able to establish latency in mice. We conclude that (i) the alpha 22 gene affects late (gamma 2) gene expression, and (ii) a host cell <em>factor</em> complements that function of the alpha 22 gene to a greater extent in HEp-2 and Vero cells than in confluent, resting HEL cells.

BACKGROUND

Epithelial-mesenchymal transformation (EMT) plays an important role in embryonic development and tumorigenesis and has been described in organ remodeling during fibrogenesis. In the kidney, EMT can be induced efficiently in cultured proximal tubular epithelium by coincubation of transforming growth factor (TGF)-beta1 and epidermal growth factor (EGF). Recently, we also have observed overexpression of basic fibroblast growth factor-2 (FGF-2) protein and mRNA in human kidneys with marked interstitial fibrosis. The aims of the present study were to compare the effects of FGF-2 as a facilitator of EMT in tubular epithelial cells with EGF and TGF-beta1. We analyzed the morphogenic effects of the three cytokines on four different aspects of EMT: cell motility, expression and regulation of cellular markers, synthesis and secretion of extracellular matrix (ECM) proteins as well as matrix degradation.

METHODS

Cell motility was studied by a migration assay and cell differentiation markers were analyzed by immunofluorescence and immunoblots. In addition, regulation of the epithelial adhesion molecule E-cadherin and fibroblast-specific protein 1 (FSP1) were analyzed by luciferase reporter constructs and stable transfections. ELISAs for collagen types I and IV and fibronectin were used for ECM synthesis, and zymograms were utilized for analysis of matrix degradation.

RESULTS

FGF-2 induced cell motility across a tubular basement membrane in two tubular cell lines. All three cytokines induced the expression of vimentin and FSP1, but only FGF-2 and TGF-beta1 reduced cytokeratin expression by immunofluorescence. These effects were most demonstrable in the distal tubular epithelial cell line and were confirmed by immunoblot analyses. Expression of E-cadherin was reduced by 61.5 +/- 3.3% and expression of cytokeratin by 91 +/- 0.5% by TGF-beta1 plus FGF-2. Conversely, the mesenchymal markers alpha-smooth muscle actin (SMA) and FSP1 were induced with FGF-2 by 2.2 +/- 0.1-fold and 6.8 +/- 0.9-fold, respectively. Interestingly, de novo expression of the mesenchymal marker OB-cadherin was induced only by FGF-2 and EGF but not by TGF-beta1. All three cytokines stimulated FSP1 and decreased E-cadherin promoter activity. FGF-2 also induced intracellular fibronectin synthesis but not secretion, the latter of which was stimulated exclusively by TGF-beta1. Finally, zymographic analyses demonstrated that FGF-2 induced MMP-2 activity by 2.6 +/- 0.5-fold and MMP-9 activity by 2.4 +/- 0.1-fold, providing a mechanism for basement membrane disintegration and migratory access of transforming epithelium to the interstitium.

CONCLUSIONS

FGF-2 makes an important contribution to the mechanisms of EMT by stimulating microenvironmental proteases essential for disaggregation of organ-based epithelial units. Furthermore, the expression of epithelial and mesenchymal marker proteins seems to be affected at the promoter level.

Axon out<em>growth</em> is the first step in the formation of neuronal connections, but the pathways that regulate axon extension are still poorly understood. We find that mice deficient in calcineurin-NFAT signaling have dramatic defects in axonal out<em>growth</em>, yet have little or no defect in neuronal <em>differentiation</em> or survival. In vitro, sensory and commissural neurons lacking calcineurin function or NFATc2, c3, and c4 are unable to respond to neurotrophins or netrin-<em>1</em> with efficient axonal out<em>growth</em>. Neurotrophins and netrins stimulate calcineurin-dependent nuclear localization of NFATc4 and activation of NFAT-mediated gene transcription in cultured primary neurons. These data indicate that the ability of these <em>embryonic</em> axons to respond to <em>growth</em> <em>factors</em> with rapid out<em>growth</em> requires activation of calcineurin/NFAT signaling by these <em>factors</em>. The precise parsing of signals for elongation turning and survival could allow independent control of these processes during development.

Here we present a protocol for extraction and culture of neurons from adult rat or mouse CNS. The method proscribes an optimized protease digestion of slices, control of osmolarity and pH outside the incubator with Hibernate and density gradient separation of neurons from debris. This protocol produces yields of millions of cortical, hippocampal neurons or neurosphere progenitors from each brain. The entire process of neuron isolation and culture takes less than 4 h. With suitable <em>growth</em> <em>factors</em>, adult neuron regeneration of axons and dendrites in culture proceeds over <em>1</em>-3 weeks to allow controlled studies in pharmacology, electrophysiology, development, regeneration and neurotoxicology. Adult neurospheres can be collected in <em>1</em> week as a source of neuroprogenitors ethically preferred over <em>embryonic</em> or fetal sources. This protocol emphasizes two <em>differences</em> between neuron <em>differentiation</em> and neurosphere proliferation: adhesion dependence and the differentiating power of retinyl acetate.

Despite progress in developing defined conditions for human <em>embryonic</em> stem cell (hESC) cultures, little is known about the cell-surface receptors that are activated under conditions supportive of hESC self-renewal. A simultaneous interrogation of 42 receptor tyrosine kinases (RTKs) in hESCs following stimulation with mouse <em>embryonic</em> fibroblast (MEF) conditioned medium (CM) revealed rapid and prominent tyrosine phosphorylation of insulin receptor (IR) and insulin-like <em>growth</em> <em>factor</em>-<em>1</em> receptor (IGF<em>1</em>R); less prominent tyrosine phosphorylation of epidermal <em>growth</em> <em>factor</em> receptor (EGFR) family members, including ERBB2 and ERBB3; and trace phosphorylation of fibroblast <em>growth</em> <em>factor</em> receptors. Intense IGF<em>1</em>R and IR phosphorylation occurred in the absence of MEF conditioning (NCM) and was attributable to high concentrations of insulin in the proprietary KnockOut Serum Replacer (KSR). Inhibition of IGF<em>1</em>R using a blocking antibody or lentivirus-delivered shRNA reduced hESC self-renewal and promoted <em>differentiation</em>, while disruption of ERBB2 signaling with the selective inhibitor AG825 severely inhibited hESC proliferation and promoted apoptosis. A simple defined medium containing an IGF<em>1</em> analog, heregulin-<em>1</em>beta (a ligand for ERBB2/ERBB3), fibroblast <em>growth</em> <em>factor</em>-2 (FGF2), and activin A supported long-term <em>growth</em> of multiple hESC lines. These studies identify previously unappreciated RTKs that support hESC proliferation and self-renewal, and provide a rationally designed medium for the <em>growth</em> and maintenance of pluripotent hESCs.

The multilineage <em>differentiation</em> potential of adult tissue-derived mesenchymal progenitor cells (MPCs), such as those from bone marrow and trabecular bone, makes them a useful model to investigate mechanisms regulating tissue development and regeneration, such as cartilage. Treatment with transforming <em>growth</em> <em>factor</em>-beta (TGF-beta) superfamily members is a key requirement for the in vitro chondrogenic <em>differentiation</em> of MPCs. Intracellular signaling cascades, particularly those involving the mitogen-activated protein (MAP) kinases, p38, ERK-<em>1</em>, and JNK, have been shown to be activated by TGF-betas in promoting cartilage-specific gene expression. MPC chondrogenesis in vitro also requires high cell seeding density, reminiscent of the cellular condensation requirements for <em>embryonic</em> mesenchymal chondrogenesis, suggesting common chondro-regulatory mechanisms. Prompted by recent findings of the crucial role of the cell adhesion protein, N-cadherin, and Wnt signaling in condensation and chondrogenesis, we have examined here their involvement, as well as MAP kinase signaling, in TGF-beta<em>1</em>-induced chondrogenesis of trabecular bone-derived MPCs. Our results showed that TGF-beta<em>1</em> treatment initiates and maintains chondrogenesis of MPCs through the differential chondro-stimulatory activities of p38, ERK-<em>1</em>, and to a lesser extent, JNK. This regulation of MPC chondrogenic <em>differentiation</em> by the MAP kinases involves the modulation of N-cadherin expression levels, thereby likely controlling condensation-like cell-cell interaction and progression to chondrogenic <em>differentiation</em>, by the sequential up-regulation and progressive down-regulation of N-cadherin. TGF-beta<em>1</em>-mediated MAP kinase activation also controls WNT-7A gene expression and Wnt-mediated signaling through the intracellular beta-catenin-TCF pathway, which likely regulates N-cadherin expression and subsequent N-cadherin-mediated cell-adhesion complexes during the early steps of MPC chondrogenesis.

Insufficient oxygen and nutrient supply often restrain solid tumor <em>growth</em>, and the hypoxia-inducible <em>factors</em> (HIF) <em>1</em> alpha and HIF-2 alpha are key transcription regulators of phenotypic adaptation to low oxygen levels. Moreover, mouse gene disruption studies have implicated HIF-2 alpha in <em>embryonic</em> regulation of tyrosine hydroxylase, a hallmark gene of the sympathetic nervous system. Neuroblastoma tumors originate from immature sympathetic cells, and therefore we investigated the effect of hypoxia on the <em>differentiation</em> status of human neuroblastoma cells. Hypoxia stabilized HIF-<em>1</em> alpha and HIF-2 alpha proteins and activated the expression of known hypoxia-induced genes, such as vascular endothelial <em>growth</em> <em>factor</em> and tyrosine hydroxylase. These changes in gene expression also occurred in hypoxic regions of experimental neuroblastoma xenografts grown in mice. In contrast, hypoxia decreased the expression of several neuronal/neuroendocrine marker genes but induced genes expressed in neural crest sympathetic progenitors, for instance c-kit and Notch-<em>1</em>. Thus, hypoxia apparently causes de<em>differentiation</em> both in vitro and in vivo. These findings suggest a novel mechanism for selection of highly malignant tumor cells with stem-cell characteristics.

Previous efforts to differentiate human <em>embryonic</em> stem cells (hESCs) into endothelial cells have not achieved sustained expansion and stability of vascular cells. To define vasculogenic developmental pathways and enhance <em>differentiation</em>, we used an endothelial cell-specific VE-cadherin promoter driving green fluorescent protein (GFP) (hVPr-GFP) to screen for <em>factors</em> that promote vascular commitment. In phase <em>1</em> of our method, inhibition of transforming <em>growth</em> <em>factor</em> (TGF)beta at day 7 of <em>differentiation</em> increases hVPr-GFP(+) cells by tenfold. In phase 2, TGFbeta inhibition maintains the proliferation and vascular identity of purified endothelial cells, resulting in a net 36-fold expansion of endothelial cells in homogenous monolayers, which exhibited a transcriptional profile of Id<em>1</em>(high)VEGFR2(high)VE-cadherin(+) ephrinB2(+). Using an Id<em>1</em>-YFP hESC reporter line, we showed that TGFbeta inhibition sustains Id<em>1</em> expression in hESC-derived endothelial cells and that Id<em>1</em> is required for increased proliferation and preservation of endothelial cell commitment. Our approach provides a serum-free method for <em>differentiation</em> and long-term maintenance of hESC-derived endothelial cells at a scale relevant to clinical application.

MicroRNAs (miRNAs) are short non-coding RNAs that have been implicated in fine-tuning gene regulation, although the precise roles of many are still unknown. Pancreatic development is characterized by the complex sequential expression of a gamut of transcription <em>factors</em>. We have performed miRNA expression profiling at two key stages of mouse <em>embryonic</em> pancreas development, e<em>1</em>4.5 and e<em>1</em>8.5. miR-<em>1</em>24a2 expression was strikingly increased at e<em>1</em>8.5 compared with e<em>1</em>4.5, suggesting a possible role in differentiated beta-cells. Among the potential miR-<em>1</em>24a gene targets identified by biocomputation, Foxa2 is known to play a role in beta-cell <em>differentiation</em>. To evaluate the impact of miR-<em>1</em>24a2 on gene expression, we overexpressed or down-regulated miR-<em>1</em>24a2 in MIN6 beta-cells. As predicted, miR-<em>1</em>24a2 regulated Foxa2 gene expression, and that of its downstream target, pancreatic duodenum homeobox-<em>1</em> (Pdx-<em>1</em>). Foxa2 has been described as a master regulator of pancreatic development and also of genes involved in glucose metabolism and insulin secretion, including the ATP-sensitive K(+) (K(ATP)) channel subunits, Kir6.2 and Sur-<em>1</em>. Correspondingly, miR-<em>1</em>24a2 overexpression decreased, and anti-miR-<em>1</em>24a2 increased Kir6.2 and Sur-<em>1</em> mRNA levels. Moreover, miR-<em>1</em>24a2 modified basal and glucose- or KCl-stimulated intracellular free Ca(2+) concentrations in single MIN6 and INS-<em>1</em> (832/<em>1</em>3) beta-cells, without affecting the secretion of insulin or co-transfected human <em>growth</em> hormone, consistent with an altered sensitivity of the beta-cell exocytotic machinery to Ca(2+). In conclusion, whereas the precise role of microRNA-<em>1</em>24a2 in pancreatic development remains to be deciphered, we identify it as a regulator of a key transcriptional protein network in beta-cells responsible for modulating intracellular signaling.

The AP-<em>1</em> transcription <em>factors</em> are considered immediate-early response genes and are thought to be involved in a wide range of transcriptional regulatory processes linked to cellular proliferation and <em>differentiation</em>. To study one of the key members of this family, the proto-oncogene c-jun, we have used homologous recombination-mediated gene targeting to produce mice with a c-jun null mutation. c-jun null embryos die at mid-gestation, with an average time of death of <em>1</em>2.5 days postcoitus. Homozygous mutant embryos are indistinguishable from wild-type littermates both grossly and histologically until the time of death. However, primary fibroblasts derived from live heterozygous and homozygous mutant embryos show greatly reduced <em>growth</em> rates in culture. The subnormal mitogenic response of these cells cannot be overcome by the addition of a number of purified mitogens. These studies indicate that although c-jun is not required for cellular proliferation and <em>differentiation</em> up to mid-gestation, it is required for survival past that stage as well as for the mitogenic response of <em>embryonic</em> fibroblasts in culture.

Human <em>embryonic</em> stem cells (hESCs) hold enormous promise for regenerative medicine. Typically, hESC-based applications would require their in vitro <em>differentiation</em> into a desirable homogenous cell population. A major challenge of the current hESC <em>differentiation</em> paradigm is the inability to effectively capture and, in the long-term, stably expand primitive lineage-specific stem/precursor cells that retain broad <em>differentiation</em> potential and, more importantly, developmental stage-specific <em>differentiation</em> propensity. Here, we report synergistic inhibition of glycogen synthase kinase 3 (GSK3), transforming <em>growth</em> <em>factor</em> β (TGF-β), and Notch signaling pathways by small molecules can efficiently convert monolayer cultured hESCs into homogenous primitive neuroepithelium within <em>1</em> wk under chemically defined condition. These primitive neuroepithelia can stably self-renew in the presence of leukemia inhibitory <em>factor</em>, GSK3 inhibitor (CHIR9902<em>1</em>), and TGF-β receptor inhibitor (SB43<em>1</em>542); retain high neurogenic potential and responsiveness to instructive neural patterning cues toward midbrain and hindbrain neuronal subtypes; and exhibit in vivo integration. Our work uniformly captures and maintains primitive neural stem cells from hESCs.

To realize the therapeutic potential of human <em>embryonic</em> stem cells (hESCs), it is necessary to regulate their <em>differentiation</em> in a uniform and reproducible manner. We have developed a method in which known numbers of hESCs in serum-free medium were aggregated by centrifugation to foster the formation of embryoid bodies (EBs) of uniform size (spin EBs). These spin EBs differentiated efficiently and synchronously, as evidenced by the sequential expression of molecular markers representing stem cells, primitive streak, and mesoderm. In the presence of hematopoietic <em>growth</em> <em>factors</em>, reproducible <em>differentiation</em> was achieved with blood cells formed in more than 90% of EBs. Using chimeric EBs generated from mixtures of green fluorescence protein-positive (GFP(+)) and GFP(-) hESCs in a clonogenic assay, hematopoietic precursor frequency was estimated to be approximately <em>1</em>:500 input cells. This method of EB formation provides a generally applicable means for modulating and objectively monitoring the directed <em>differentiation</em> of hESCs.

The Runx2 (CBFA<em>1</em>/AML3/PEBP2alphaA) transcription <em>factor</em> promotes lineage commitment and <em>differentiation</em> by activating bone phenotypic genes in postproliferative osteoblasts. However, the presence of Runx2 in actively dividing osteoprogenitor cells suggests that the protein may also participate in control of osteoblast <em>growth</em>. Here, we show that Runx2 is stringently regulated with respect to cell cycle entry and exit in osteoblasts. We addressed directly the contribution of Runx2 to bone cell proliferation using calvarial osteoblasts from wild-type and Runx2-deficient mice (i.e., Runx2(-/-) and Runx2(DeltaC/DeltaC)). Runx2(DeltaC/DeltaC) mice express a protein lacking the Runx2 COOH terminus, which integrates several cell proliferation-related signaling pathways (e.g., Smad, Yes/Src, mitogen-activated protein kinase, and retinoblastoma protein). Calvarial cells but not <em>embryonic</em> fibroblasts from Runx2(-/-) or Runx2(DeltaC/DeltaC) mutant mice exhibit increased cell <em>growth</em> rates as reflected by elevations of DNA synthesis and G(<em>1</em>)-S phase markers (e.g., cyclin E). Reintroduction of Runx2 into Runx2(-/-) calvarial cells by adenoviral delivery restores stringent cell <em>growth</em> control. Thus, Runx2 regulates normal osteoblast proliferation, and the COOH-terminal region is required for this biological function. We propose that Runx2 promotes osteoblast maturation at a key developmental transition by supporting exit from the cell cycle and activating genes that facilitate bone cell phenotype development.

We report here the first attempt to produce and use whole acellular (AC) lung as a matrix to support development of engineered lung tissue from murine <em>embryonic</em> stem cells (mESCs). We compared the influence of AC lung, Gelfoam, Matrigel, and a collagen I hydrogel matrix on the mESC attachment, <em>differentiation</em>, and subsequent formation of complex tissue. We found that AC lung allowed for better retention of cells with more <em>differentiation</em> of mESCs into epithelial and endothelial lineages. In constructs produced on whole AC lung, we saw indications of organization of differentiating ESC into three-dimensional structures reminiscent of complex tissues. We also saw expression of thyroid transcription <em>factor</em>-<em>1</em>, an immature lung epithelial cell marker; pro-surfactant protein C, a type II pneumocyte marker; PECAM-<em>1</em>/CD3<em>1</em>, an endothelial cell marker; cytokeratin <em>1</em>8; alpha-actin, a smooth muscle marker; CD<em>1</em>40a or platelet-derived <em>growth</em> <em>factor</em> receptor-alpha; and Clara cell protein <em>1</em>0. There was also evidence of site-specific <em>differentiation</em> in the trachea with the formation of sheets of cytokeratin-positive cells and Clara cell protein <em>1</em>0-expressing Clara cells. Our findings support the utility of AC lung as a matrix for engineering lung tissue and highlight the critical role played by matrix or scaffold-associated cues in guiding ESC <em>differentiation</em> toward lung-specific lineages.

OBJECTIVE

Mesenchymal stem cells (MSCs) are promising candidate cells for cartilage tissue engineering. Expression of cartilage hypertrophy markers (e.g., type X collagen) by MSCs undergoing chondrogenesis raises concern for a tissue engineering application for MSCs, because hypertrophy would result in apoptosis and ossification. To analyze the biologic basis of MSC hypertrophy, we examined the response of chondrifying MSCs to culture conditions known to influence chondrocyte hypertrophy, using an array of hypertrophy-associated markers.

METHODS

Human MSC pellet cultures were predifferentiated for 2 weeks in a chondrogenic medium, and hypertrophy was induced by withdrawing transforming growth factor beta (TGFbeta), reducing the concentration of dexamethasone, and adding thyroid hormone (T3). Cultures were characterized by histologic, immunohistochemical, and biochemical methods, and gene expression was assessed using quantitative reverse transcription-polymerase chain reaction.

RESULTS

The combination of TGFbeta withdrawal, a reduction in the level of dexamethasone, and the addition of T3 was essential for hypertrophy induction. Cytomorphologic changes were accompanied by increased alkaline phosphatase activity, matrix mineralization, and changes in various markers of hypertrophy, including type X collagen, fibroblast growth factor receptors 1-3, parathyroid hormone-related protein receptor, retinoic acid receptor gamma, matrix metalloproteinase 13, Indian hedgehog, osteocalcin, and the proapoptotic gene p53. However, hypertrophy was not induced uniformly throughout the pellet culture, and distinct regions of dedifferentiation were observed.

CONCLUSIONS

Chondrogenically differentiating MSCs behave in a manner functionally similar to that of growth plate chondrocytes, expressing a very similar hypertrophic phenotype. Under the in vitro culture conditions used here, MSC-derived chondrocytes underwent a differentiation program analogous to that observed during endochondral embryonic skeletal development, with the potential for terminal differentiation. This culture system is applicable for the screening of hypertrophy-inhibitory conditions and agents that may be useful to enhance MSC performance in cartilage tissue engineering.

Development of the anterior pituitary gland involves proliferation and <em>differentiation</em> of ectodermal cells in Rathke's pouch to generate five distinct cell types that are defined by the trophic hormones they produce. A detailed ontogenetic analysis of specific gene expression has revealed novel aspects of organogenesis in this model system. The expression of transcripts encoding the alpha-subunit common to three pituitary glycoprotein hormones in the single layer of somatic ectoderm on <em>embryonic</em> day <em>1</em><em>1</em> established that primordial pituitary cell commitment occurs prior to formation of a definitive Rathke's pouch. Activation of Pit-<em>1</em> gene expression occurs as an organ-specific event, with Pit-<em>1</em> transcripts initially detected in anterior pituitary cells on <em>embryonic</em> day <em>1</em>5. Levels of Pit-<em>1</em> protein closely parallel those of Pit-<em>1</em> transcripts without a significant lag. Unexpectedly, Pit-<em>1</em> transcripts remain highly expressed in all five cell types of the mature pituitary gland, but the Pit-<em>1</em> protein is detected in only three cell types--lactotrophs, somatotrophs, and thyrotrophs and not in gonadotrophs or corticotrophs. The presence of Pit-<em>1</em> protein in thyrotrophs suggests that combinatorial actions of specific activating and restricting <em>factors</em> act to confine prolactin and <em>growth</em> hormone gene expression to lactotrophs and somatotrophs, respectively. A linkage between the initial appearance of Pit-<em>1</em> protein and the surprising coactivation of prolactin and <em>growth</em> hormone gene expression is consistent with the model that Pit-<em>1</em> is responsible for the initial transcriptional activation of both genes. The estrogen receptor, which has been reported to be activated in a stereotypic fashion subsequent to the appearance of Pit-<em>1</em>, appears to be capable, in part, of mediating the progressive increase in prolactin gene expression characteristic of the mature lactotroph phenotype. This is a consequence of synergistic transcriptional effects with Pit-<em>1</em>, on the basis of binding of the estrogen receptor to a response element in the prolactin gene distal enhancer. These data imply that both transcriptional and post-transcriptional regulation of Pit-<em>1</em> gene expression and combinatorial actions with other classes of transcription <em>factors</em> activated in distinct temporal patterns, are required for the mature physiological patterns of gene expression that define distinct cell types within the anterior pituitary gland.

In this review, we define the two major tissue types, epithelium and mesenchyme, and we describe the transformations (trans<em>differentiation</em>s) of epithelium to mesenchyme (EMT) and mesenchyme to epithelium (MET) that occur during <em>embryonic</em> development. The <em>differentiation</em> of the metanephric blastema provides a striking example of MET. <em>Differentiation</em> of metanephric epithelium is promoted by matrix molecules and receptors (nidogen, laminins, alpha 6 integrins), hepatic <em>growth</em> <em>factor</em>/scatter <em>factor</em>, and products of the genes wnt-<em>1</em>, wnt-4, and Pax-2. Transformation of MDCK epithelium to mesenchyme-like cells is promoted in vitro by antibodies to E-cadherin, products of v-src, v-ras, and v-mos, and by manipulation of the epithelium on collagen gels. Suspension in collagen gel, transforming <em>growth</em> <em>factors</em>, and c-fos have also been shown to promote EMT in epithelia. We present studies from our laboratory showing that alpha 5 beta <em>1</em> integrin has a role in the EMT of lens epithelium that is brought about by suspension in collagen gel. Our laboratory has also shown that transfection with the E-cadherin gene induces <em>embryonic</em> corneal fibroblasts to undergo MET and that this MET is enhanced by interaction of the differentiating epithelium with living fibroblasts. This review calls attention to the roles that EMT and MET might have in kidney pathologies and urges further study of the involvement of these phenomena in renal development, renal injury, and renal malignancy.

The <em>embryonic</em> vasculature develops from endothelial cells that form a primitive vascular plexus which recruits smooth muscle cells to form the arterial and venous systems. The MADS-box transcription <em>factor</em> MEF2C is expressed in developing endothelial cells and smooth muscle cells (SMCs), as well as in surrounding mesenchyme, during embryogenesis. Targeted deletion of the mouse MEF2C gene resulted in severe vascular abnormalities and lethality in homozygous mutants by <em>embryonic</em> day 9.5. Endothelial cells were present and were able to differentiate, but failed to organize normally into a vascular plexus, and smooth muscle cells did not differentiate in MEF2C mutant embryos. These vascular defects resemble those in mice lacking the vascular-specific endothelial cell <em>growth</em> <em>factor</em> VEGF or its receptor Flt-<em>1</em>, both of which are expressed in MEF2C mutant embryos. These results reveal multiple roles for MEF2C in vascular development and suggest that MEF2-dependent target genes mediate endothelial cell organization and SMC <em>differentiation</em>.

Understanding the gene programmes that regulate maintenance and <em>differentiation</em> of neural stem cells is a central question in stem cell biology. Virtually all neural stem cells maintain an undifferentiated state and the capacity to self-renew in response to fibroblast <em>growth</em> <em>factor</em>-2 (FGF2). Here we report that a repressor of transcription, the nuclear receptor co-repressor (N-CoR), is a principal regulator in neural stem cells, as FGF2-treated <em>embryonic</em> cortical progenitors from N-CoR gene-disrupted mice display impaired self-renewal and spontaneous <em>differentiation</em> into astroglia-like cells. Stimulation of wild-type neural stem cells with ciliary neurotrophic <em>factor</em> (CNTF), a <em>differentiation</em>-inducing cytokine, results in phosphatidylinositol-3-OH kinase/Akt<em>1</em> kinase-dependent phosphorylation of N-CoR, and causes a temporally correlated redistribution of N-CoR to the cytoplasm. We find that this is a critical strategy for cytokine-induced astroglia <em>differentiation</em> and lineage-characteristic gene expression. Recruitment of protein phosphatase-<em>1</em> to a specific binding site on N-CoR exerts a reciprocal effect on the cellular localization of N-CoR. We propose that repression by N-CoR, modulated by opposing enzymatic activities, is a critical mechanism in neural stem cells that underlies the inhibition of glial <em>differentiation</em>.