The <em>growth</em>-<em>factor</em>-like phospholipid lysophosphatidic acid (LPA) mediates a wide variety of biological functions. We recently reported the cloning of the first G-protein-coupled receptor for LPA, called ventricular zone gene-<em>1</em> (vzg-<em>1</em>/lpA<em>1</em>/edg-2) because its <em>embryonic</em> central nervous system (CNS) expression is restricted to the neocortical ventricular zone (Hecht et al. [<em>1</em>996] J. Cell Biol. <em>1</em>35:<em>1</em>07<em>1</em>-<em>1</em>083). Vzg-<em>1</em> neural expression diminishes at the end of the cortical neurogenetic period, just before birth. Here, we have investigated the subsequent reappearance of vzg-<em>1</em> expression in the postnatal murine brain, by using in situ hybridization and northern blot analyses. Vzg-<em>1</em> expression was undetectable by in situ hybridization at birth, but reappeared in the hindbrain during the <em>1</em>st postnatal week. Subsequently, expression expanded from caudal to rostral, with peak expression observed around postnatal day <em>1</em>8. At all postnatal ages, vzg-<em>1</em> expression was concentrated in and around developing white matter tracts, and its expansion, peak, and subsequent downregulation closely paralleled the progress of myelination. Double-label in situ hybridization studies demonstrated that vzg-<em>1</em>-expressing cells co-expressed mRNA encoding proteolipid protein (PLP), a mature oligodendrocyte marker, but not glial fibrillary acidic protein (GFAP), an astrocyte marker. Consistent with this, vzg-<em>1</em> mRNA expression was reduced by 40% in the brains of jimpy mice, which exhibit aberrant oligodendrocyte <em>differentiation</em> and cell death. Together with our characterization of vzg-<em>1</em> during cortical neurogenesis, these data suggest distinct pre- and postnatal roles for LPA in the development of neurons and oligodendrocytes and implicate lysophospholipid signaling as a potential regulator of myelination.

Mice lacking the vascular endothelial <em>growth</em> <em>factor</em> (VEGF) receptor flt-<em>1</em> die of vascular over<em>growth</em>, and we are interested in how flt-<em>1</em> normally prevents this outcome. Our results support a model whereby aberrant endothelial cell division is the cellular mechanism resulting in vascular over<em>growth</em>, and they suggest that VEGF-dependent endothelial cell division is normally finely modulated by flt-<em>1</em> to produce blood vessels. Flt-<em>1</em>(-/-) <em>embryonic</em> stem cell cultures had a 2-fold increase in endothelial cells by day 8, and the endothelial cell mitotic index was significantly elevated before day 8. Flt-<em>1</em> mutant embryos also had an increased endothelial cell mitotic index, indicating that aberrant endothelial cell division occurs in vivo in the absence of flt-<em>1</em>. The flt-<em>1</em> mutant vasculature of the cultures was partially rescued by mitomycin C treatment, consistent with a cell division defect in the mutant background. Analysis of cultures at earlier time points showed no significant <em>differences</em> until day 5, when flt-<em>1</em> mutant cultures had increased beta-galactosidase(+) cells, indicating that the expansion of flt-<em>1</em> responsive cells occurs after day 4. Mitomycin C treatment blocked this early expansion, suggesting that aberrant division of angioblasts and/or endothelial cells is a hallmark of the flt-<em>1</em> mutant phenotype throughout vascular development. Consistent with this model is the finding that expansion of platelet and endothelial cell adhesion molecule(+) and VE-cadherin(+) vascular cells in the flt-<em>1</em> mutant background first occurs between day 5 and day 6. Taken together, these data show that flt-<em>1</em> normally modulates vascular <em>growth</em> by controlling the rate of endothelial cell division both in vitro and in vivo.

The thyroid-stimulating hormone/thyrotropin (TSH) is the most relevant hormone in the control of thyroid gland physiology in adulthood. TSH effects on the thyroid gland are mediated by the interaction with a specific TSH receptor (TSHR). We studied the role of TSHTSHR signaling on gland morphogenesis and <em>differentiation</em> in the mouse embryo using mouse lines deprived either of TSH (pit(dw)pit(dw)) or of a functional TSHR (tshr(hyt)tshr(hyt) and TSHR-knockout lines). The results reported here show that in the absence of either TSH or a functional TSHR, the thyroid gland develops to a normal size, whereas the expression of thyroperoxidase and the sodium/iodide symporter are reduced greatly. Conversely, no relevant changes are detected in the amounts of thyroglobulin and the thyroid-enriched transcription <em>factors</em> TTF-<em>1</em>, TTF-2, and Pax8. These data suggest that the major role of the TSH/TSHR pathway is in controlling genes involved in iodide metabolism such as sodium/iodide symporter and thyroperoxidase. Furthermore, our data indicate that in <em>embryonic</em> life TSH does not play an equivalent role in controlling gland <em>growth</em> as in the adult thyroid.

Insulin-like <em>growth</em> <em>factor</em>-<em>1</em> (IGF-<em>1</em>) has been shown to play a key role during <em>embryonic</em> and postnatal development of the CNS, but its effect on a sensory organ has not been studied in vivo. Therefore, we examined cochlear <em>growth</em>, <em>differentiation</em>, and maturation in Igf-<em>1</em> gene knock-out mice at postnatal days 5 (P5), P8, and P20 by using stereological methods and immunohistochemistry. Mutant mice showed reduction in size of the cochlea and cochlear ganglion. An immature tectorial membrane and a significant decrease in the number and size of auditory neurons were also evident at P20. IGF-<em>1</em>-deficient cochlear neurons showed increased caspase-3-mediated apoptosis, along with aberrant expression of the early neural markers nestin and Islet <em>1</em>/2. Cochlear ganglion and fibers innervating the sensory cells of the organ of Corti presented decreased levels of neurofilament and myelin P(0) in P20 mouse mutants. In addition, an abnormal synaptophysin expression in the somata of cochlear ganglion neurons and sensory hair cells suggested the persistence of an immature pattern of synapses distribution in the organ of Corti of these animals. These results demonstrate that lack of IGF-<em>1</em> in mice severely affects postnatal survival, <em>differentiation</em>, and maturation of the cochlear ganglion cells and causes abnormal innervation of the sensory cells in the organ of Corti.

Fibroblast <em>growth</em> <em>factors</em> and receptors are intimately connected to the extracellular matrix by their affinity to heparan sulfate proteoglycans. They mediate multiple processes during <em>embryonic</em> development and adult life. In this study, <em>embryonic</em> stem cell-derived embryoid bodies were used to model fibroblast <em>growth</em> <em>factor</em> signaling during early epithelial morphogenesis. To avoid redundancy caused by multiple receptors, we employed a dominant negative mutation of Fgfr2. Mutant-derived embryoid bodies failed to form endoderm, ectoderm, and basement membrane and did not cavitate. However, in mixed cultures they displayed complete <em>differentiation</em> induced by extracellular products of the normal cell. Evidence will be presented here that at least one of these products is the basement membrane or <em>factors</em> connected to it. It will be shown that in the mutant, collagen IV and laminin-<em>1</em> synthesis is coordinately suppressed. We will demonstrate that the basement membrane is required for embryoid body <em>differentiation</em> by rescuing columnar ectoderm <em>differentiation</em> and cavitation in the mutant by externally added basement membrane proteins. This treatment induced transcription of Eomesodermin, an early developmental gene, suggesting that purified basement membrane proteins can activate inherent developmental programs. Our results provide a new paradigm for the role of fibroblast <em>growth</em> <em>factor</em> signaling in basement membrane formation and epithelial <em>differentiation</em>.

Various types of feeder cells have been adopted for the culture of human <em>embryonic</em> stem cells (hESCs) to improve their attachment and provide them with stemness-supporting <em>factors</em>. However, feeder cells differ in their capacity to support the <em>growth</em> of undifferentiated hESCs. Here, we compared the expression and secretion of four well-established regulators of hESC pluripotency and/or <em>differentiation</em> among five lines of human foreskin fibroblasts and primary mouse <em>embryonic</em> fibroblasts throughout a standard hESC culture procedure. We found that human and mouse feeder cells secreted comparable levels of TGF beta <em>1</em>. However, mouse feeder cells secreted larger quantities of activin A than human feeder cells. Conversely, FGF-2, which was produced by human feeder cells, could not be detected in culture media from mouse feeder cells. The quantity of BMP-4 was at about the level of detectability in media from all feeder cell types, although BMP-4 dimers were present in all feeder cells. Production of TGF beta <em>1</em>, activin A, and FGF-2 varied considerably among the human-derived feeder cell lines. Low- and high-producing human feeder cells as well as mouse feeder cells were evaluated for their ability to support the undifferentiated <em>growth</em> of hESCs. We found that a significantly lower proportion of hESCs maintained on human feeder cell types expressed SSEA3, an undifferentiated cell marker. Moreover, SSEA3 expression and thus the pluripotent hESC compartment could be partially rescued by addition of activin A. Cumulatively, these results suggest that the ability of a feeder layer to promote the undifferentiated <em>growth</em> of hESCs is attributable to its characteristic <em>growth</em> <em>factor</em> production.

The expression of vascular endothelial <em>growth</em> <em>factor</em> (VEGF) and its receptors Flt-<em>1</em> and Flk-<em>1</em> in the rat kidney was examined during ontogeny using Northern blot analysis and immunocytochemistry. In prevascular <em>embryonic</em> kidneys (<em>embryonic</em> day <em>1</em>4 [E<em>1</em>4]), immunoreactive Flt-<em>1</em> and Flk-<em>1</em> were observed in isolated angioblasts, whereas VEGF was not detected. Angioblasts aligned forming cords before morphologically differentiating into endothelial cells. In late fetal kidneys (E<em>1</em>9), immunoreactive VEGF was detected in glomerular epithelial and tubular cells, whereas Flt-<em>1</em> and Flk-<em>1</em> were expressed in contiguous endothelial cells. To determine whether VEGF induces endothelial cell <em>differentiation</em> and vascular development in the kidney, the effect of recombinant human VEGF (5 ng/ml) was examined on rat metanephric organ culture, a model known to recapitulate nephrogenesis in the absence of vessels. After 6 d in culture in serum-free, defined media, metanephric kidney <em>growth</em> and morphology were assessed. DNA content was higher in VEGF-treated explants (<em>1</em>.9 +/- 0.<em>1</em>7 microg/kidney, n = 9) than in paired control explants (<em>1</em>.4 +/- 0.<em>1</em>0 microg/kidney, n = 9) (P < 0.05). VEGF induced proliferation of tubular epithelial cells, as indicated by an increased number of tubules and tubular proliferating cell nuclear antigen-containing cells. VEGF induced upregulation of Flk-<em>1</em> and Flt-<em>1</em> expression, as assessed by Western blot analysis. Developing endothelial cells were identified and localized using immunocytochemistry and electron microscopy. Flt-<em>1</em>, Flk-<em>1</em>, and angiotensin-converting enzyme-containing cells were detected in VEGF-treated explants, whereas control explants were negative. These studies confirmed previous reports indicating that the expression of VEGF and its receptors is temporally and spatially associated with kidney vascularization and identified angioblasts expressing Flt-<em>1</em> and Flk-<em>1</em> in prevascular <em>embryonic</em> kidneys. The data indicate that VEGF expression is downregulated in standard culture conditions and that VEGF stimulates <em>growth</em> of <em>embryonic</em> kidney explants by expanding both endothelium and epithelium, resulting in vasculogenesis and enhanced tubulogenesis. These data suggest that VEGF plays a critical role in renal development by promoting endothelial cell <em>differentiation</em>, capillary formation, and proliferation of tubular epithelia.

Fibroblast <em>growth</em> <em>factors</em> (FGFs), such as FGF-<em>1</em>, have been shown to induce <em>differentiation</em> of lens epithelial cells both in tissue culture and in transgenic mice. In the present study, using the alpha A-crystallin promoter, we generated transgenic mice that express different FGFs (FGF-4, FGF-7, FGF-8, FGF-9) specifically in the lens. All four FGFs induced changes in ocular development. Microphthalmic eyes were evident in transgenic mice expressing FGF-8, FGF-9 and some lines expressing FGF-4. A developmental study of the microphthalmic eyes revealed that, by <em>embryonic</em> day <em>1</em>5, expression of these FGFs induced lens epithelial cells to undergo premature fiber <em>differentiation</em>. In less severely affected lines expressing FGF-4 or FGF-7, the lens epithelial cells exhibited a premature exit from the cell cycle and underwent a fiber <em>differentiation</em> response later in development, leading to cataract formation. The responsiveness of lens cells to different FGFs indicates that these proteins stimulate the same or overlapping downstream signalling pathway(s). These overlapping effects of different FGFs on a common cell type indicate that the normal developmental roles for these genes are determined by the temporal and spatial regulation of their expression patterns. The fact that any of these FGFs can induce ocular defects and loss of lens transparency implies that it is essential for the normal eye to maintain very specific spatial control over FGF expression in order to prevent cataract induction.

Glucagon-like peptide-<em>1</em> (GLP-<em>1</em>) is an intestinal incretin hormone, derived from the processing of proglucagon, that exerts insulinotropic actions on insulin-producing pancreatic islet beta-cells. Recently GLP-<em>1</em> was shown to stimulate the <em>growth</em> and <em>differentiation</em> (neogenesis) of beta-cells and appears to do so by inducing the expression of the homeodomain protein IDX-<em>1</em> (islet duodenum homeobox-<em>1</em>; also known as PDX-<em>1</em>, pancreatic and duodenal homeobox gene; and as IPF-<em>1</em>, insulin promoter <em>factor</em>), which is required for pancreas development and the expression of beta-cell-specific genes. Earlier we identified multipotential progenitor cells in the islet and ducts of the pancreas, termed nestin-positive islet-derived progenitor cells (NIPs). Here we report the expression of functional GLP-<em>1</em> receptors on NIPs and that GLP-<em>1</em> stimulates the <em>differentiation</em> of NIPs into insulin-producing cells. Furthermore, confluent NIP cultures express the proglucagon gene and secrete GLP-<em>1</em>. These findings suggest a model of islet development in which pancreatic progenitor cells express both GLP-<em>1</em> receptors and proglucagon with the formation of GLP-<em>1</em>. Locally produced GLP-<em>1</em> may act as an autocrine/paracrine developmental morphogen on receptors on NIPs, resulting in the activation of IDX-<em>1</em> and the expression of the proinsulin gene conferring a beta-cell phenotype. GLP-<em>1</em> may be an important morphogen both for the <em>embryonic</em> development of the pancreas and for the neogenesis of beta-cells in the islets of the adult pancreas.

The transforming <em>growth</em> <em>factor</em>-beta (TGF-beta) superfamily encompasses a large group of structurally related polypeptides that are capable of regulating cell <em>growth</em> and <em>differentiation</em> in a wide range of <em>embryonic</em> and adult tissues. <em>Growth</em>/<em>differentiation</em> <em>factor</em>-<em>1</em> (Gdf-<em>1</em>, encoded by Gdf<em>1</em>) is a TGF-beta family member of unknown function that was originally isolated from an early mouse embryo cDNA library and is expressed specifically in the nervous systemin late-stage embryos and adult mice. Here we show that at early stages of mouse development, Gdfl is expressed initially throughout the embryo proper and then most prominently in the primitive node, ventral neural tube, and intermediate and lateral plate mesoderm. To examine its biological function, we generated a mouse line carrying a targeted mutation in Gdf<em>1</em>. Gdf<em>1</em>-/- mice exhibited a spectrum of defects related to left-right axis formation, including visceral situs inversus, right pulmonary isomerism and a range of cardiac anomalies. In most Gdf<em>1</em>-/- embryos, the expression of Ebaf (formerly lefty-<em>1</em>) in the left side of the floor plate and Leftb (formerly lefty-2), nodal and Pitx2 in the left lateral plate mesoderm was absent, suggesting that Gdf<em>1</em> acts upstream of these genes either directly or indirectly to activate their expression. Our findings suggest that Gdf<em>1</em> acts early in the pathway of gene activation that leads to the establishment of left-right asymmetry.

Polycystic ovarian syndrome (PCOS) is a common endocrinopathy characterized by oligo/anovulatiaon and elevated circulating androgens or evidence of hyperandrogenism after all known potential causes have been excluded. In addition, insulin resistance and accompanying hyperinsulinemia commonly occur in women with PCOS. There is increasing evidence that the endocrinologic and metabolic abnormalities in PCOS may have complex effects on the endometrium, contributing to the infertility and endometrial disorders observed in women with this syndrome. Androgen receptors and steroid receptor co-activators are over-expressed in the endometrium of women with PCOS. Also, biomarkers of endometrial receptivity to <em>embryonic</em> implantation-such as alpha(v)beta3-integrin and glycodelin-are decreased, and epithelial expression of estrogen receptor alpha (ERalpha) abnormally persists in the window of implantation in endometrium in women with PCOS. In addition to being responsive to the steroid hormones estradiol, progesterone, and androgens, the endometrium is also a target for insulin, the receptor for which is cyclically regulated in normo-ovulatory women. In vitro, insulin inhibits the normal process of endometrial stromal <em>differentiation</em> (decidualization). In addition, insulin-like <em>growth</em> <em>factors</em> (IGFs) and their binding proteins are regulated in and act on endometrial cellular constituents, and hyperinsulinemia down-regulates hepatic IGFBP-<em>1</em>, resulting in elevated free IGF-I in the circulation. Thus, elevated estrogen (without the opposing effects of progesterone in the absence of ovulation), hyperinsulinemia, elevated free IGF-I and androgens, and obesity all likely contribute to endometrial dysfunction, infertility, increased miscarriage rate, endometrial hyperplasia, and endometrial cancer common in women with PCOS. The potential mechanisms underlying these disorders, specifically in women with PCOS, are complex and await additional transdisciplinary research for their complete elucidation.

OBJECTIVE

To develop a simple and efficient method for producing homogeneous populations of monocytes and macrophages from human embryonic stem cells (hES).

METHODS

Human embryonic stem cell lines KCL001, KCL002, and HUES-2 were differentiated into monocytes by coculture-free differentiation with two growth factors using a three-step method. The method involved embryoid body (EB) formation in hES media, directed differentiation with macrophage colony-stimulating factor and interleukin (IL)-3, and harvest of nonadherent monocytes from the culture supernatants. hES monocytes (esMCs) were analyzed by microscopy, flow cytometry, transcriptome analysis, and tested for the ability to differentiate into macrophages. hES monocyte-derived macrophages (esMDM) were analyzed for phagocytosis and endocytosis by microscopy and flow cytometry, cytokine secretion by multiplex cytokine assay, and for interferon (IFN)-gamma and IL-4 activation by flow cytometry.

RESULTS

Homogeneous esMCs (>90% CD14-positive) that did not require any additional purification steps were produced after 18.7 +/- 7.7 days (mean +/- SD, n = 19). Production continued for several months when growth factors were replaced, with a total yield of 3.4 x 10(5) +/- 2.0 esMCs (mean +/- SD, n = 9) per EB. Transcriptome analysis of the esMC and the esMDM revealed a distinct myeloid signature that correlated with primary adult blood-derived monocytes and spleen tissue samples but not with other tissue samples tested. We found that esMCs and esMDMs expressed well-defined markers of the mononuclear phagocyte system including PU-1, C/EBPalpha, EMR1, and EMR2, MPEG1, CD1c, CD4, CD18, CD32, CD33, CD68, cathepsins and serine carboxypeptidase. Finally, esMCs differentiated into functional macrophages that could endocytose acetylated low-density lipoprotein, phagocytose opsonized yeast particles, secrete specific cytokines in response to lipopolysaccharide, and be activated differentially with IFN-gamma and IL-4.

CONCLUSIONS

We have developed a simple and efficient method for producing homogeneous populations of monocytes and macrophages from hES cells. esMCs have a myeloid signature and can differentiate into functional macrophages. The method should prove useful in answering experimental questions regarding monocyte and macrophage development and biology.

Angiopoietin-2 (Ang-2) modulates <em>embryonic</em> vascular <em>differentiation</em> primarily by inhibiting the antiapoptotic effects of Ang-<em>1</em> on endothelia that express the Tie-2 receptor. Ang-2 is transiently expressed by developing glomeruli but is downregulated with normal maturation. Glomerular Ang-2 expression is, however, markedly upregulated in animal models of diabetic nephropathy and glomerulonephritis, both leading causes of human chronic renal disease, affecting <em>1</em>0% of the world population. It was hypothesized that Ang-2 might have significant roles in the pathobiology of glomerular disease. Mice with inducible podocyte-specific Ang-2 overexpression were generated. When the transgene was induced in adults for up to <em>1</em>0 wk, mice had significant increases in both albuminuria and glomerular endothelial apoptosis, with significant decreases of both vascular endothelial <em>growth</em> <em>factor</em>-A and nephrin proteins, critical for maintenance of glomerular endothelia and filtration barrier functional integrity, respectively. There was, however, no significant change of systemic BP, creatinine clearance, or markers of renal fibrosis, and podocytes appeared structurally intact. In kidneys of young animals in which Ang-2 had been upregulated during organogenesis, increased apoptosis occurred in just-formed glomeruli. In vitro, short-term exposure of isolated wild-type murine glomeruli to exogenous Ang-2 led to decreased levels of vascular endothelial <em>growth</em> <em>factor</em>-A protein. These novel results provide insight into molecular mechanisms underlying proteinuric disorders, highlight potentially complex interactions between subsets of glomerular cells, and emphasize how a vascular <em>growth</em> <em>factor</em> that has critical roles in normal development may be harmful when re-expressed in the context of adult disease.

<em>Differentiation</em> of astrocytes from human stem cells has significant potential for analysis of their role in normal brain function and disease, but existing protocols generate only immature astrocytes. Using early neuralization, we generated spinal cord astrocytes from mouse or human <em>embryonic</em> or induced pluripotent stem cells with high efficiency. Remarkably, short exposure to fibroblast <em>growth</em> <em>factor</em> <em>1</em> (FGF<em>1</em>) or FGF2 was sufficient to direct these astrocytes selectively toward a mature quiescent phenotype, as judged by both marker expression and functional analysis. In contrast, tumor necrosis <em>factor</em> alpha and interleukin-<em>1</em>β, but not FGFs, induced multiple elements of a reactive inflammatory phenotype but did not affect maturation. These phenotypically defined, scalable populations of spinal cord astrocytes will be important both for studying normal astrocyte function and for modeling human pathological processes in vitro.

<em>Embryonic</em> stem (ES) cells are able to differentiate in vitro into endodermal, mesodermal, and ectodermal cell types. However. the spontaneous development of neuronal cells from ES cells is rather limited. Therefore, specific protocols to increase the <em>differentiation</em> of neuronal cells have been established, such as retinoic acid (RA) induction and lineage selection of neuronal cells. High concentrations of RA resulted in efficient neuronal <em>differentiation</em> paralleled by the expression of tissue-specific genes, proteins, ion channels, and receptors in a developmentally controlled manner. Because the developmental pattern and survival capacity of RA-induced neuronal cells were limited, specific <em>differentiation</em> protocols by lineage selection of neuronal cells have been established using <em>growth</em> and extracellular matrix <em>factors</em>. After formation of cells of the three primary germ layers, mesodermal <em>differentiation</em> was inhibited by serum depletion, and neural precursor cells were generated by addition of basic fibroblast <em>growth</em> <em>factor</em>, followed by <em>differentiation</em> induction by neuronal <em>differentiation</em> <em>factors</em>. Further application of survival-promoting <em>factors</em> such as neurotrophic <em>factors</em> and cytokines at terminal stages resulted in a significant increase, survival, and maintenance of dopaminergic neurons. In the future, these cellular systems will be applicable: (<em>1</em>) for studying commitment and neuronal specification in vitro, (2) as pharmacological assays for drug screening, and (3) for the selective isolation of differentiated neuronal cells which may be used as a source for cell and tissue grafts.

The study of how human <em>embryonic</em> stem cells (hESCs) differentiate into insulin-producing beta cells has twofold significance: first, it provides an in vitro model system for the study of human pancreatic development, and second, it serves as a platform for the ultimate production of beta cells for transplantation into patients with diabetes. The delineation of <em>growth</em> <em>factor</em> interactions regulating pancreas specification from hESCs in vitro is critical to achieving these goals. In this study, we describe the roles of <em>growth</em> <em>factors</em> bFGF, BMP4 and Activin A in early hESC fate determination. The entire <em>differentiation</em> process is carried out in serum-free chemically-defined media (CDM) and results in reliable and robust induction of pancreatic endoderm cells, marked by PDX<em>1</em>, and cell clusters co-expressing markers characteristic of beta cells, including PDX<em>1</em> and insulin/C-peptide. Varying the combinations of <em>growth</em> <em>factors</em>, we found that treatment of hESCs with bFGF, Activin A and BMP4 (FAB) together for 3-4days resulted in strong induction of primitive-streak and definitive endoderm-associated genes, including MIXL<em>1</em>, GSC, SOX<em>1</em>7 and FOXA2. Early proliferative foregut endoderm and pancreatic lineage cells marked by PDX<em>1</em>, FOXA2 and SOX9 expression are specified in EBs made from FAB-treated hESCs, but not from Activin A alone treated cells. Our results suggest that important tissue interactions occur in EB-based suspension culture that contribute to the complete induction of definitive endoderm and pancreas progenitors. Further <em>differentiation</em> occurs after EBs are embedded in Matrigel and cultured in serum-free media containing insulin, transferrin, selenium, FGF7, nicotinamide, islet neogenesis associated peptide (INGAP) and exendin-4, a long acting GLP-<em>1</em> agonist. 2<em>1</em>-28days after embedding, PDX<em>1</em> gene expression levels are comparable to those of human islets used for transplantation, and many PDX<em>1</em>(+) clusters are formed. Almost all cells in PDX<em>1</em>(+) clusters co-express FOXA2, HNF<em>1</em>ß, HNF6 and SOX9 proteins, and many cells also express CPA<em>1</em>, NKX6.<em>1</em> and PTF<em>1</em>a. If cells are then switched to medium containing B27 and nicotinamide for 7-<em>1</em>4days, then the number of insulin(+) cells increases markedly. Our study identifies a new chemically defined culture protocol for inducing endoderm- and pancreas-committed cells from hESCs and reveals an interplay between FGF, Activin A and BMP signaling in early hESC fate determination.

We directly isolated neural stem cells and lineage-restricted neuronal and glial progenitors from the <em>embryonic</em> rat telencephalon using a novel strategy of surface labeling and fluorescence-activated cell sorting. Neural stem cells, which did not express surface epitopes characteristic of <em>differentiation</em> or apoptosis, were sorted by negative selection. These cells predominantly expressed fibroblast <em>growth</em> <em>factor</em> receptor type <em>1</em> (FGFR-<em>1</em>), and a minority exhibited basic fibroblast <em>growth</em> <em>factor</em> (bFGF), whereas few expressed epidermal <em>growth</em> <em>factor</em> receptor (EGFR) or EGF. Clonal analyses revealed that these cells primarily self-renewed without differentiating in bFGF-containing medium, whereas few survived or expanded in EGF-containing medium. Culturing of neural stem cells in bFGF- and EGF-containing medium permitted both self-renewal and <em>differentiation</em> into neuronal, astroglial, and oligodendroglial phenotypes. In contrast, lineage-restricted progenitors were directly sorted by positive selection using a combination of surface epitopes identifying neuronal or glial phenotypes or both. These cells were also primarily FGFR-<em>1</em>(+), with few EGFR(+), and most expanded and progressed along their expected lineages in bFGF-containing medium but not in EGF-containing medium. Ca(2+) imaging of self-renewing neural stem cells cultured in bFGF-containing medium revealed that bFGF, but not EGF, induced cytosolic Ca(2+) (Ca(2+)c) responses in these cells, whereas in bFGF- and EGF-containing medium, both bFGF and EGF evoked Ca(2+)c signals only in differentiating progeny of these cells. The results demonstrate that bFGF, but not EGF, sustains a calcium-dependent self-renewal of neural stem cells and early expansion of lineage-restricted progenitors, whereas together the two <em>growth</em> <em>factors</em> permit the initial commitment of neural stem cells into neuronal and glial phenotypes.

In this study, we report a serum-free culture system for primary neonatal pulmonary cells that can support the <em>growth</em> of octamer-binding transcription <em>factor</em> 4+ (Oct-4+) epithelial colonies with a surrounding mesenchymal stroma. In addition to Oct-4, these cells also express other stem cell markers such as stage-specific <em>embryonic</em> antigen <em>1</em> (SSEA-<em>1</em>), stem cell antigen <em>1</em> (Sca-<em>1</em>), and Clara cell secretion protein (CCSP) but not c-Kit, CD34, and p63, indicating that they represent a subpopulation of Clara cells that have been implicated as lung stem/progenitor cells in lung injury models. These colony cells can be kept for weeks in primary cultures and undergo terminal <em>differentiation</em> to alveolar type-2- and type-<em>1</em>-like pneumocytes sequentially when removed from the stroma. In addition, we have demonstrated the presence of Oct-4+ long-term BrdU label-retaining cells at the bronchoalveolar junction of neonatal lung, providing a link between the Oct-4+ cells in vivo and in vitro and strengthening their identity as putative neonatal lung stem/progenitor cells. Lastly, these Oct-4+ epithelial colony cells, which also express angiotensin-converting enzyme 2, are the target cells for severe acute respiratory syndrome coronavirus infection in primary cultures and support active virus replication leading to their own destruction. These observations imply the possible involvement of lung stem/progenitor cells, in addition to pneumocytes, in severe acute respiratory syndrome coronavirus infection, accounting for the continued deterioration of lung tissues and apparent loss of capacity for lung repair.

Recent studies have suggested a pivotal role for autophagy in stem cell maintenance and <em>differentiation</em>. Reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) has been also suggested to bio-energetically take advantage of mitochondrial autophagy (mitophagy). We have preliminary addressed how mitophagy might play a role in the regulation of induced pluripotency using mdivi-<em>1</em> (for mitochondrial division inhibitor), a highly efficacious small molecule that selectively inhibits the self-assembly of DRP<em>1</em>, a member of the dynamin family of large GTPases that mediates mitochondrial fission. At mdivi-<em>1</em> concentrations that rapidly induced the formation of mitochondrial net-like or collapsed perinuclear mitochondrial structures, we observed that the reprogramming efficiency of mouse <em>embryonic</em> fibroblasts transduced with the Yamanaka three-<em>factor</em> cocktail (OCT4, KLF4, and SOX2) is drastically reduced by more than 95%. Treatment of MEFs with mdivi-<em>1</em> at the early stages of reprogramming before the appearance of iPSC colonies was sufficient to completely inhibit somatic cell reprogramming. Therefore, the observed effects on reprogramming efficiencies were due likely to the inhibition of the process of reprogramming itself and not to an impairment of iPSC colony survival or <em>growth</em>. Moreover, the typical morphology of established iPSC colonies with positive alkaline phosphatase staining was negatively affected by mdivi-<em>1</em> exposure. In the presence of mdivi-<em>1</em>, the colony morphology of the iPSCs was lost, and they somewhat resembled fibroblasts. The alkaline phosphatase staining was also significantly reduced, a finding that is indicative of <em>differentiation</em>. Our current findings provide new insight into how mitochondrial division is integrated into the reprogramming <em>factors</em>-driven transcriptional network that specifies the unique pluripotency of stem cells.

Kallmann syndrome (KS) combines hypogonadotropic hypogonadism and anosmia. Anosmia is related to the absence or hypoplasia of the ol<em>factor</em>y bulbs and tracts. Hypogonadism is due to gonadotropin-releasing hormone (GnRH) deficiency, which presumably results from a failure of the <em>embryonic</em> migration of neuroendocrine GnRH cells from the ol<em>factor</em>y epithelium to the forebrain. This failure could be a consequence of the early degeneration of ol<em>factor</em>y nerve and terminal nerve fibres, because the latter normally act as guiding cues for the migration of GnRH cells. Defects in GnRH cell fate specification, <em>differentiation</em>, axon elongation or axon targeting to the hypothalamus median eminence may, however, also contribute to GnRH deficiency, at least in some genetic forms of the disease. To date, five KS genes have been identified, namely, FGFR<em>1</em>, FGF8, PROKR2, PROK2, and KAL<em>1</em>. Mutations in these genes, however, account for barely 30% of all KS cases. Mutations in FGFR<em>1</em>, encoding fibroblast <em>growth</em> <em>factor</em> receptor <em>1</em>, underlie an autosomal dominant form of the disease. Mutations in PROKR2 and PROK2, encoding prokineticin receptor-2 and prokineticin-2, have been found in heterozygous, homozygous or compound heterozygous states. These two genes are likely to be involved both in monogenic recessive and digenic or oligogenic KS transmission modes. Finally, KAL<em>1</em>, encoding the extracellular glycoprotein anosmin-<em>1</em>, is responsible for the X chromosome-linked form of the disease. It is believed that anosmin-<em>1</em> acts as an enhancer of FGF signalling and perhaps of prokineticin signalling too.

Recent investigations have suggested an active role for endothelial cells in organ development, including the lung. Herein, we investigated some of the molecular mechanisms underlying normal pulmonary vascular development and their influence on epithelial branching morphogenesis. Because the lung in utero develops in a relative hypoxic environment, we first investigated the influence of low oxygen on epithelial and vascular branching morphogenesis. Two transgenic mouse models, the C<em>1</em>0<em>1</em>-LacZ (epithelial-LacZ marker) and the Tie2-LacZ (endothelial-LacZ marker), were used. At <em>embryonic</em> day <em>1</em><em>1</em>.5, primitive lung buds were dissected and cultured at either 20 or 3% oxygen. At 24-h intervals, epithelial and endothelial LacZ gene expression was visualized by X-galactosidase staining. The rate of branching of both tissue elements was increased in explants cultured at 3% oxygen compared with 20% oxygen. Low oxygen increased expression of VEGF, but not that of the VEGF receptor (Flk-<em>1</em>). Expression of two crucial epithelial branching <em>factors</em>, fibroblast <em>growth</em> <em>factor</em>-<em>1</em>0 and bone morphogenetic protein-4, were not affected by low oxygen. Epithelial <em>differentiation</em> was maintained at low oxygen as shown by surfactant protein C in situ hybridization. To explore epithelial-vascular interactions, we inhibited vascular development with antisense oligonucleotides targeted against either hypoxia inducible <em>factor</em>-<em>1</em> alpha or VEGF. Epithelial branching morphogenesis in vitro was dramatically abrogated when pulmonary vascular development was inhibited. Collectively, the in vitro data show that a low-oxygen environment enhances branching of both distal lung epithelium and vascular tissue and that pulmonary vascular development appears to be rate limiting for epithelial branching morphogenesis.

Experimental evidence indicates that shear stress (SS) exerts a morphogenetic function during cardiac development of mouse and zebrafish embryos. However, the molecular basis for this effect is still elusive. Our previous work described that in adult endothelial cells, SS regulates gene expression by inducing epigenetic modification of histones and activation of transcription complexes bearing acetyltransferase activity. In this study, we evaluated whether SS treatment could epigenetically modify histones and influence cell <em>differentiation</em> in mouse <em>embryonic</em> stem (ES) cells. Cells were exposed to a laminar SS of <em>1</em>0 dyne per cm2/s(-<em>1</em>), or kept in static conditions in the presence or absence of the histone deacetylase inhibitor trichostatin A (TSA). These experiments revealed that SS enhanced lysine acetylation of histone H3 at position <em>1</em>4 (K<em>1</em>4), as well as serine phosphorylation at position <em>1</em>0 (S<em>1</em>0) and lysine methylation at position 79 (K79), and cooperated with TSA, inducing acetylation of histone H4 and phosphoacetylation of S<em>1</em>0 and K<em>1</em>4 of histone H3. In addition, ES cells exposed to SS strongly activated transcription from the vascular endothelial <em>growth</em> <em>factor</em> (VEGF) receptor 2 promoter. This effect was paralleled by an early induction of cardiovascular markers, including smooth muscle actin, smooth muscle protein 22-alpha, platelet-endothelial cell adhesion molecule-<em>1</em>, VEGF receptor 2, myocyte enhancer <em>factor</em>-2C (MEF2C), and alpha-sarcomeric actin. In this condition, transcription <em>factors</em> MEF2C and Sma/MAD homolog protein 4 could be isolated from SS-treated ES cells complexed with the cAMP response element-binding protein acetyltransferase. These results provide molecular basis for the SS-dependent cardiovascular commitment of mouse ES cells and suggest that laminar flow may be successfully applied for the in vitro production of cardiovascular precursors.

The importance of mesenchymal-epithelial interactions for normal development of the pancreas was recognized in the early <em>1</em>960s, and mesenchymal signals have been shown to control the proliferation of early pancreatic progenitor cells. The mechanisms by which the mesenchyme coordinates cell proliferation and <em>differentiation</em> to produce the normal number of differentiated pancreatic cells are not fully understood. Here, we demonstrate that the mesenchyme positively controls the final number of beta-cells that develop from early pancreatic progenitor cells. In vitro, the number of beta-cells that developed from rat <em>embryonic</em> pancreatic epithelia was larger in cultures with mesenchyme than without mesenchyme. The effect of mesenchyme was not due to an increase in beta-cell proliferation but was due to increased proliferation of early pancreatic duodenal homeobox-<em>1</em> (PDX<em>1</em>)-positive progenitor cells, as confirmed by bromodeoxyuridine incorporation. Consequently, the window during which early PDX<em>1</em>(+) pancreatic progenitor cells differentiated into endocrine progenitor cells expressing Ngn3 was extended. Fibroblast <em>growth</em> <em>factor</em> <em>1</em>0 mimicked mesenchyme effects on proliferation of early PDX<em>1</em>(+) progenitor cells and induction of Ngn3 expression. Taken together, our results indicate that expansion of early PDX<em>1</em>(+) pancreatic progenitor cells represents a way to increase the final number of beta-cells developing from early <em>embryonic</em> pancreas.

The generation of human ventricular cardiomyocytes from human <em>embryonic</em> stem cells and/or induced pluripotent stem cells could fulfill the demand for therapeutic applications and in vitro pharmacological research; however, the production of a homogeneous population of ventricular cardiomyocytes remains a major limitation. By combining small molecules and <em>growth</em> <em>factors</em>, we developed a fully chemically defined, directed <em>differentiation</em> system to generate ventricular-like cardiomyocytes (VCMs) from human <em>embryonic</em> stem cells and induced pluripotent stem cells with high efficiency and reproducibility. Molecular characterization revealed that the <em>differentiation</em> recapitulated the developmental steps of cardiovascular fate specification. Electrophysiological analyses further illustrated the generation of a highly enriched population of VCMs. These chemically induced VCMs exhibited the expected cardiac electrophysiological and calcium handling properties as well as the appropriate chronotropic responses to cardioactive compounds. In addition, using an integrated computational and experimental systems biology approach, we demonstrated that the modulation of the canonical Wnt pathway by the small molecule IWR-<em>1</em> plays a key role in cardiomyocyte subtype specification. In summary, we developed a reproducible and efficient experimental platform that facilitates a chemical genetics-based interrogation of signaling pathways during cardiogenesis that bypasses the limitations of genetic approaches and provides a valuable source of ventricular cardiomyocytes for pharmacological screenings as well as cell replacement therapies.