<em>Differentiation</em> of human <em>embryonic</em> stem (hES) cells into cells for regenerative medicine is often initiated by embryoid body (EB) formation. EBs may be treated with soluble biochemicals such as cytokines, <em>growth</em> <em>factors</em> and vitamins to induce <em>differentiation</em>. A scanning electron microscopy analysis, conducted over <em>1</em>4 days, revealed time-dependent changes in EB structure which led to the formation of a shell that significantly reduced the diffusive transport of a model molecule (374 Da) by >80%. We found that the shell consists of <em>1</em>) an extracellular matrix (ECM) comprised of collagen type I; 2) a squamous cellular layer with tight cell-cell adhesions associated with E-cadherin; and 3) a collagen type IV lining indicative of a basement membrane. Disruption of the basement membrane, by either inhibiting its formation with noggin or permeabilizing it with collagenase, resulted in recovery of diffusive transport. Increasing the diffusive transport of retinoic acid (RA) and serum in EBs by a <em>1</em>5-min collagenase digestion on days 4, 5, 6 and 7 promoted neuronal <em>differentiation</em>. Flow cytometry and quantitative RT-PCR analysis of collagenase-treated EBs revealed 68% of cells expressing neural cell adhesion molecule (NCAM) relative to 28% for untreated EBs. Our results suggest that limitations in diffusive transport of biochemicals need to be considered when formulating EB <em>differentiation</em> strategies.

<em>Growth</em> <em>factors</em>, hormones, and neurotransmitters have been implicated in the regulation of stem cell fate. Since various neural precursors express functional neurotransmitter receptors, which include G protein-coupled receptors, it is anticipated that they are involved in cell fate decisions. We detected mu-opioid receptor (MOR-<em>1</em>) and kappa-opioid receptor (KOR-<em>1</em>) expression and immunoreactivity in <em>embryonic</em> stem (ES) cells and in retinoic acid-induced ES cell-derived, nestin-positive, neural progenitors. Moreover, these G protein-coupled receptors are functional, since [D-Ala(2),MePhe(4),Gly-ol(5)]enkephalin, a MOR-selective agonist, and U69,593, a KOR-selective agonist, induce a sustained activation of extracellular signal-regulated kinase (ERK) signaling throughout a 24-h treatment period in undifferentiated, self-renewing ES cells. Both opioids promote limited proliferation of undifferentiated ES cells via the ERK/MAP kinase signaling pathway. Importantly, biochemical and immunofluorescence data suggest that [D-Ala(2),MePhe(4),Gly-ol(5)]enkephalin and U69,593 divert ES cells from self-renewal and coax the cells to differentiate. In retinoic acid-differentiated ES cells, opioid-induced signaling features a biphasic ERK activation profile and an opioid-induced, ERK-independent inhibition of proliferation in these neural progenitors. Collectively, the data suggest that opioids may have opposite effects on ES cell self-renewal and ES cell <em>differentiation</em> and that ERK activation is only required by the latter. Finally, opioid modulation of ERK activity may play an important role in ES cell fate decisions by directing the cells to specific lineages.

Previously, based on high ALDH activity, we showed that cancer stem cells (CSCs) could be identified as ALDH(br) cells from an aggressive human osteosarcoma OS99-<em>1</em> cell line. In this study, we evaluate the impact of BMP-2 on CSCs.Three types of BMP receptors were expressed in freshly sorted ALDH(br) cells. In vitro, <em>growth</em> of the sorted ALDH(br) cells was inhibited by BMP-2. Using RT-PCR analysis, BMP-2 was found to down-regulate the expression of <em>embryonic</em> stem cell markers Oct3/4, Nanog, and Sox-2, and up-regulate the transcription of osteogenic markers Runx-2 and Collagen Type I. In vivo, all animals receiving ALDH(br) cells treated with BMP-2 did not form significant tumors, while untreated ALDH(br) cells developed large tumor masses in NOD/SCID mice. Immunostaining confirmed few Ki-67 positive cells were present in the sections of tumor containing ALDH(br) cells treated with BMP-2. These results suggest that BMP-2 suppresses tumor <em>growth</em> by reducing the gene expression of tumorigenic <em>factors</em> and inducing the <em>differentiation</em> of CSCs in osteosarcoma. BMP-2 or BMP-2-mimetic drugs, if properly delivered to tumor and combined with traditional therapies, may therefore provide a new therapeutic option for treatment of osteosarcoma.

Human <em>embryonic</em> stem cells (hESCs) rely on fibroblast <em>growth</em> <em>factor</em> and Activin-Nodal signaling to maintain their pluripotency. However, Activin-Nodal signaling is also known to induce mesendoderm <em>differentiation</em>. The mechanisms by which Activin-Nodal signaling can achieve these contradictory functions remain unknown. Here, we demonstrate that Smad-interacting protein <em>1</em> (SIP<em>1</em>) limits the mesendoderm-inducing effects of Activin-Nodal signaling without inhibiting the pluripotency-maintaining effects exerted by SMAD2/3. In turn, Activin-Nodal signaling cooperates with NANOG, OCT4, and SOX2 to control the expression of SIP<em>1</em> in hESCs, thereby limiting the neuroectoderm-promoting effects of SIP<em>1</em>. Similar results were obtained with mouse epiblast stem cells, implying that these mechanisms are evolutionarily conserved and may operate in vivo during mammalian development. Overall, our results reveal the mechanisms by which Activin-Nodal signaling acts through SIP<em>1</em> to regulate the cell-fate decision between neuroectoderm and mesendoderm in the progression from pluripotency to primary germ layer <em>differentiation</em>.

Human amniotic fluid-derived stem (AFS) cells possess several advantages over <em>embryonic</em> and adult stem cells, as evidenced by expression of both types of stem cell markers and ability to differentiate into cells of all three germ layers. Herein, we examine endothelial <em>differentiation</em> of AFS cells in response to <em>growth</em> <em>factors</em>, shear force, and hypoxia. We isolated human AFS cells from amniotic fluid samples (<em>1</em>-4 cc/specimen) obtained from patients undergoing amniocentesis at <em>1</em>5-<em>1</em>8 weeks of gestation (n = <em>1</em>0). Isolates maintained in nondifferentiating medium expressed the stem cell markers CD<em>1</em>3, CD29, CD44, CD90, CD<em>1</em>05, OCT-4, and SSEA-4 through passage 8. After 3 weeks of culture in endothelial <em>growth</em> media-2 (EGM-2), the stem cells exhibited an endothelial-like morphology, formed cord-like structures when plated on Matrigel, and uptook acetylated LDL/lectin. Additionally, mRNA and protein levels of CD3<em>1</em> and von Willebrand <em>factor</em> (vWF) significantly increased in response to culture in EGM-2, with further up-regulation when stimulated by physiological levels (<em>1</em>2 dyne/cm(2)) of shear force. Culture in hypoxic conditions (5% O(2)) resulted in significant expression of vascular endothelial <em>growth</em> <em>factor</em> (VEGF) and placental <em>growth</em> <em>factor</em> (PGF) mRNA. This study suggests that AFS cells, isolated from minute amounts of amniotic fluid, acquire endothelial cell characteristics when stimulated by <em>growth</em> <em>factors</em> and shear force, and produce angiogenic <em>factors</em> (VEGF, PGF, and hepatocyte <em>growth</em> <em>factor</em> [HGF]) in response to hypoxia. Thus, amniotic fluid represents a rich source of mesenchymal stem cells potentially suitable for use in cardiovascular regenerative medicine.

The generation of human induced pluripotent stem cells (hiPSCs) with a high <em>differentiation</em> potential provided a new source for hepatocyte generation not only for drug discovery and in vitro disease models, but also for cell replacement therapy. However, the reported hiPSC-derived hepatocyte-like cells (HLCs) were not well characterized and their transplantation, as the most promising clue of cell function was not reported. Here, we performed a <em>growth</em> <em>factor</em>-mediated <em>differentiation</em> of functional HLCs from hiPSCs and evaluated their potential for recovery of a carbon tetrachloride (CCl4)-injured mouse liver following transplantation. The hiPSC-derived hepatic lineage cells expressed hepatocyte-specific markers, showed glycogen and lipid storage activity, secretion of albumin (ALB), alpha-fetoprotein (AFP), urea, and CYP450 metabolic activity in addition to low-density lipoprotein (LDL) and indocyanin green (ICG) uptake. Similar results were observed with human <em>embryonic</em> stem cell (hESC)-derived HLCs. The transplantation of hiPSC-HLCs into a CCl4-injured liver showed incorporation of the hiPSC-HLCs into the mouse liver which resulted in a significant enhancement in total serum ALB after <em>1</em> week. A reduction of total serum LDH and bilirubin was seen when compared with the control and sham groups <em>1</em> and 5 weeks post-transplantation. Additionally, we detected human serum ALB and ALB-positive transplanted cells in both the host serum and livers, respectively, which showed functional integration of transplanted cells within the mouse livers. Therefore, our results have opened up a proof of concept that functional HLCs can be generated from hiPSCs, thus improving the general condition of a CCl4-injured mouse liver after their transplantation. These results may bring new insights in the clinical applications of hiPSCs once safety issues are overcome.

Blastocyst-derived pluripotent mouse <em>embryonic</em> stem cells can differentiate in vitro to form so-called embryoid bodies (EBs), which recapitulate several aspects of murine embryogenesis. We used this in vitro model to study oxygen supply and consumption as well as the response to reduced oxygenation during the earliest stages of development. EBs were found to grow equally well when cultured at 20% (normoxia) or <em>1</em>% (hypoxia) oxygen during the first 5 days of <em>differentiation</em>. Microelectrode measurements of pericellular oxygen tension within <em>1</em>3- to <em>1</em>4-day-old EBs (diameter 5<em>1</em>0-890 micron) done at 20% oxygen revealed efficient oxygenation of the EBs' core region. Confocal laser scanning microscopy analysis of EBs incubated with fluorescent dyes that specifically stain living cells confirmed that the cells within an EB were viable. To determine the EBs' capability to sense low oxygen tension and to specifically respond to low ambient oxygen by modulating gene expression we quantified aldolase A and vascular endothelial <em>growth</em> <em>factor</em> (VEGF) mRNAs, since expression of these genes is upregulated by hypoxia in a variety of cells. Compared with the normoxic controls, we found increased aldolase A and VEGF mRNA levels after exposing 8- to 9-day-old EBs to <em>1</em>% oxygen. We propose that EBs represent a powerful tool to study oxygen-regulated gene expression during the early steps of embryogenesis, where the preimplantation conceptus resides in a fluid environment with low oxygen tension until implantation and vascularization allow efficient oxygenation.

We have isolated the rat, mouse and human genes of a distant member of the TGF-beta superfamily, <em>growth</em>/<em>differentiation</em> <em>factor</em>-<em>1</em>5/macrophage inhibiting cytokine-<em>1</em> (GDF-<em>1</em>5/MIC-<em>1</em>) by screening of genomic libraries. All three genes are composed of two exons, and contain one single intron that interrupts the coding sequences at identical positions within the prepro-domain of the corresponding proteins. The predicted proteins contain the structural hallmarks of members of the TGF-beta superfamily, including the seven conserved carboxy-terminal cysteine residues that form the cystine knot. The orthologous molecules show the lowest sequence conservation of all members of the TGF-beta superfamily. RT-PCR reveals an abundant expression of GDF-<em>1</em>5/MIC-<em>1</em> mRNA in numerous <em>embryonic</em> and adult organs and tissues. Promoter analysis of the rat promoter indicates the presence of multiple regulatory elements, including a TATA-like sequence as well as several SP<em>1</em>, AP-<em>1</em> and AP-2 sites. Deletion analysis suggests that a 350 bp region upstream of the start of the open reading frame appears to be the most important for regulation of transcription.

The transforming <em>growth</em> <em>factor</em>-beta (TGF-beta) superfamily consists of a group of secreted signaling molecules that perform important roles in the regulation of cell <em>growth</em> and <em>differentiation</em>. TGF-beta activated kinase-<em>1</em> binding protein-<em>1</em> (TAB<em>1</em>) was identified as a molecule that activates TGF-beta activated kinase-<em>1</em> (TAK<em>1</em>). Recent studies have revealed that the TAB<em>1</em>-TAK<em>1</em> interaction plays an important role in signal transduction in vitro, but little is known about the role of these molecules in vivo. To investigate the role of TAB<em>1</em> during development, we cloned the murine Tab<em>1</em> gene and disrupted it by homologous recombination. Homozygous Tab<em>1</em> mutant mice died, exhibiting a bloated appearance with extensive edema and hemorrhage at the late stages of gestation. By histological examinations, it was revealed that mutant embryos exhibited cardiovascular and lung dysmorphogenesis. Tab<em>1</em> mutant <em>embryonic</em> fibroblast cells displayed drastically reduced TAK<em>1</em> kinase activities and decreased sensitivity to TGF-beta stimulation. These results indicate a possibility that TAB<em>1</em> plays an important role in mammalian embryogenesis and is required for TAK<em>1</em> activation in TGF-beta signaling.

Fibroblast <em>growth</em> <em>factors</em> (FGFs) and receptors (FGFRs) are expressed in the developing lung and appear to be major regulators of lung <em>growth</em> and <em>differentiation</em>. By using mesenchyme-free lung epithelial cultures we show that FGF-<em>1</em> (aFGF) and FGF-7 (KGF) produce different effects in the developing lung. FGF-<em>1</em> stimulates epithelial proliferation that results in bud formation (branching), while FGF-7 promotes epithelial proliferation that leads to formation of cyst-like structures. In addition, FGF-7 stimulates epithelial <em>differentiation</em>, stimulating expression of SP-A and SP-B mRNA throughout the explant, and inducing formation of focal areas of highly differentiated cells. The FGF-<em>1</em> effects on <em>differentiation</em> are limited to induction of surfactant protein SP-B mRNA at the tips of the explant. The FGF-induced patterns of <em>growth</em> appear to correlate with the distribution of epithelial FGFRs mRNAs; FGFR-2 IIIb (KGFR) is diffusely expressed in the day <em>1</em><em>1</em> lung epithelium, while FGFR-4 appears in distal but not in proximal sites. We propose that cyst-like structures may result from FGF-7 binding to the uniformly distributed FGFR-2-IIIb. Lung bud formation may be regulated by FGF-<em>1</em> and/or other ligands binding to FGFR-2 and a distally located FGFR, such as FGFR-4, leading to an increasing binding and activation of FGFRs at the tips of the explant. Thus, in the <em>embryonic</em> lung epithelium, <em>growth</em> effects of FGFs appear to be dependent on location of FGFRs, while effects on <em>differentiation</em> are ligand-dependent.

The <em>growth</em> <em>factor</em> receptors expressed on endothelial cells are of special interest because of their potential to program endothelial cell <em>growth</em> and <em>differentiation</em> during development and neovascularization in various pathological states, such as wound healing and angiogenesis associated with tumorigenesis. Vascular endothelial <em>growth</em> <em>factor</em> ([VEGF] also known as vascular permeability <em>factor</em>) is a potent mitogen and permeability <em>factor</em>, which has been suggested to play a role in <em>embryonic</em> and tumor angiogenesis. The newly cloned FLT4 receptor tyrosine kinase gene encodes a protein related to the VEGF receptors FLT<em>1</em> and KDR/FLK-<em>1</em>. We have here studied the expression of FLT4 and the other two members of this receptor family in human fetal tissues by Northern and in situ hybridization. These results were also compared with the sites of expression of VEGF and the related placenta <em>growth</em> <em>factor</em> (PlGF). Our results reveal FLT4 mRNA expression in vascular endothelial cells in developing vessels of several organs. A comparison of FLT4, FLT<em>1</em> and KDR/FLK-<em>1</em> receptor mRNA signals shows overlapping, but distinct expression patterns in the tissues studied. Certain endothelia lack one or two of the three receptor mRNAs. These data suggest that the receptor tyrosine kinases encoded by the FLT gene family may have distinct functions in the regulation of the <em>growth</em>/<em>differentiation</em> of blood vessels.

Bone fractures and segmental bone defects are a significant source of patient morbidity and place a staggering economic burden on the healthcare system. The annual cost of treating bone defects in the US has been estimated to be $5 billion, while enormous costs are spent on bone grafts for bone injuries, tumors, and other pathologies associated with defective fracture healing. Autologous bone grafts represent the gold standard for the treatment of bone defects. However, they are associated with variable clinical outcomes, postsurgical morbidity, especially at the donor site, and increased surgical costs. In an effort to circumvent these limitations, tissue engineering and cell-based therapies have been proposed as alternatives to induce and promote bone repair. This review focuses on the recent advances in bone tissue engineering (BTE), specifically looking at its role in treating delayed fracture healing (non-unions) and the resulting segmental bone defects. Herein we discuss: (<em>1</em>) the processes of endochondral and intramembranous bone formation; (2) the role of stem cells, looking specifically at mesenchymal (MSC), <em>embryonic</em> (ESC), and induced pluripotent (iPSC) stem cells as viable building blocks to engineer bone implants; (3) the biomaterials used to direct tissue <em>growth</em>, with a focus on ceramic, biodegradable polymers, and composite materials; (4) the <em>growth</em> <em>factors</em> and molecular signals used to induce <em>differentiation</em> of stem cells into the osteoblastic lineage, which ultimately leads to active bone formation; and (5) the mechanical stimulation protocols used to maintain the integrity of the bone repair and their role in successful cell engraftment. Finally, a couple clinical scenarios are presented (non-unions and avascular necrosis-AVN), to illustrate how novel cell-based therapy approaches can be used. A thorough understanding of tissue engineering and cell-based therapies may allow for better incorporation of these potential therapeutic approaches in bone defects allowing for proper bone repair and regeneration.

Blood vessel <em>growth</em> by angiogenesis plays an essential role in <em>embryonic</em> development, wound healing, and tumor <em>growth</em>. To understand the molecular cues underlying this process we have used the PCR-based subtractive hybridization method, representational <em>difference</em> analysis, to identify genes upregulated in endothelial cells (EC) forming tubes in 3D collagen gels, compared to migrating and proliferating cells in 2D cultures. We identified several previously characterized angiogenic markers, including the alpha(v) chain of the alpha(v)beta3 integrin and plasminogen activator inhibitor-<em>1</em>, suggesting overlap in gene expression between tube-forming cells in vitro and in vivo. We also found a 2- to <em>1</em>0-fold upregulation of (beta)ig-h3 (a collagen-binding extracellular matrix protein), NrCAM (a "neural" cell adhesion molecule), Annexin II (a tPA receptor), ESM-<em>1</em> (an EC-specific molecule of unknown function), and Id2 (an inhibitory bHLH transcription <em>factor</em>). We identified a novel splice variant of the ESM-<em>1</em> gene and also detected dramatically enhanced expression of ESM-<em>1</em> and (beta)ig-h3 in several tumors. Antisense oligonucleotides to (beta)ig-h3 blocked both gene expression and tube formation in vitro, suggesting that (beta)ig-h3 may play a critical role in EC-matrix interactions. These data expand the suite of genes implicated in vascular remodeling and angiogenesis.

The recent success in restoring normoglycemia in type <em>1</em> diabetes by islet cell transplantation indicates that cell replacement therapy of this severe disease is achievable. However, the severe lack of donor islets has increased the demand for alternative sources of beta-cells, such as adult and <em>embryonic</em> stem cells. Here, we investigate the potential of human <em>embryonic</em> stem cells (hESCs) to differentiate into beta-cells. Spontaneous <em>differentiation</em> of hESCs under two-dimensional <em>growth</em> conditions resulted in <em>differentiation</em> of Pdx<em>1</em>(+)/Foxa2(+) pancreatic progenitors and Pdx<em>1</em>(+)/Isl<em>1</em>(+) endocrine progenitors but no insulin-producing cells. However, cotransplantation of differentiated hESCs with the dorsal pancreas, but not with the liver or telencephalon, from mouse embryos resulted in <em>differentiation</em> of beta-cell-like cell clusters. Comparative analysis of the basic characteristics of hESC-derived insulin(+) cell clusters with human adult islets demonstrated that the insulin(+) cells share important features with normal beta-cells, such as synthesis (proinsulin) and processing (C-peptide) of insulin and nuclear localization of key beta-cell transcription <em>factors</em>, including Foxa2, Pdx<em>1</em>, and Isl<em>1</em>.

Derivation of human neural progenitors (hNP) from human <em>embryonic</em> stem (hES) cells in culture has been reported with the use of feeder cells or conditioned media. This introduces undefined components into the system, limiting the ability to precisely investigate the requirement for <em>factors</em> that control the process. Also, the use of feeder cells of non-human origin introduces the potential for zoonotic transmission, limiting its clinical usefulness. Here we report a feeder-free system to produce hNP from hES cells and test the effects of various media components involved in the process. Five protocols using defined media components were compared for efficiency of hNP generation. Based on this analysis, we discuss the role of basic fibroblast <em>growth</em> <em>factor</em> (FGF2), N2 supplement, non-essential amino acids (NEAA), and knock-out serum replacement (KSR) on the process of hNP generation. All protocols led to down-regulation of Oct4/POU5F<em>1</em> expression (from 90.5% to <3%), and up-regulation of neural progenitor markers to varying degrees. Media with N2 but not KSR and NEAA produced cultures with significantly higher (p<0.05) expression of the neural progenitor marker Musashi <em>1</em> (MSI<em>1</em>). Approximately 89% of these cells were Nestin (NES)+ after 3 weeks, but they did not proliferate. In contrast, <em>differentiation</em> media supplemented with KSR and NEAA produced fewer NES+ (75%) cells, but these cells were proliferative, and by five passages the culture consisted of >97% NES+ cells. This suggests that KSR and NEAA supplements did not enhance early <em>differentiation</em> but did promote proliferating of hNP cell cultures. This resulted in an efficient, robust, repeatable <em>differentiation</em> system suitable for generating large populations of hNP cells. This will facilitate further study of molecular and biochemical mechanisms in early human neural <em>differentiation</em> and potentially produce uniform neuronal cells for therapeutic uses without concern of zoonotic transmission from feeder layers.

Initial steps in establishing an optimal strategy for functional bioengineered tissues is generation of three-dimensional constructs containing cells with the appropriate organization and phenotype. To effectively utilize rhesus monkey decellularized kidney scaffolds, these studies evaluated two key parameters: (<em>1</em>) residual scaffold components after decellularization including proteomics analysis, and (2) the use of undifferentiated human <em>embryonic</em> stem cells (hESCs) for recellularization in order to explore cellular <em>differentiation</em> in a tissue-specific manner. Sections of kidney and lung were selected for a comparative evaluation because of their similar pattern of organogenesis. Proteomics analysis revealed the presence of <em>growth</em> <em>factors</em> and antimicrobial proteins as well as stress proteins and complement components. Immunohistochemistry of recellularized kidney scaffolds showed the generation of Cytokeratin+ epithelial tubule phenotypes throughout the scaffold that demonstrated a statistically significant increase in expression of kidney-associated genes compared to baseline hESC gene expression. Recellularization of lung scaffolds showed that cells lined the alveolar spaces and demonstrated statistically significant upregulation of key lung-associated genes. However, overall expression of kidney and lung-associated markers was not statistically different when the kidney and lung recellularized scaffolds were compared. These results suggest that decellularized scaffolds have an intrinsic spatial ability to influence hESC <em>differentiation</em> by physically shaping cells into tissue-appropriate structures and phenotypes, and that additional approaches may be needed to ensure consistent recellularization throughout the matrix.

Human small cell lung cancers might be derived from pulmonary cells with a neuroendocrine phenotype. They are driven to proliferate by autocrine and paracrine neuropeptide <em>growth</em> <em>factor</em> stimulation. The molecular basis of the neuroendocrine phenotype of lung carcinomas is relatively unknown. The Achaete-Scute Homologue-<em>1</em> (ASH<em>1</em>) transcription <em>factor</em> is critically required for the formation of pulmonary neuroendocrine cells and is a marker for human small cell lung cancers. The Drosophila orthologues of ASH<em>1</em> (Achaete and Scute) and the <em>growth</em> <em>factor</em> independence-<em>1</em> (GFI<em>1</em>) oncoprotein (Senseless) genetically interact to inhibit Notch signaling and specify fly sensory organ development. Here, we show that GFI<em>1</em>, as with ASH<em>1</em>, is expressed in neuroendocrine lung cancer cell lines and that GFI<em>1</em> in lung cancer cell lines functions as a DNA-binding transcriptional repressor protein. Forced expression of GFI<em>1</em> potentiates tumor formation of small-cell lung carcinoma cells. In primary human lung cancer specimens, GFI<em>1</em> expression strongly correlates with expression of ASH<em>1</em>, the neuroendocrine <em>growth</em> <em>factor</em> gastrin-releasing peptide, and neuroendocrine markers synaptophysin and chromogranin A (P < 0.000000<em>1</em>). GFI<em>1</em> colocalizes with chromogranin A and calcitonin-gene-related peptide in <em>embryonic</em> and adult murine pulmonary neuroendocrine cells. In addition, mice with a mutation in GFI<em>1</em> display abnormal development of pulmonary neuroendocrine cells, indicating that GFI<em>1</em> is important for neuroendocrine <em>differentiation</em>.

The low-density lipoprotein receptor-related protein (LRP<em>1</em>) is a multifunctional cell surface receptor that belongs to the LDL receptor (LDLR) gene family and that is widely expressed in several tissues. LRP<em>1</em> consists of an 85-kDa membrane-bound carboxyl fragment (β chain) and a non-covalently attached 5<em>1</em>5-kDa (α chain) amino-terminal fragment. Through its extracellular domain, LRP<em>1</em> binds at least 40 different ligands ranging from lipoprotein and protease inhibitor complex to <em>growth</em> <em>factors</em> and extracellular matrix proteins. LRP-<em>1</em> has also been shown to interact with scaffolding and signaling proteins via its intracellular domain in a phosphorylation-dependent manner and to function as a co-receptor partnering with other cell surface or integral membrane proteins. LRP-<em>1</em> is thus implicated in two major physiological processes: endocytosis and regulation of signaling pathways, which are both involved in diverse biological roles including lipid metabolism, cell <em>growth</em>/<em>differentiation</em> processes, degradation of proteases, and tissue invasion. The <em>embryonic</em> lethal phenotype obtained after target disruption of the LRP-<em>1</em> gene in the mouse highlights the biological importance of this receptor and revealed a critical, but yet undefined role in development. Tissue-specific gene deletion studies also reveal an important contribution of LRP<em>1</em> in vascular remodeling, foam cell biology, the central nervous system, and in the molecular mechanisms of atherosclerosis.

Gap junction-mediated intercellular coupling is higher in the equatorial region of the lens than at either pole, a property believed to be essential for lens transparency. We show that fibroblast <em>growth</em> <em>factor</em> (FGF) upregulates gap junctional intercellular dye transfer in primary cultures of <em>embryonic</em> chick lens cells without detectably increasing either gap junction protein (connexin) synthesis or assembly. Insulin and insulin-like <em>growth</em> <em>factor</em> <em>1</em>, as potent as FGF in inducing lens cell <em>differentiation</em>, had no effect on gap junctions. FGF induced sustained activation of extracellular signal-regulated kinase (ERK) in lens cells, an event necessary and sufficient to increase gap junctional coupling. We also identify vitreous humor as an in vivo source of an FGF-like intercellular communication-promoting activity and show that FGF-induced ERK activation in the intact lens is higher in the equatorial region than in polar and core fibers. These findings support a model in which regional <em>differences</em> in FGF signaling through the ERK pathway lead to the asymmetry in gap junctional coupling required for proper lens function. Our results also identify upregulation of intercellular communication as a new function for sustained ERK activation and change the current paradigm that ERKs only negatively regulate gap junction channel activity.

Notch receptors play an essential role in the regulation of central cellular processes during <em>embryonic</em> and postnatal development. The mammalian genome encodes for four Notch paralogs (Notch <em>1</em>-4), which are activated by three Delta-like (Dll<em>1</em>/3/4) and two Serrate-like (Jagged<em>1</em>/2) ligands. Further, non-canonical Notch ligands such as epidermal <em>growth</em> <em>factor</em> like protein 7 (EGFL7) have been identified and serve mostly as antagonists of Notch signaling. The Notch pathway prevents neuronal <em>differentiation</em> in the central nervous system by driving neural stem cell maintenance and commitment of neural progenitor cells into the glial lineage. Notch is therefore often implicated in the development of brain tumors, as tumor cells share various characteristics with neural stem and progenitor cells. Notch receptors are overexpressed in gliomas and their oncogenicity has been confirmed by gain- and loss-of-function studies in vitro and in vivo. To this end, special attention is paid to the impact of Notch signaling on stem-like brain tumor-propagating cells as these cells contribute to <em>growth</em>, survival, invasion, and recurrence of brain tumors. Based on the outcome of ongoing studies in vivo, Notch-directed therapies such as γ-secretase inhibitors and blocking antibodies have entered and completed various clinical trials. This review summarizes the current knowledge on Notch signaling in brain tumor formation and therapy.

Liver regeneration is known to be a process involving highly organized and ordered tissue <em>growth</em> triggered by the loss of liver tissue, and remains a fascinating topic. A large number of genes are involved in this process, and there exists a sequence of stages that results in liver regeneration, while at the same time inhibitors control the size of the regenerated liver. The initiation step is characterized by priming of quiescent hepatocytes by <em>factors</em> such as TNF-α, IL-6 and nitric oxide. The proliferation step is the step during which hepatocytes enter into the cell cycle's G<em>1</em> phase and are stimulated by complete mitogens including HGF, TGF-α and EGF. Hepatic stimulator substance, glucagon, insulin, TNF-α, IL-<em>1</em> and IL-6 have also been implicated in regulating the regeneration process. Inhibitors and stop signals of hepatic regeneration are not well known and only limited information is available. Furthermore, the effects of other <em>factors</em> such as VEGF, PDGF, hypothyroidism, proliferating cell nuclear antigen, heat shock proteins, ischemic-reperfusion injury, steatosis and granulocyte colony-stimulating <em>factor</em> on liver regeneration are also systematically reviewed in this article. A tissue engineering approach using isolated hepatocytes for in vitro tissue generation and heterotopic transplantation of liver cells has been established. The use of stem cells might also be very attractive to overcome the limitation of donor liver tissue. Liver-specific <em>differentiation</em> of <em>embryonic</em>, fetal or adult stem cells is currently under investigation.

MicroRNA-<em>1</em>22 (miR-<em>1</em>22), a highly abundant and liver-specific miRNA, acts as a tumor suppressor against hepatocellular carcinoma (HCC). Decreased expression of miR-<em>1</em>22 in HCC is frequently observed and is associated with poor <em>differentiation</em>, larger tumor size, metastasis and invasion, and poor prognosis. Mutant mice with knockout (KO) of the miR-<em>1</em>22 locus developed steatohepatitis due to increased triglyceride (TG) synthesis and decreased TG secretion from hepatocytes, and eventually developed HCC. Exogenic miR-<em>1</em>22 introduction into miR-<em>1</em>22 KO mice inhibited the development of HCC. Target genes of miR-<em>1</em>22, including cyclin G<em>1</em>, a disintegrin and metalloprotease (ADAM)<em>1</em>0, serum response <em>factor</em>, insulin-like <em>growth</em> <em>factor</em>-<em>1</em> receptor, ADAM<em>1</em>7, transcription <em>factor</em> CUTL<em>1</em>, the <em>embryonic</em> isoform of pyruvate kinase (Pkm2), Wnt<em>1</em>, pituitary tumor-transforming gene <em>1</em> binding <em>factor</em>, Cut-like homeobox <em>1</em>, and c-myc, are involved in hepatocarcinogenesis, epithelial mesenchymal transition, and angiogenesis. MiR-<em>1</em>22 expression is regulated by liver-enriched transcription <em>factors</em> such as hepatocyte nuclear <em>factor</em> (HNF)<em>1</em>α, HNF3β, HNF4α, HNF6, and CCAAT/enhancer-binding protein (C/EBP)α. A positive feedback loop exists between C/EBPα and miR-<em>1</em>22 and between HNF6 and miR-<em>1</em>22, whereas a negative feedback loop exists between c-myc and miR-<em>1</em>22. Since cotreatment of 5-Aza-Cd and histone deacetylase inhibitor restored miR-<em>1</em>22 expression in HCC cells, epigenetic modulation of miR-<em>1</em>22 expression is involved in the suppression of miR-<em>1</em>22 in HCC. Several experiments suggest that increasing miR-<em>1</em>22 levels in HCC with or without antitumor agents may be a promising strategy for HCC treatment.

How self-renewal versus <em>differentiation</em> of neural progenitor cells is temporally controlled during early development remains ill-defined. We show that mouse Lin4<em>1</em> (mLin4<em>1</em>) is highly expressed in neural progenitor cells and its expression declines during neural <em>differentiation</em>. Loss of mLin4<em>1</em> function in mice causes reduced proliferation and premature <em>differentiation</em> of <em>embryonic</em> neural progenitor cells. mLin4<em>1</em> was recently implicated as the E3 ubiquitin ligase that mediates degradation of Argonaute 2 (AGO2), a key effector of the microRNA pathway. However, our mechanistic studies of neural progenitor cells indicate mLin4<em>1</em> is not required for AGO2 ubiquitination or stability. Instead, mLin4<em>1</em>-deficient neural progenitors exhibit hyposensitivity for fibroblast <em>growth</em> <em>factor</em> (FGF) signaling. We show that mLin4<em>1</em> promotes FGF signaling by directly binding to and enhancing the stability of Shc SH2-binding protein <em>1</em> (SHCBP<em>1</em>) and that SHCBP<em>1</em> is an important component of FGF signaling in neural progenitor cells. Thus, mLin4<em>1</em> acts as a temporal regulator to promote neural progenitor cell maintenance, not via the regulation of AGO2 stability, but through FGF signaling.

Type <em>1</em> and type 2 diabetes result from an absolute or relative reduction in functional β-cell mass. One approach to replacing lost β-cell mass is transplantation of cadaveric islets; however, this approach is limited by lack of adequate donor tissue. Therefore, there is much interest in identifying <em>factors</em> that enhance β-cell <em>differentiation</em> and proliferation in vivo or in vitro. Connective tissue <em>growth</em> <em>factor</em> (CTGF) is a secreted molecule expressed in endothelial cells, pancreatic ducts, and <em>embryonic</em> β cells that we previously showed is required for β-cell proliferation, <em>differentiation</em>, and islet morphogenesis during development. The current study investigated the tissue interactions by which CTGF promotes normal pancreatic islet development. We found that loss of CTGF from either endothelial cells or β cells results in decreased <em>embryonic</em> β-cell proliferation, making CTGF unique as an identified β cell-derived <em>factor</em> that regulates <em>embryonic</em> β-cell proliferation. Endothelial CTGF inactivation was associated with decreased islet vascularity, highlighting the proposed role of endothelial cells in β-cell proliferation. Furthermore, CTGF overexpression in β cells during embryogenesis using an inducible transgenic system increased islet mass at birth by promoting proliferation of immature β cells, in the absence of changes in islet vascularity. Together, these findings demonstrate that CTGF acts in an autocrine manner during pancreas development and suggest that CTGF has the potential to enhance expansion of immature β cells in directed <em>differentiation</em> or regeneration protocols.