Human pluripotent stem cells, such as <em>embryonic</em> stem cells (hESCs) and induced pluripotent stem cells (iPSCs), have the unique abilities of <em>differentiation</em> into any cell type of the organism (pluripotency) and indefinite self-renewal. Here, we show that the Rem2 GTPase, a suppressor of the p53 pathway, is up-regulated in hESCs and, by loss- and gain-of-function studies, that it is a major player in the maintenance of hESC self-renewal and pluripotency. We show that Rem2 mediates the fibroblastic <em>growth</em> <em>factor</em> 2 (FGF2) signaling pathway to maintain proliferation of hESCs. We demonstrate that Rem2 effects are mediated by suppressing the transcriptional activity of p53 and cyclin D(<em>1</em>) to maintain survival of hESCs. Importantly, Rem2 does this by preventing protein degradation during DNA damage. Given that Rem2 maintains hESCs, we also show that it is as efficient as c-Myc by enhancing reprogramming of human somatic cells into iPSCs eightfold. Rem2 does this by accelerating the cell cycle and protecting from apoptosis via its effects on cyclin D(<em>1</em>) expression/localization and suppression of p53 transcription. We show that the effects of Rem2 on cyclin D(<em>1</em>) are independent of p53 function. These results define the cell cycle and apoptosis as a rate-limiting step during the reprogramming phenomena. Our studies highlight the possibility of reprogramming somatic cells by imposing hESC-specific cell cycle features for making safer iPSCs for cell therapy use.

The tyrosine kinase receptor fetal liver kinase <em>1</em> (Flk-<em>1</em>) plays a crucial role in vasculogenesis and angiogenesis, but its target genes remain elusive. Comparing Flk-<em>1</em>(+/+) with Flk-<em>1</em>(-/-) <em>embryonic</em> stem (ES) cells, we identified transcripts regulated by the vascular endothelial <em>growth</em> <em>factor</em> A (VEGF-A)/Flk-<em>1</em> pathway at an early stage of their <em>differentiation</em> to endothelial and mural precursors. Further analysis of a number of these genes (Nm23-M<em>1</em>, Nm23-M2, Slug, Set, pp32, Cbp, Ship-<em>1</em>, Btk, and Pim-<em>1</em>) showed that their products were transiently up-regulated in vivo in endothelial cells (ECs) during angiogenesis of the ovary, and their mRNA was rapidly induced in vitro by VEGF-A in human umbilical cord vein endothelial cells (HUVECs). Functional analysis by RNA interference (RNAi) in ES cells induced to differentiate demonstrated that Pim-<em>1</em> is required for their <em>differentiation</em> into ECs and smooth muscle cells (SMCs). In HUVECs, RNAi showed that Pim-<em>1</em> is required in ECs for VEGF-A-dependent proliferation and migration. The identification of Flk-<em>1</em> target genes should help in elucidating the molecular pathways that govern the vasculogenesis and angiogenesis processes.

<em>Embryonic</em> stem (ES) cells fluctuate between self-renewal and the threshold of <em>differentiation</em>. Signalling via the fibroblast <em>growth</em> <em>factor</em> (Fgf)/Erk pathway is required to progress from this dynamic state and promote mouse ES cell <em>differentiation</em>. Retinoic acid also induces <em>differentiation</em> in many cellular contexts, but its mechanism of action in relation to Fgf/Erk signalling in ES cells is poorly understood. Here, we show for the first time that endogenous retinoid signalling is required for the timely acquisition of somatic cell fate in mouse ES cells and that exposure to retinoic acid advances <em>differentiation</em> by a dual mechanism: first increasing, but in the long-term decreasing, Fgf signalling. Rapid retinoid induction of Fgf8 and downstream Erk activity on day <em>1</em> in <em>differentiation</em> conditions may serve to ensure loss of self-renewal. However, more gradual repression of Fgf4 by retinoic acid is accompanied by an overall reduction in Erk activity on day 2, and the acquisition of neural and non-neural fates is now advanced by inhibition of Fgf signalling. So, although blocking Fgf/Erk activity is known to promote ES cell self-renewal, once cells have experienced a period of such signals, subsequent inhibition of Fgf signalling has the opposite effect and drives <em>differentiation</em>. We further show in the embryo that retinoid repression of Fgf signalling promotes neural <em>differentiation</em> onset in an analogous step in the extending <em>embryonic</em> body axis and so identify attenuation of Fgf signalling by retinoic acid as a conserved fundamental mechanism driving <em>differentiation</em> towards somatic cell fates.

Nerves and blood vessels have similar branching patterns and use common morphogenic molecules during development. Recent studies show that sonic hedgehog (Shh), a traditional neurogenic morphogen, is required for <em>embryonic</em> arterial <em>differentiation</em> and can induce angiogenesis. We investigated whether Shh regulates the expression of angiogenic <em>factors</em>. Using NIH3T3 <em>embryonic</em> fibroblast cells, we demonstrated that Shh increased the mRNA levels of angiopoietin-<em>1</em> (Ang-<em>1</em>), a secreted ligand that regulates endothelial interaction with mural cells (pericytes and smooth muscle cells) and promotes blood vessel maturation. In contrast, Shh decreased mRNA levels of angiopoietin-2 (Ang-2), a negative modulator of Ang-<em>1</em>. By contrast, Shh did not change the expression of vascular endothelial <em>growth</em> <em>factor</em> (VEGF) mRNA, a potent endothelial mitogen. The effect of Shh appeared to be cell-type specific as the addition of Shh to neural progenitor cells or neurons did not alter Ang-<em>1</em>, Ang-2 or VEGF mRNA levels. The addition of cyclopamine, an inhibitor of Shh signaling, to NIH3T3 cells, suppressed the regulation of Ang-<em>1</em> and Ang-2 mRNA levels in the presence of Shh. Collectively, our results suggest that Shh may contribute to blood vessel <em>growth</em>, maturation and stabilization in a neurovascular network by reciprocally regulating the vascular morphogens Ang-<em>1</em> and Ang-2 in a cell-type-specific manner.

Phospholipid scramblase <em>1</em> (PLSCR<em>1</em>) is an interferon (IFN)- and <em>growth</em> <em>factor</em>-inducible, calcium-binding protein that either inserts into the plasma membrane or binds DNA in the nucleus depending on its state of palmyitoylation. In certain hematopoietic cells, PLSCR<em>1</em> is required for normal maturation and terminal <em>differentiation</em> from progenitor cells as regulated by select <em>growth</em> <em>factors</em>, where it promotes recruitment and activation of Src kinases. PLSCR<em>1</em> is a substrate of Src (and Abl) kinases, and transcription of the PLSCR<em>1</em> gene is regulated by the same <em>growth</em> <em>factor</em> receptor pathways in which PLSCR<em>1</em> potentiates afferent signaling. The marked transcriptional upregulation of PLSCR<em>1</em> by IFNs led us to explore whether PLSCR<em>1</em> plays an analogous role in cellular responses to IFN, with specific focus on antiviral activities. Accordingly, human cells in which PLSCR<em>1</em> expression was decreased with short interfering RNA were rendered relatively insensitive to the antiviral activity of IFNs, resulting in higher titers of vesicular stomatitis virus (VSV) and encephalomyocarditis virus. Similarly, VSV replicated to higher titers in mouse PLSCR<em>1</em>(-/-) <em>embryonic</em> fibroblasts than in identical cells transduced to express PLSCR<em>1</em>. PLSCR<em>1</em> inhibited accumulation of primary VSV transcripts, similar to the effects of IFN against VSV. The antiviral effect of PLSCR<em>1</em> correlated with increased expression of a subset of IFN-stimulated genes (ISGs), including ISG<em>1</em>5, ISG54, p56, and guanylate binding proteins. Our results suggest that PLSCR<em>1</em>, which is itself an ISG-encoded protein, provides a mechanism for amplifying and enhancing the IFN response through increased expression of a select subset of potent antiviral genes.

Oxygen is vital to nearly all forms of life on Earth via its role in energy homeostasis and other cell functions. Until recently, the effects of oxygen on the proliferation and <em>differentiation</em> of neural stem cells (NSCs) have been largely ignored. Some studies have been carried out on the basis of the fact that NSCs exists within a "physiological hypoxic" environment at <em>1</em> to 5% O2 in both <em>embryonic</em> and adult brains. The results showed that hypoxia could promote the <em>growth</em> of NSCs and maintain its survival in vitro. In vivo studies also showed that ischemia/hypoxia increased the number of endogenous NSCs in the subventricular zone and dentate gyrus. In addition, hypoxia could influence the <em>differentiation</em> of NSCs. More neurons, especially more doparminergic neurons, were produced under hypoxic condition. The effects of hypoxia on the other kind of stem cell were briefly introduced as additional evidence. The mechanism of these responses might be primarily involved in the hypoxic inducible <em>factor</em>-<em>1</em> (HIF-<em>1</em>) signal pathway. The present review summarizes recent works on the role of hypoxia in the proliferation and <em>differentiation</em> of NSCs both in vitro and in vivo, and the mechanism involved in HIF-<em>1</em> signaling pathway behind this response was also discussed.

Types of <em>growth</em> include <em>embryonic</em>, fetal, neonatal, juvenile and mature. Until full <em>differentiation</em> is achieved, cells grow through proliferation from progenitor cells. At maturity, the cellular genome is fixed with committed patterns of cell cycle duration and adaptation, ranging from static to renewing type 3. The static cell type cannot proliferate and adapts through hypertrophy. The renewing type continuously proliferates even without stimulus. In all cell types the processes of <em>differentiation</em> and proliferation are mutually exclusive. Cellular kinetics involve (a) the duration of the cell cycle, (b) the birth rate of cells, and (c) the <em>growth</em> rate fractions. The duration of the cell cycle is 2-4 days. All <em>growth</em> <em>factors</em> (GF) exert their influence during G<em>1</em> phase. Release a GF by one cell type can influence the proliferation of another (= paracrine stimulation). At the end of G<em>1</em> is the point of highest sensitivity to toxicity. Tumor suppressor genes act here through tyrosine phosphorylation. During S, the cell replicates its chromosomes. During G2 the immune surveillance and DNA damage repair mechanisms operate. Injured cells stay here longer enabling repair of their damaged DNA. Cell division involves both nuclear (mitosis) and cytoplasmic (cytokinesis) phases giving rise to 2 new cells. The cell cycle has 2 checkpoints. The first involves the G<em>1</em>-S transition and the second the G2-M transition. The types of cell cycle inhibition include (a) cycle- and phase-specific inhibition; (b) cycle-and nonphase-specific inhibition; (c) noncycle-and nonphase-specific inhibition, and finally (d) noncycle, nonphase-, and nonorgan-specific inhibition. Proliferation is a circadian process and it is stimulated by a variety of stimuli which include (<em>1</em>) interference with hormonal feedback pathways; (2) inhibition of the tissue trophic activity; (3) sustained presence of antigenic substances; (4) tissue ischemia; (5) changes of conditions luminally or on surfaces of tissues; (6) sustained cytotoxicity; (7) cell death; and (8) surgical resection. Proliferation can be arrested through senescence, apoptosis, injury or even during the development of immune cells. In the past, tissue/cell kinetics have been studied by tritiated thymidine histoautoradiography. Recently, monoclonal antibodies to proliferation-associated antigens, have been successfully employed. These antigens are cycle-associated proteins and include (<em>1</em>) PCNA; (2) p53; (3) Ki67; (4) AGNOR; (5) Statin; and (6) BrdU. Practical examples are given comparing PCNA and BrdU markers from 3 tissues, i.e. liver, glandular stomach, and uterus, across 2 or 3 strains of rats. Mean values of labeling indices are cited. Within the PCNA marker, 2 different clones are compared from the glandular stomach of SD rats of 2 different ages. Gender and across species comparisons are also made. All these comparisons denote that in every study where markers are used (a) there is a need for a concurrent study control group of the same age; (b) there is a need for in-house control data for this particular organ by species, strain, gender and age; (c) there is ancillary assessment of the trophic status of the target tissue; (d) there is a need for at least 2 different time points during assessment; (e) there is a need for such proliferation data prior to commencing the 2 year rodent bioassay; and (f) that PCNA is the most reliable and versatile of all markers used, capable of rendering good results even from archival specimens.

Human umbilical cord mesenchymal stromal cells (hUCMSCs) have recently shown the capacity to differentiate into multiple cell lineages in all three <em>embryonic</em> germ layers. The osteogenic <em>differentiation</em> of hUCMSCs in monolayer culture has been reported, while the <em>differentiation</em> in three-dimensional biomaterials has not yet been reported for tissue-engineering applications. Thus, the aim of this study was to evaluate the feasibility of using hUCMSCs for bone tissue engineering. hUCMSCs were cultured in poly(L-lactic acid) (PLLA) scaffolds in osteogenic medium (OM) for 3 weeks, after which the scaffolds were exposed to several different media, including the OM, a mineralization medium (MM) and the MM with either <em>1</em>0 or <em>1</em>00 ng/ml insulin-like <em>growth</em> <em>factor</em> (IGF)-<em>1</em>. The osteogenic <em>differentiation</em> was confirmed by the up-regulation of Runx2 and OCN, calcium quantification and bone histology. Switching from the OM to the MM promoted collagen synthesis and calcium content per cell, while continuing in the OM retained more cells in the constructs and promoted higher osteogenic gene expression. The addition of IGF-<em>1</em> into the MM had no effect on cell proliferation, <em>differentiation</em> and matrix synthesis. In conclusion, hUCMSCs show significant potential for bone tissue engineering and culturing in the OM throughout the entire period is beneficial for osteogenic <em>differentiation</em> of these cells.

The number of patients worldwide suffering from the chronic disease diabetes mellitus is <em>growing</em> at an alarming rate. Insulin-secreting beta-cells in the islet of Langerhans are damaged to different extents in diabetic patients, either through an autoimmune reaction present in type <em>1</em> diabetic patients or through inherent changes within beta-cells that affect their function in patients suffering from type 2 diabetes. Cell replacement strategies via islet transplantation offer potential therapeutic options for diabetic patients. However, the discrepancy between the limited number of donor islets and the high number of patients who could benefit from such a treatment reflects the dire need for renewable sources of high-quality beta-cells. Human <em>embryonic</em> stem cells (hESCs) are capable of self-renewal and can differentiate into components of all three germ layers, including all pancreatic lineages. The ability to differentiate hESCs into beta-cells highlights a promising strategy to meet the shortage of beta-cells. Here, we review the different approaches that have been used to direct <em>differentiation</em> of hESCs into pancreatic and beta-cells. We will focus on recent progress in the understanding of signaling pathways and transcription <em>factors</em> during <em>embryonic</em> pancreas development and how this knowledge has helped to improve the methodology for high-efficiency beta-cell <em>differentiation</em> in vitro.

Human <em>embryonic</em> stem (HES) cells can give rise to cardiomyocytes in vitro. However, whether undifferentiated HES cells also feature a myocardial regenerative capacity after in vivo engraftment has not been established yet. We compared two HES cell lines (HUES-<em>1</em> and I6) that were specified toward a cardiac lineage by exposure to bone morphogenetic protein-2 (BMP2) and SU5402, a fibroblast <em>growth</em> <em>factor</em> receptor inhibitor. Real-time polymerase chain reaction (PCR) revealed that the cardiogenic inductive <em>factor</em> turned on expression of mesodermal and cardiac genes (Tbx6, Isl<em>1</em>, FoxH<em>1</em>, Nkx2.5, Mef2c, and alpha-actin). Thirty immunosuppressed rats underwent coronary artery ligation and, 2 weeks later, were randomized and received in-scar injections of either culture medium (controls) or BMP2 (+/-SU5402)-treated HES cells. After 2 months, human cells were detected by anti-human lamin immunostaining, and their cardiomyocytic <em>differentiation</em> was evidenced by their expression of cardiac markers by reverse transcription-PCR and immunofluorescence using an anti-beta myosin antibody. No teratoma was observed in hearts or any other organ of the body. The ability of cardiac-specified HES cells to differentiate along the cardiomyogenic pathway following transplantation into infarcted myocardium raises the hope that these cells might become effective candidates for myocardial regeneration.

Two recurring problems with stem/neural progenitor cell (NPC) transplantation therapies for spinal cord injury (SCI) are poor cell survival and uncontrolled cell <em>differentiation</em>. The current study evaluated the viability and <em>differentiation</em> of <em>embryonic</em> stem cell-derived neural progenitor cells (ESNPCs) transplanted within fibrin scaffolds containing <em>growth</em> <em>factors</em> (GFs) and a heparin-binding delivery system (HBDS) to enhance cell survival and direct <em>differentiation</em> into neurons. Mouse ESNPCs were generated from mouse <em>embryonic</em> stem cells (ESCs) using a 4-/4+ retinoic acid (RA) induction protocol that resulted in a population of cells that was 70% nestin positive NPCs. The ESNPCs were transplanted directly into a rat subacute dorsal hemisection lesion SCI model. ESNPCs were either encapsulated in a fibrin scaffold; encapsulated in fibrin containing the HBDS, neurotrophin-3 (NT-3) and platelet derived <em>growth</em> <em>factor</em> (PDGF-AA); or encapsulated in fibrin scaffolds with NT-3 and PDGF-AA without the HBDS. We report that the combination of GFs and fibrin scaffold (without HBDS) enhanced the total number of ESNPCs present in the treated spinal cords and increased the number of ESNPC-derived NeuN positive neurons 8 weeks after transplantation. All experimental groups treated with ESNPCs exhibited an increase in behavioral function 4 weeks after transplantation. In a subset of animals, the ESNPCs over-proliferated as evidenced by SSEA-<em>1</em> positive/Ki67 positive ESCs found at 4 and 8 weeks. These results demonstrate the potential of tissue-engineered fibrin scaffolds to enhance the survival of NPCs and highlight the need to purify cell populations used in therapies for SCI.

The forkhead box f<em>1</em> (Foxf<em>1</em>) transcription <em>factor</em> is expressed in the visceral (splanchnic) mesoderm, which is involved in mesenchymal-epithelial signaling required for development of organs derived from foregut endoderm such as lung, liver, gall bladder, and pancreas. Our previous studies demonstrated that haploinsufficiency of the Foxf<em>1</em> gene caused pulmonary abnormalities with perinatal lethality from lung hemorrhage in a subset of Foxf<em>1</em>+/- newborn mice. During mouse <em>embryonic</em> development, the liver and biliary primordium emerges from the foregut endoderm, invades the septum transversum mesenchyme, and receives inductive signaling originating from both the septum transversum and cardiac mesenchyme. In this study, we show that Foxf<em>1</em> is expressed in <em>embryonic</em> septum transversum and gall bladder mesenchyme. Foxf<em>1</em>+/- gall bladders were significantly smaller and had severe structural abnormalities characterized by a deficient external smooth muscle cell layer, reduction in mesenchymal cell number, and in some cases, lack of a discernible biliary epithelial cell layer. This Foxf<em>1</em>+/- phenotype correlates with decreased expression of vascular cell adhesion molecule-<em>1</em> (VCAM-<em>1</em>), alpha(5) integrin, platelet-derived <em>growth</em> <em>factor</em> receptor alpha (PDGFRalpha) and hepatocyte <em>growth</em> <em>factor</em> (HGF) genes, all of which are critical for cell adhesion, migration, and mesenchymal cell <em>differentiation</em>.

The aberrant <em>differentiation</em> of pericytes along the adipogenic, chondrogenic, and osteogenic lineages may contribute to the development and progression of several vascular diseases, including atherosclerosis and calcific vasculopathies. However, the mechanisms controlling pericyte <em>differentiation</em> and, in particular, adipogenic and chondrogenic <em>differentiation</em> are poorly defined. Wnt/beta-catenin signaling regulates cell <em>differentiation</em> during <em>embryonic</em> and postnatal development, and there is increasing evidence that it is involved in vascular pathology. Therefore, this study tested the hypothesis that Wnt/beta-catenin signaling regulates the chondrogenic and adipogenic <em>differentiation</em> of pericytes. We demonstrate that pericytes express several Wnt receptors, including LDL receptor-related proteins 5 and 6, and Frizzled <em>1</em> to 4 and 7, 8, and <em>1</em>0, and that Wnt/beta-catenin signaling is stimulated by both Wnt3a and LiCl. Furthermore, induction of Wnt/beta-catenin signaling by LiCl enhances chondrogenesis in pericyte pellet cultures in the presence of transforming <em>growth</em> <em>factor</em>-beta3, as demonstrated by increased Sox-9 expression and glycosaminoglycan accumulation into the matrix. In contrast, transduction of pericytes with a recombinant adenovirus encoding dominant-negative T-cell <em>factor</em>-4 (RAd/dnTCF), which blocks Wnt/beta-catenin signaling, inhibited chondrogenesis, leading to reduced Sox-9 and type II collagen expression and less glycosaminoglycan accumulation. Together, these data demonstrate that transforming <em>growth</em> <em>factor</em>-beta3 induces the chondrogenic <em>differentiation</em> of pericytes by inducing Wnt/beta-catenin signaling and T-cell <em>factor</em>-induced gene transcription. Induction of Wnt/beta-catenin signaling also attenuates adipogenic <em>differentiation</em> of pericytes in both pellet and monolayer cultures, as demonstrated by decreased staining with oil red O and reduced peroxisome proliferator-activated receptor gamma2 expression. This effect was negated by transduction of pericytes with RAd/dnTCF. Together, these results demonstrate that Wnt/beta-catenin signaling inhibits adipogenic and enhances chondrogenic <em>differentiation</em> of pericytes.

Neural stem cell (NSC) has emerged as a potential source for cell replacement therapy following traumatic injuries and degenerative diseases of the central nervous system. However, clinical applications of NSC further require technological advances especially for controlling <em>differentiation</em> of NSC. This study aimed at developing biomaterials that serve to expand undifferentiated NSC or to induce cells with specific phenotypes. Our approach is to construct composite biomaterials that consist of extracellular matrix components and <em>growth</em> <em>factors</em>. In order to optimize matrix-<em>growth</em> <em>factor</em> combinations, we conducted the parallel and rapid screening of composite biomaterials through assays using cell-based arrays. The photo-assisted patterning of an alkanethiol self-assembled monolayer was employed to achieve site-addressable combinatorial immobilization of natural and synthetic matrices incorporated with <em>growth</em> <em>factors</em> including epidermal <em>growth</em> <em>factor</em> (EGF), ciliary neurotrophic <em>factor</em> (CNTF), nerve <em>growth</em> <em>factor</em> (NGF), and neurotrophin-3 (NT-3). NSC obtained from the rat <em>embryonic</em> striatum was cultured directly on the array to screen for cell adhesion, proliferation, and promotion of neuronal and glial specification. The results showed that the significant number of cells adhered to laminin-<em>1</em>, fibronectin, ProNectin, and poly(ethyleneimine). It was found that cells proliferated most extensively on a spot with immobilized EGF among the spots with different matrix-<em>growth</em> <em>factor</em> combinations. The results also showed that neuronal <em>differentiation</em> was promoted on the spots with immobilized NGF or NT-3, and astroglial <em>differentiation</em> with CNTF. Importantly, observed effects of <em>growth</em> <em>factors</em> were frequently altered depending on the type of co-immobilized matrices, suggesting synergic effects of adhesion and <em>growth</em> <em>factor</em> signals.

Adult bone marrow-derived very small <em>embryonic</em>-like stem cells (VSEL-SCs) exhibit a Sca-<em>1</em>(+)/Lin(-)/CD45(-) phenotype and can differentiate into various cell types, including cardiomyocytes and endothelial cells. We have previously reported that transplantation of a small number (<em>1</em> × <em>1</em>0(6)) of freshly isolated, non-expanded VSEL-SCs into infarcted mouse hearts resulted in improved left ventricular (LV) function and anatomy. Clinical translation, however, will require large numbers of cells. Because the frequency of VSEL-SCs in the marrow is very low, we examined whether VSEL-SCs can be expanded in culture without loss of therapeutic efficacy. Mice underwent a 30 min. coronary occlusion followed by reperfusion and, 48 hrs later, received an intramyocardial injection of vehicle (group I, n = <em>1</em><em>1</em>), <em>1</em> × <em>1</em>0(5) enhanced green fluorescent protein (EGFP)-labelled expanded untreated VSEL-SCs (group II, n = 7), or <em>1</em> × <em>1</em>0(5) EGFP-labelled expanded VSEL-SCs pre-incubated in a cardiogenic medium (group III, n = 8). At 35 days after myocardial infarction (MI), mice treated with pre-incubated VSEL-SCs exhibited better global and regional LV systolic function and less LV hypertrophy compared with vehicle-treated controls. In contrast, transplantation of expanded but untreated VSEL-SCs did not produce appreciable reparative benefits. Scattered EGFP(+) cells expressing α-sarcomeric actin, platelet endothelial cell adhesion molecule (PECAM)-<em>1</em>, or von Willebrand <em>factor</em> were present in VSEL-SC-treated mice, but their numbers were very small. No tumour formation was observed. We conclude that VSEL-SCs expanded in culture retain the ability to alleviate LV dysfunction and remodelling after a reperfused MI provided that they are exposed to a combination of cardiomyogenic <em>growth</em> <em>factors</em> and cytokines prior to transplantation. Counter intuitively, the mechanism whereby such pre-incubation confers therapeutic efficacy does not involve <em>differentiation</em> into new cardiac cells. These results support the potential therapeutic utility of VSEL-SCs for cardiac repair.

SOX2 is a well-known core transcription <em>factor</em> in <em>embryonic</em> stem cells (ESCs) and has an important role in the maintenance of pluripotency. Recently, SOX2 expression has also been reported in adult stem cells (ASCs), but the role of SOX2 in ASCs remains unknown. In this study, we examined the molecular mechanisms of SOX2 in human mesenchymal stem cells (hMSCs), a type of ASCs, by performing inhibition studies. SOX2 inhibition resulted in altered cell <em>growth</em> and <em>differentiation</em> capabilities. These changes coincided with a decrease in Dickkopf-<em>1</em> (DKK<em>1</em>), a soluble inhibitor of WNT signaling. Chromatin immunoprecipitation and luciferase assays showed that SOX2 binds to DKK<em>1</em> and has a positive regulatory role in transcription. The enforced expression of DKK<em>1</em> in SOX2-inhibited hMSCs reversed the <em>differentiation</em> deformities, but could not abrogate the cell proliferation defect. Proliferation was regulated by c-MYC, whose expression can also be controlled by SOX2. Our study shows that SOX2 directly regulates DKK<em>1</em> expression and, as a consequence, determines the <em>differentiation</em> lineage of hMSCs. Moreover, SOX2 also regulates proliferation by affecting c-MYC. Therefore, these results suggest that SOX2 might have a specific function by regulating DKK<em>1</em> and c-MYC in the <em>differentiation</em> and <em>growth</em> of ASCs, which is separate from its roles in ESCs.

The calcium-calmodulin-activated protein phosphatase calcineurin functions as a key mediator of diverse biologic processes, including <em>differentiation</em>, apoptosis, <em>growth</em>, and adaptive responses, in part through dephosphorylation and activation of nuclear <em>factor</em> of activated T-cell (NFAT) transcription <em>factors</em>. Apoptosis signal-regulating kinase <em>1</em> (ASK<em>1</em>) is an upstream component of the mitogen-activated protein kinases that serves as a pivotal regulator of cytokine-, oxidative-, and stress-induced cell death. Here, we performed a yeast two-hybrid screen with calcineurin B as bait, which identified ASK<em>1</em> as a direct physical interacting partner. The C-terminal 2<em>1</em>8 amino acids of ASK<em>1</em> were sufficient to mediate interaction with calcineurin B in yeast, as well as in mammalian cell lysates. Importantly, endogenous calcium binding B subunit (CnB) protein interacted with endogenous ASK<em>1</em> protein in cardiomyocytes at baseline, suggesting that the interaction observed in yeast was of potential biologic relevance. Indeed, calcineurin directly dephosphorylated ASK<em>1</em> at serine 967 using purified proteins or mammalian cell lysates. Dephosphorylation of ASK<em>1</em> serine 967 by calcineurin promoted its disassociation from <em>1</em>4-3-3 proteins, resulting in ASK<em>1</em> activation. Calcineurin and ASK<em>1</em> cooperatively enhanced cardiomyocyte apoptosis, while expression of a dominant negative ASK<em>1</em> blocked calcineurin-induced apoptosis. Mouse <em>embryonic</em> fibroblasts deficient in ask<em>1</em> were also partially resistant to calcineurin- or ionomycin-induced apoptosis. Finally, ASK<em>1</em> negatively regulated calcineurin-NFAT signaling indirectly through c-Jun NH2-terminal kinase (JNK)- and p38-mediated phosphorylation of NFAT, which blocked calcineurin- and agonist-dependent hypertrophic <em>growth</em> of cardiomyocytes. Thus, ASK<em>1</em> and calcineurin-NFAT constitute a feedback regulatory circuit in which calcineurin positively regulates ASK<em>1</em> through direct dephosphorylation, while ASK<em>1</em> negatively regulates calcineurin-NFAT signaling through p38- and JNK-mediated NFAT phosphorylation.

Systemic derangements and perinatal death of generalized insulin-like <em>growth</em> <em>factor</em> <em>1</em> (IGF-<em>1</em>) and IGF-<em>1</em> receptor (IGF-<em>1</em>R) knockout mice preclude definitive assessment of IGF-<em>1</em>R actions in <em>growth</em>-plate (GP) chondrocytes. We generated cartilage-specific Igf<em>1</em>r knockout ((Cart) Igf<em>1</em>r(-/-)) mice to investigate local control of chondrocyte <em>differentiation</em> in the GP by this receptor. These mice died shortly after birth and showed disorganized chondrocyte columns, delayed ossification and vascular invasion, decreased cell proliferation, increased apoptosis, and increased expression of parathyroid hormone-related protein (Pthrp) RNA and protein in their GPs. The increased Pthrp expression in the knockout GPs likely was due to an increase in gene transcription, as determined by the increased activity of a LacZ reporter that was inserted downstream of the endogenous PTHrP promoter and bred into the knockout mice. To circumvent the early death of (Cart) Igf<em>1</em>r(-/-) mice and investigate the role of IGF-<em>1</em>R during postnatal <em>growth</em>, we made tamoxifen (Tam)-inducible, cartilage-specific Igf<em>1</em>r knockout ((TamCart) Igf<em>1</em>r(-/-)) mice. At 2 weeks of age and 7 to 8 days after Tam injection, the (TamCart) Igf<em>1</em>r(-/-) mice showed <em>growth</em> retardation with a disorganized GP, reduced chondrocyte proliferation, decreased type 2 collagen and Indian Hedgehog (Ihh) expression, but increased expression of PTHrP. Consistent with in vivo observations, in vitro knockout of the Igf<em>1</em>r gene by adenoviral expression of Cre recombinase suppressed cell proliferation, promoted apoptosis, and increased Pthrp expression. Our data indicate that the IGF-<em>1</em>R in chondrocytes controls cell <em>growth</em>, survival, and <em>differentiation</em> in <em>embryonic</em> and postnatal GPs in part by suppression of Pthrp expression.

Chondrogenesis during <em>embryonic</em> skeletal development involves the condensation of mesenchymal cells followed by their <em>differentiation</em> into chondrocytes. We describe herein a previously unrecognized regulator of mammalian chondrogenesis encoded by a murine <em>growth</em> <em>factor</em>-inducible immediate-early gene, cyr6<em>1</em>. The Cyr6<em>1</em> protein is a secreted, heparin-binding protein (379 amino acids with 38 conserved cysteines) that promotes cell adhesion, migration, and proliferation. The expression pattern of the cyr6<em>1</em> gene during embryogenesis is tissue specific and temporally regulated. Most notably, cyr6<em>1</em> is transiently expressed in mesenchymal cells of both mesodermal and neuroectodermal origins undergoing chondrogenesis, suggesting that Cyr6<em>1</em> may play a role in the development of the <em>embryonic</em> skeleton. In this communication, we demonstrate that the Cyr6<em>1</em> protein promotes chondrogenesis in micromass cultures of limb bud mesenchymal cells in vitro and is likely to play a similar role in vivo based on the following observations: (<em>1</em>) Cyr6<em>1</em> is present in the <em>embryonic</em> limb mesenchyme during chondrogenesis in vivo and in vitro; (2) purified recombinant Cyr6<em>1</em> protein added exogenously to micromass cultures promotes chondrogenesis as judged by precocious expression of type II collagen, increased [35S]sulfate incorporation, and larger Alcian blue-staining cartilage nodules; (3) Cyr6<em>1</em> enhances cell-cell aggregation, an initial step in chondrogenesis, and promotes chondrogenic <em>differentiation</em> in cultures plated at subthreshold cell densities that are otherwise unable to support <em>differentiation</em>; and (4) neutralization of the endogenous Cyr6<em>1</em> with specific antibodies inhibits chondrogenesis. Taken together, these results identify Cyr6<em>1</em> as a novel player in chondrogenesis that contributes to the development of the mammalian <em>embryonic</em> skeleton.

Nkx2.5 (also known as Csx) is an evolutionarily conserved cardiac transcription <em>factor</em> of the homeobox gene family. Nkx2.5 is required for early heart development, since Nkx2.5-null mice die before completion of cardiac looping. To identify genes regulated by Nkx2.5 in the developing heart, we performed subtractive hybridization by using RNA isolated from wild-type and Nkx2.5-null hearts at <em>embryonic</em> day 8.5. We isolated a mouse cDNA encoding myocardin A, which is an alternative spliced isoform of myocardin and the most abundant isoform in the heart from embryo to adult. The expression of myocardin A and myocardin was markedly downregulated in Nkx2.5-null mouse hearts. Transient-cotransfection analysis showed that Nkx2.5 transactivates the myocardin promoter. Inhibition of myocardin function in the teratocarcinoma cell line P<em>1</em>9CL6 prevented <em>differentiation</em> into cardiac myocytes after dimethyl sulfoxide treatment. Myocardin A transactivated the promoter of the atrial natriuretic <em>factor</em> gene through the serum response element, which was augmented by bone morphogenetic protein 2 and transforming <em>growth</em> <em>factor</em> beta-activated kinase <em>1</em>. These results suggest that myocardin expression is regulated by Nkx2.5 and that its function is required for cardiomyogenesis.

The prevailing concept has been that an FGF induces epithelial-to-fiber <em>differentiation</em> in the mammalian lens, whereas chick lens cells are unresponsive to FGF and are instead induced to differentiate by IGF/insulin-type <em>factors</em>. We show here that when treated for periods in excess of those used in previous investigations (>5 h), purified recombinant FGFs stimulate proliferation of primary cultures of <em>embryonic</em> chick lens epithelial cells and (at higher concentrations) expression of the fiber <em>differentiation</em> markers delta-crystallin and CP49. Surprisingly, upregulation of proliferation and delta-crystallin synthesis by FGF does not require activation of ERK kinases. ERK function is, however, essential for stimulation of delta-crystallin expression in response to insulin or IGF-<em>1</em>. Vitreous humor, the presumptive source of <em>differentiation</em>-promoting activity in vivo, contains a <em>factor</em> capable of diffusing out of the vitreous body and inducing delta-crystallin and CP49 expression in chick lens cultures. This <em>factor</em> binds heparin with high affinity and increases delta-crystallin expression in an ERK-insensitive manner, properties consistent with an FGF but not insulin or IGF. Our findings indicate that <em>differentiation</em> in the chick lens is likely to be mediated by an FGF and provide the first insights into the role of the ERK pathway in <em>growth</em> <em>factor</em>-induced signal transduction in the lens.

Protein kinase C lambda (PKClambda) is an atypical member of the PKC family of serine/threonine kinases with high similarity to the other atypical family member, PKCzeta. This similarity has made it difficult to determine specific roles for the individual atypical isoforms. Both PKClambda and PKCzeta have been implicated in the signal transduction, initiated by mediators of innate immunity, that culminates in the activation of MAPKs and NF-kappaB. In addition, work from invertebrates shows that atypical PKC molecules play a role in embryo development and cell polarity. To determine the unique functions of PKClambda, mice deficient for PKClambda were generated by gene targeting. The ablation of PKClambda results in abnormalities early in gestation with lethality occurring by <em>embryonic</em> day 9. The role of PKClambda in cytokine-mediated cellular activation was studied by making mouse chimeras from PKClambda-deficient <em>embryonic</em> stem cells and C57BL/6 or Rag2-deficient blastocysts. Cell lines derived from these chimeric animals were then used to dissect the role of PKClambda in cytokine responses. Although the mutant cells exhibited alterations in actin stress fibers and focal adhesions, no other phenotypic <em>differences</em> were noted. Contrary to experiments using dominant interfering forms of PKClambda, mutant cells responded normally to TNF, serum, epidermal <em>growth</em> <em>factor</em>, IL-<em>1</em>, and LPS. In addition, no abnormalities were found in T cell development or T cell activation. These data establish that, in vertebrates, the two disparate functions of atypical PKC molecules have been segregated such that PKCzeta mediates signal transduction of the innate immune system and PKClambda is essential for early embryogenesis.

The role of FGF signaling in early epithelial <em>differentiation</em> was investigated in ES (<em>embryonic</em> stem) cell derived embryoid bodies. A dominant negative fibroblast <em>growth</em> <em>factor</em> receptor (FGFR) mutation was created by stably introducing into ES cells an Fgfr2 cDNA, truncated in its enzymatic domains. These cells failed to differentiate into cystic embryoid bodies. No epithelial <em>differentiation</em> and cavitation morphogenesis could be observed, in the mutant, although its rate of cell proliferation remained unchanged. This phenotype was associated with a significant decrease in the activation of Akt/PKB and PLCgamma-<em>1</em>, as compared to the wild type, while the activation of MAPK/Erk was less affected. Requirement for PI 3-kinase signaling in embryoid body <em>differentiation</em> was demonstrated by specific inhibitors. Akt/PKB activation was abrogated by wortmannin in short-term experiments. In long-term cultures Ly294002 inhibited the <em>differentiation</em> of ES cells into embryoid bodies. Our data demonstrate that for early epithelial <em>differentiation</em> FGF signaling is required through the PI 3-kinase-Akt/ PKB pathway.

Combinations of hematopoietic cytokines and the ventral mesoderm inducer BMP-4 have recently been shown to augment hematopoietic cell fate of human <em>embryonic</em> stem cells (hESCs) during embryoid body (EB) development. However, <em>factors</em> capable of regulating lineage commitment of hESC-derived hematopoiesis have yet to be reported. Here we show that vascular endothelial <em>growth</em> <em>factor</em> (VEGF-A<em>1</em>65) selectively promotes erythropoietic development from hESCs. Effects of VEGF-A<em>1</em>65 were dependent on the presence of hematopoietic cytokines and BMP-4, and could be augmented by addition of erythropoietin (EPO). Treatment of human EBs with VEGF-A<em>1</em>65 increased the frequency of cells coexpressing CD34 and the VEGF-A<em>1</em>65 receptor KDR, as well as cells expressing erythroid markers. Although fetal/adult globins were unaffected, VEGF-A<em>1</em>65 induced the expression of <em>embryonic</em> zeta (zeta) and epsilon (epsilon) globins, and was accompanied by expression of the hematopoietic transcription <em>factor</em> SCL/Tal-<em>1</em>. In addition to promoting erythropoietic <em>differentiation</em> from hESCs, the presence of VEGF-A<em>1</em>65 enhanced the in vitro self-renewal potential of primitive hematopoietic cells capable of erythroid progenitor capacity. Our study demonstrates a role for VEGF-A<em>1</em>65 during erythropoiesis of differentiating hESCs, thereby providing the first evidence for a <em>factor</em> capable of regulating hematopoietic lineage development of hESCs.