Blocking autocrine VEGF signaling by sunitinib,an anti-cancer drug,promotes embryonic stem cell self-renewal and somatic cell reprogramming

Maintaining the self-renewal of embryonic stem cells (ESCs) could be achieved by activating the extrinsic signaling, i.e., the use of leukemia inhibitory factor (LIF), or blocking the intrinsic differentiation pathways, i.e., the use of GSK3 and MEK inhibitors (2i). Here we found that even in medium supplemented with LIF, mESCs still tend to differentiate toward meso-endoderm lineages after long-term culture and the culture spontaneously secretes vascular endothelial growth factors (VEGFs). Blocking VEGF signaling with sunitinib, an anti-cancer drug and a receptor tyrosine kinase (RTK) inhibitor mainly targeting VEGF receptors (VEGFRs), is capable of maintaining the mESCs in the undifferentiated state without the need for feeder cells or LIF. Sunitinib facilitates the derivation of mESCs from blastocysts, and the mESCs maintained in sunitinib-containing medium remain pluripotent and are able to contribute to chimeric mice. Sunitinib also promotes iPSC generation from MEFs with only Oct4. Knocking down VEGFR2 or blocking it with neutralizing antibody mimicks the effect of sunitinib, indicating that blocking VEGF/VEGFR signaling is indeed beneficial to the self-renewal of mESCs. We also found that hypoxia-inducible factor alpha (HIF1α) and endoplasmic reticulum (ER) stress are involved in the production of VEGF in mESCs. Blocking both pathways inhibits the expression of VEGF and prevents spontaneous differentiation of mESCs. Interestingly, LIF may also exert its effect by downregulating HIF1α and ER stress pathways and subsequent VEGF expression. These results indicate the existence of an intrinsic differentiation pathway in mESCs by activating the autocrine VEGF signaling. Blocking VEGF signaling with sunitinib or other small molecules help to maintain the mESCs in the ground state of pluripotency.     

Unfolded Protein Response Is Required for the Definitive Endodermal Specification of Mouse Embryonic Stem Cells via Smad2 and β-Catenin Signaling

Tremendous efforts have been made to elucidate the molecular mechanisms that control the specification of definitive endoderm cell fate in gene knockout mouse models and ES cell (ESC) differentiation models. However, the impact of the unfolded protein response (UPR), because of the stress of the endoplasmic reticulum on endodermal specification, is not well addressed. We employed UPR-inducing agents, thapsigargin and tunicamycin, in vitro to induce endodermal differentiation of mouse ESCs. Apart from the endodermal specification of ESCs, Western blotting demonstrated the enhanced phosphorylation of Smad2 and nuclear translocation of β-catenin in ESC-derived cells. The inclusion of the endoplasmic reticulum stress inhibitor tauroursodeoxycholic acid to the induction cultures prevented the differentiation of ESCs into definitive endodermal cells even when Activin A was supplemented. Also, the addition of the TGF-β inhibitor SB431542 and the Wnt/β-catenin antagonist IWP-2 negated the endodermal differentiation of ESCs mediated by thapsigargin and tunicamycin. These data suggest that the activation of the UPR appears to orchestrate the induction of the definitive endodermal cell fate of ESCs via both the Smad2 and β-catenin signaling pathways. The prospective regulatory machinery may be helpful for directing ESCs to differentiate into definitive endodermal cells for cellular therapy in the future.     

Sexual Dimorphism in MAPK-Activated Protein Kinase-2 (MK2) Regulation of RANKL-Induced Osteoclastogenesis in Osteoclast Progenitor Subpopulations

Osteoclasts (OCs) are bone-resorptive cells critical for maintaining skeletal integrity through coupled bone turnover. OC differentiation and activation requires receptor activator of NF-kB ligand (RANKL) signaling through the p38 MAPK pathway. However the role of the p38 MAPK substrate, MAPK-activated protein kinase 2 (MK2), is not clearly delineated. Within the bone marrow exists a specific subpopulation of defined osteoclast progenitor cells (dOCPs) with surface expression of B220^-Gr1^-CD11b^lo/-CD115⁺ (dOCP^lo/-). In this study, we isolated dOCPs from male and female mice to determine sex-specific effects of MK2 signaling in osteoclastogenesis (OCgen). Male Mk2^-/- mice display an increase in the dOCP^lo cell population when compared to Mk2^+/+ mice, while female Mk2^-/- and Mk2^+/+ mice exhibit no difference. Defined OCPs from male and female Mk2^+/+ and Mk2^-/- bone marrow were treated with macrophage colony stimulation factor (M-CSF) and RANKL cytokines to promote OCgen. RANKL treatment of dOCP^lo cells stimulated p38 and MK2 phosphorylation. Tartrate-resistant acid phosphatase (TRAP) assays were used to quantify OC number, size, and TRAP enzyme activity post-RANKL stimulation. MK2 signaling was critical for male dOCP^lo OCgen, yet MK2 signaling regulated OCgen from female dOCP^- and CD11b^hi subpopulations as well. The functional gene, Ctsk, was attenuated in both male and female Mk2^-/- dOCP^lo-derived OCs. Conversely, MK2 signaling was only critical for gene expression of pre-OC fusion genes, Oc-stamp andTm7sf4, in male OCgen. Therefore, these data suggest there is a sexual dimorphism in MK2 signaling of OCP subpopulations.     

CD34+VEGFR‐3+ progenitor cells have a potential to differentiate towards lymphatic endothelial cells

Endothelial progenitor cells (EPCs) play an important role in postnatal neovascularization. However, it is poorly understood whether EPCs contribute to lymphangiogenesis. Here, we assessed differentiation of a novel population of EPCs towards lymphatic endothelial cells and their lymphatic formation. CD34⁺VEGFR‐3⁺ EPCs were isolated from mononuclear cells of human cord blood by fluorescence‐activated cell sorting. These cells expressed CD133 and displayed the phenotype of the endothelial cells. Cell colonies appeared at 7–10 days after incubation. The cells of the colonies grew rapidly and could be repeatedly subcultured. After induction with VEGF‐C for 2 weeks, CD34⁺VEGFR‐3⁺ EPCs could differentiate into lymphatic endothelial cells expressing specific markers 5′‐nucleotidase, LYVE‐1 and Prox‐1. The cells also expressed hyaluronan receptor CD44. The differentiated cells had properties of proliferation, migration and formation of lymphatic capillary‐like structures in three‐dimensional collagen gel and Matrigel. VEGF‐C enhanced VEGFR‐3 mRNA expression. After interfering with VEGFR‐3 siRNA, the effects of VEGF‐C were diminished. These results demonstrate that there is a population of CD34⁺VEGFR‐3⁺ EPCs with lymphatic potential in human cord blood. VEGF‐C/VEGFR‐3 signalling pathway mediates differentiation of CD34⁺VEGFR‐3⁺ EPCs towards lymphatic endothelial cells and lymphangiogenesis. Cord blood‐derived CD34⁺VEGFR‐3⁺ EPCs may be a reliable source in transplantation therapy for lymphatic regenerative diseases.     

Glycyrrhizin and its derivatives promote hepatic differentiation via sweet receptor,Wnt, and Notch signaling

The acute liver disease is involved in aberrant release of high-mobility group box 1 (HMGB1). Glycyrrhizin (GL), a traditional Chinese medicine for liver disease, binds to HMGB1, thereby inhibits tissue injury. However the mode of action of GL for chronic liver disease remains unclear.We investigated the effects of glycyrrhizin (GL) and its derivatives on liver differentiation using human iPS cells by using a flow cytometric analysis.GL promoted hepatic differentiation at the hepatoblast formation stage. The GL derivatives, 3-O-mono-glucuronyl 18β-glycyrrhetinic acid (Mono) and 3-O-[glucosyl (1 → 2)-glucuronyl] 18β-glycyrrhetinic acid increased AFP⁺ cell counts and albumin⁺ cell counts. Glucuronate conjugation seemed to be a requirement for hepatic differentiation. Mono exhibited the most significant hepatic differentiation effect.We evaluated the effects of (±)-2-(2,4-dichlorophenoxy) propionic acid (DP), a T1R3 antagonist, and sucralose, a T1R3 agonist, on hepatic differentiation, and found that DP suppressed Mono-induced hepatic differentiation, while sucralose promoted hepatic differentiation. Thus, GL promoted hepatic differentiation via T1R3 signaling. In addition, Mono increased β-catenin⁺ cell count and decreased Hes5⁺ cell count suggesting the involvement of Wnt and Notch signaling in GL-induced hepatic differentiation.In conclusion, GL exerted a hepatic differentiation effect via sweet receptor (T1R3), canonical Wnt, and Notch signaling.     

Role of voltage‐gated potassium channels in the fate determination of embryonic stem cells

Embryonic stem cells (ESCs) possess two unique characteristics: self‐renewal and pluripotency. In this study, roles of voltage‐gated potassium channels (K_v) in maintaining mouse (m) ESC characteristics were investigated. Tetraethylammonium (TEA⁺), a K_v blocker, attenuated cell proliferation in a concentration‐dependent manner. Possible reasons for this attenuation, including cytotoxicity, cell cycle arrest and differentiation, were examined. Blocking K_v did not change the viability of mESCs. Interestingly, K_v inhibition increased the proportion of cells in G₀/G₁ phase and decreased that in S phase. This change in cell cycle distribution can be attributed to cell cycle arrest or differentiation. Loss of pluripotency as determined at both molecular and functional levels was detected in mESCs with K_v blockade, indicating that K_v inhibition in undifferentiated mESCs directs cells to differentiate instead of to self‐renew and progress through the cell cycle. Membrane potential measurement revealed that K_v blockade led to depolarization, consistent with the role of K_v as the key determinant of membrane potential. The present results suggest that membrane potential changes may act as a “switch” for ESCs to decide whether to proliferate or to differentiate: hyperpolarization at G₁ phase would favor ESCs to enter S phase while depolarization would favor ESCs to differentiate. Consistent with this notion, S‐phase‐synchronized mESCs were found to be more hyperpolarized than G₀/G₁‐phase‐synchronized mESCs. Moreover, when mESCs differentiated, the differentiation derivatives depolarized at the initial stage of differentiation. This investigation is the first study to provide evidence that K_v and membrane potential affect the fate determination of ESCs. J. Cell. Physiol. 224:165–177, 2010 © 2010 Wiley‐Liss, Inc.     

Endothelial Cell Co-culture Mediates Maturation of Human Embryonic Stem Cell to Pancreatic Insulin Producing Cells in a Directed Differentiation Approach

Embryonic stem cells (ESC) have two main characteristics: they can be indefinitely propagated in vitro in an undifferentiated state and they are pluripotent, thus having the potential to differentiate into multiple lineages. Such properties make ESCs extremely attractive for cell based therapy and regenerative treatment applications ¹. However for its full potential to be realized the cells have to be differentiated into mature and functional phenotypes, which is a daunting task. A promising approach in inducing cellular differentiation is to closely mimic the path of organogenesis in the in vitro setting. Pancreatic development is known to occur in specific stages ², starting with endoderm, which can develop into several organs, including liver and pancreas. Endoderm induction can be achieved by modulation of the nodal pathway through addition of Activin A ³ in combination with several growth factors ^4-7. Definitive endoderm cells then undergo pancreatic commitment by inhibition of sonic hedgehog inhibition, which can be achieved in vitro by addition of cyclopamine ⁸. Pancreatic maturation is mediated by several parallel events including inhibition of notch signaling; aggregation of pancreatic progenitors into 3-dimentional clusters; induction of vascularization; to name a few. By far the most successful in vitro maturation of ESC derived pancreatic progenitor cells have been achieved through inhibition of notch signaling by DAPT supplementation ⁹. Although successful, this results in low yield of the mature phenotype with reduced functionality. A less studied area is the effect of endothelial cell signaling in pancreatic maturation, which is increasingly being appreciated as an important contributing factor in in-vivo pancreatic islet maturation ^10,11.The current study explores such effect of endothelial cell signaling in maturation of human ESC derived pancreatic progenitor cells into insulin producing islet-like cells. We report a multi-stage directed differentiation protocol where the human ESCs are first induced towards endoderm by Activin A along with inhibition of PI3K pathway. Pancreatic specification of endoderm cells is achieved by inhibition of sonic hedgehog signaling by Cyclopamine along with retinoid induction by addition of Retinoic Acid. The final stage of maturation is induced by endothelial cell signaling achieved by a co-culture configuration. While several endothelial cells have been tested in the co-culture, herein we present our data with rat heart microvascular endothelial Cells (RHMVEC), primarily for the ease of analysis.     

Single Mechanosensitive and Ca2+-Sensitive Channel Currents Recorded from Mouse and Human Embryonic Stem Cells

Cell-attached and inside-out patch clamp recording was used to compare the functional expression of membrane ion channels in mouse and human embryonic stem cells (ESCs). Both ESCs express mechanosensitive Ca²⁺ permeant cation channels (MscCa) and large conductance (200 pS) Ca²⁺-sensitive K⁺ (BK_Ca2+) channels but with markedly different patch densities. MscCa is expressed at higher density in mESCs compared with hESCs (70 % vs. 3 % of patches), whereas the BK_Ca2+ channel is more highly expressed in hESCs compared with mESCs (~50 % vs. 1 % of patches). ESCs of both species express a smaller conductance (25 pS) nonselective cation channel that is activated upon inside-out patch formation but is neither mechanosensitive nor strictly Ca²⁺-dependent. The finding that mouse and human ESCs express different channels that sense membrane tension and intracellular [Ca²⁺] may contribute to their different patterns of growth and differentiation in response to mechanical and chemical cues.     

Hedgehog signal activation coordinates proliferation and differentiation of fetal liver progenitor cells

Hedgehog (Hh) signaling plays crucial roles in development and homeostasis of various organs. In the adult liver, it regulates proliferation and/or viability of several types of cells, particularly under injured conditions, and is also implicated in stem/progenitor cell maintenance. However, the role of this signaling pathway during the normal developmental process of the liver remains elusive. Although Sonic hedgehog (Shh) is expressed in the ventral foregut endoderm from which the liver derives, the expression disappears at the onset of the liver bud formation, and its possible recurrence at the later stages has not been investigated. Here we analyzed the activation and functional relevance of Hh signaling during the mouse fetal liver development. At E11.5, Shh and an activation marker gene for Hh signaling, Gli1, were expressed in Dlk⁺ hepatoblasts, the fetal liver progenitor cells, and the expression was rapidly decreased thereafter as the development proceeded. In the culture of Dlk⁺ hepatoblasts isolated from the E11.5 liver, activation of Hh signaling stimulated their proliferation and this effect was cancelled by a chemical Hh signaling inhibitor, cyclopamine. In contrast, hepatocyte differentiation of Dlk⁺ hepatoblasts in vitro as manifested by the marker gene expression and acquisition of ammonia clearance activity was significantly inhibited by forced activation of Hh signaling. Taken together, these results demonstrate the temporally restricted manner of Hh signal activation and its role in promoting the hepatoblast proliferation, and further suggest that the pathway needs to be shut off for the subsequent hepatic differentiation of hepatoblasts to proceed normally.     

Effects of an endothelial cell-conditioned medium on the hematopoietic and endothelial differentiation of embryonic stem cells

We have examined the effect of mouse bone marrow endothelial cell-conditioned medium (mEC-CM) on hematopoietic and endothelial differentiation of mouse embryonic stem cells (mESCs). mEC-CM can efficiently promote the differentiation of mESCs into Flk⁺ cells and hematopoietic colony-forming cells. mEC-CM proved to be as potent as a cytokine cocktail comprised of VEGF, bFGF, IGF and EGF. After inducing mESCs with mEC-CM, cobblestone-like cells were mechanically selected and identified which had the ability to incorporate DiI-Ac-LDL. DiI-Ac-LDL-positive cells were endothelial-like cells due to their expression of CD31 and Flk1, ability to bind to UEA1 and capacity to form capillary-like tube structures on matrigel. In conclusion, mEC-CM can efficiently promote the differentiation of mESCs into endothelial cells and hematopoietic colony-forming cells. The differentiated endothelial-like cells can be isolated by using DiI-Ac-LDL labeling and mechanical selection.     

Loss of Vascular Endothelial Growth Factor A (VEGFA) Isoforms in Granulosa Cells Using pDmrt-1-Cre or Amhr2-Cre Reduces Fertility by Arresting Follicular Development and by Reducing Litter Size in Female Mice

Because VEGFA has been implicated in follicle development, the objective of this study was to determine the effects of granulosa- and germ cell-specific VEGFA loss on ovarian morphogenesis, function, and female fertility. pDmrt1-Cre mice were mated to floxed VEGFA mice to develop granulosa-/germ cell-specific knockouts (pDmrt1-Cre;Vegfa^-/-). The time from mating to first parturition was increased when pDmrt1-Cre;Vegfa^-/- females were mated to control males (P = 0.0008) and tended to be longer for heterozygous females (P < 0.07). Litter size was reduced for pDmrt1-Cre;Vegfa^-/- females (P < 0.007). The time between the first and second parturitions was also increased for heterozygous females (P < 0.04) and tended to be increased for pDmrt1-Cre;Vegfa^-/- females (P < 0.07). pDmrt1-Cre;Vegfa^-/- females had smaller ovaries (P < 0.04), reduced plasma estradiol (P < 0.007), fewer developing follicles (P < 0.008) and tended to have fewer corpora lutea (P < 0.08). Expression of Igf1r was reduced (P < 0.05); expression of Foxo3a tended to be increased (P < 0.06); and both Fshr (P < 0.1) and Sirt6 tended to be reduced (P < 0.06) in pDmrt1-Cre;Vegfa^-/- ovaries. To compare VEGFA knockouts, we generated Amhr2-Cre;Vegfa^-/- mice that required more time from mating to first parturition (P < 0.003) with variable ovarian size. Both lines had more apoptotic granulosa cells, and vascular staining did not appear different. Taken together these data indicate that the loss of all VEGFA isoforms in granulosa/germ cells (proangiogenic and antiangiogenic) causes subfertility by arresting follicular development, resulting in reduced ovulation rate and fewer pups per litter.     

The embryonic midbrain directs neuronal specification of embryonic stem cells at early stages of differentiation

Specific neuronal differentiation of Embryonic Stem Cells (ESCs) depends on their capacity to interpret environmental cues. At present, it is not clear at which stage of differentiation ESCs become competent to produce multiple neuronal lineages in response to the niche of the embryonic brain. To unfold the developmental potential of ESC-derived precursors, we transplanted these cells into the embryonic midbrain explants, where neurogenesis occurs as in normal midbrain development. Using this experimental design, we show that the transition from ESCs to Embryoid Body (EB) precursors is necessary to differentiate into Lmx1a⁺/Ptx3⁺/TH⁺ dopaminergic neurons around the ventral midline of the midbrain. In addition, EB cells placed at other dorsal-ventral levels of the midbrain give rise to Nkx6.1⁺ red nucleus (RN) neurons, Nkx2.2⁺ ventral interneurons and Pax7⁺ dorsal neurons at the correct positions. Notably, differentiation of ESCs into Neural Precursor Cells (NPCs) prior to transplantation markedly reduces specification at the Lmx1a, Nkx6.1 and Pax7 expression domains, without affecting neuronal differentiation. Finally, exposure to Fgf8 and Shh in vitro promotes commitment of some ESC-derived NPCs to differentiate into putative Lmx1a⁺ dopaminergic neurons in the midbrain. Our data demonstrate intrinsic developmental potential differences among ESC-derived precursor populations.     

Factors from Human Embryonic Stem Cell-derived Fibroblast-like Cells Promote Topology-dependent Hepatic Differentiation in Primate Embryonic and Induced Pluripotent Stem Cells

The future clinical use of embryonic stem cell (ESC)-based hepatocyte replacement therapy depends on the development of an efficient procedure for differentiation of hepatocytes from ESCs. Here we report that a high density of human ESC-derived fibroblast-like cells (hESdFs) supported the efficient generation of hepatocyte-like cells with functional and mature hepatic phenotypes from primate ESCs and human induced pluripotent stem cells. Molecular and immunocytochemistry analyses revealed that hESdFs caused a rapid loss of pluripotency and induced a sequential endoderm-to-hepatocyte differentiation in the central area of ESC colonies. Knockdown experiments demonstrated that pluripotent stem cells were directed toward endodermal and hepatic lineages by FGF2 and activin A secreted from hESdFs. Furthermore, we found that the central region of ESC colonies was essential for the hepatic endoderm-specific differentiation, because its removal caused a complete disruption of endodermal differentiation. In conclusion, we describe a novel in vitro differentiation model and show that hESdF-secreted factors act in concert with regional features of ESC colonies to induce robust hepatic endoderm differentiation in primate pluripotent stem cells.     

VEGF-induced angiogenesis ameliorates the memory impairment in APP transgenic mouse model of Alzheimer's disease

Vascular endothelial growth factor (VEGF) was investigated in the present study to see whether it could provide a therapeutic opportunity for the treatment of Alzheimer’s disease (AD). PDGF-hAPP^V717I transgenic mice were treated with VEGF or PBS by intraperitoneal injection for three consecutive days. The results showed that VEGF ameliorated the memory impairment of mice, accompanied by CD34⁺ cells increasing in peripheral blood, vWF⁺ vessels increasing in hippocampus, and CD34⁺/VEGFR2⁺, vWF⁺/VEGFR2⁺ and BrdU⁺/vWF⁺ cells expressing in hippocampus. Furthermore, the level of choline acetyltransferase (ChAT) was considerably enhanced and Aβ deposition was decreased in the brains of mice upon VEGF treatment. These observations suggest that VEGF should be pursued as a novel therapeutic agent for treatment of AD.     

Murine Embryonic Stem Cell Plasticity Is Regulated through Klf5 and Maintained by Metalloproteinase MMP1 and Hypoxia

Mouse embryonic stem cells (mESCs) are expanded and maintained pluripotent in vitro in the presence of leukemia inhibitory factor (LIF), an IL6 cytokine family member which displays pleiotropic functions, depending on both cell maturity and cell type. LIF withdrawal leads to heterogeneous differentiation of mESCs with a proportion of the differentiated cells apoptosising. During LIF withdrawal, cells sequentially enter a reversible and irreversible phase of differentiation during which LIF addition induces different effects. However the regulators and effectors of LIF–mediated reprogramming are poorly understood. By employing a LIF-dependent ‘plasticity’ test, that we set up, we show that Klf5, but not JunB is a key LIF effector. Furthermore PI3K signaling, required for the maintenance of mESC pluripotency, has no effect on mESC plasticity while displaying a major role in committed cells by stimulating expression of the mesodermal marker Brachyury at the expense of endoderm and neuroectoderm lineage markers. We also show that the MMP1 metalloproteinase, which can replace LIF for maintenance of pluripotency, mimics LIF in the plasticity window, but less efficiently. Finally, we demonstrate that mESCs maintain plasticity and pluripotency potentials in vitro under hypoxic/physioxic growth conditions at 3% O₂ despite lower levels of Pluri and Master gene expression in comparison to 20% O₂.