Endothelial cell-derived stem cell factor promotes lipid accumulation through c-Kit-mediated increase of lipogenic enzymes in brown adipocytes.

Active thermogenesis in the brown adipose tissue (BAT) facilitating the utilization of lipids and glucose is critical for maintaining body temperature and reducing metabolic diseases, whereas inactive BAT accumulates lipids in brown adipocytes (BAs), leading to BAT whitening. Although cellular crosstalk between endothelial cells (ECs) and adipocytes is essential for the transport and utilization of fatty acid in BAs, the angiocrine roles of ECs mediating this crosstalk remain poorly understood. Using single-nucleus RNA sequencing and knock-out male mice, we demonstrate that stem cell factor (SCF) derived from ECs upregulates gene expressions and protein levels of the enzymes for de novo lipogenesis, and promotes lipid accumulation by activating c-Kit in BAs. In the early phase of lipid accumulation induced by denervation or thermoneutrality, transiently expressed c-Kit on BAs increases the protein levels of the lipogenic enzymes via PI3K and AKT signaling. EC-specific SCF deletion and BA-specific c-Kit deletion attenuate the induction of the lipogenic enzymes and suppress the enlargement of lipid droplets in BAs after denervation or thermoneutrality in male mice. These data provide insight into SCF/c-Kit signaling as a regulator that promotes lipid accumulation through the increase of lipogenic enzymes in BAT when thermogenesis is inhibited.

Cell-matrix signals specify bone endothelial cells during developmental osteogenesis.

Blood vessels in the mammalian skeletal system control bone formation and support haematopoiesis by generating local niche environments. While a specialized capillary subtype, termed type H, has been recently shown to couple angiogenesis and osteogenesis in adolescent, adult and ageing mice, little is known about the formation of specific endothelial cell populations during early developmental endochondral bone formation. Here, we report that embryonic and early postnatal long bone contains a specialized endothelial cell subtype, termed type E, which strongly supports osteoblast lineage cells and later gives rise to other endothelial cell subpopulations. The differentiation and functional properties of bone endothelial cells require cell-matrix signalling interactions. Loss of endothelial integrin ß1 leads to endothelial cell differentiation defects and impaired postnatal bone growth, which is, in part, phenocopied by endothelial cell-specific laminin α5 mutants. Our work outlines fundamental principles of vessel formation and endothelial cell differentiation in the developing skeletal system.

Perivascular Progenitor Cells Derived From Human Embryonic Stem Cells Exhibit Functional Characteristics of Pericytes and Improve the Retinal Vasculature in a Rodent Model of Diabetic Retinopathy.

UNLABELLED: : Diabetic retinopathy (DR) is the leading cause of blindness in working-age people. Pericyte loss is one of the pathologic cellular events in DR, which weakens the retinal microvessels. Damage to the microvascular networks is irreversible and permanent; thus further progression of DR is inevitable. In this study, we hypothesize that multipotent perivascular progenitor cells derived from human embryonic stem cells (hESC-PVPCs) improve the damaged retinal vasculature in the streptozotocin-induced diabetic rodent models. We describe a highly efficient and feasible protocol to derive such cells with a natural selection method without cell-sorting processes. As a cellular model of pericytes, hESC-PVPCs exhibited marker expressions such as CD140B, CD146, NG2, and functional characteristics of pericytes. Following a single intravitreal injection into diabetic Brown Norway rats, we demonstrate that the cells localized alongside typical perivascular regions of the retinal vasculature and stabilized the blood-retinal barrier breakdown. Findings in this study highlight a therapeutic potential of hESC-PVPCs in DR by mimicking the role of pericytes in vascular stabilization. SIGNIFICANCE: This study provides a simple and feasible method to generate perivascular progenitor cells from human embryonic stem cells. These cells share functional characteristics with pericytes, which are irreversibly lost at the onset of diabetic retinopathy. Animal studies demonstrated that replenishing the damaged pericytes with perivascular progenitor cells could restore retinal vascular integrity and prevent fluid leakage. This provides promising and compelling evidence that perivascular progenitor cells can be used as a novel therapeutic agent to treat diabetic retinopathy patients.

Regulation of vascular endothelial growth factor receptor function in angiogenesis by numb and numb-like.

OBJECTIVE: Vascular endothelial growth factor (VEGF) signaling is a major regulator of physiological and pathological angiogenesis. VEGF receptor activity is strongly controlled by endocytosis, which can terminate or enhance signal transduction in the angiogenic endothelium, but the exact molecular regulation of these processes remains incompletely understood. We have therefore examined the function of Numb family clathrin-associated sorting proteins in angiogenesis. APPROACH AND RESULTS: We show that Numb proteins are expressed by endothelial cells during retinal angiogenesis in mice. Inducible inactivation of the Numb/Numbl genes in the postnatal endothelium led to impaired vessel growth, reduced endothelial proliferation and sprouting, and decreased VEGF receptor activation. Biochemistry and cell biology experiments established that Numb can interact with VEGFR2 and VEGFR3 and controls VEGF receptor activation in response to ligand stimulation. Experiments in cultured endothelial cells showed that Numb proteins counteract VEGF receptor degradation and promote VEGFR2 recycling back to the plasma membrane. CONCLUSIONS: Numb proteins control VEGF receptor endocytosis, signaling, and recycling in endothelial cells, which promotes the angiogenic growth of blood vessels.

Bandgap Widening of Phase Quilted, 2D MoS2 by Oxidative Intercalation.

Controllable bandgap widening from 1.8 to 2.6 eV is reported from oxidized MoS2 sheets that are composed of quilted phases of various MoSxOy flakes. The exfoliated flakes have large size (≥100 µm × 100 µm) sheets with average thickness of 1.7 nm. Remarkably, fine reversible tuning of the bandgap is achieved by postprocessing sulfurization of the MoSxOy sheets.

Stem cell factor is a potent endothelial permeability factor.

OBJECTIVE: Although stem cell factor (SCF) has been shown to play a critical role in hematopoiesis, gametogenesis, and melanogenesis, the function of SCF in the regulation of vascular integrity has not been studied. APPROACH AND RESULTS: We demonstrated that SCF binds to and activates the cKit receptor in endothelial cells, thereby increasing the internalization of vascular endothelial-cadherin and enhancing extravasation of dyes to a similar extent as vascular endothelial growth factor. SCF-mediated cKit activation in endothelial cells enhanced the phosphorylation of endothelial nitric oxide (NO) synthase via the phosphoinositide 3-kinase/Akt signaling pathway and subsequently increased the production of NO. Inhibition of endothelial NO synthase expression and NO synthesis using small interfering RNA knockdown and chemical inhibitors substantially diminished the ability of SCF to increase the internalization of vascular endothelial-cadherin and in vitro endothelial permeability. SCF-induced increase in extravasation of the dyes was abrogated in endothelial NO synthase knockout mice, which indicates that endothelial NO synthase-mediated NO production was responsible for the SCF-induced vascular leakage. Furthermore, we demonstrated that the expression of SCF and cKit was significantly higher in the retina of streptozotocin-injected diabetic mice than in the nondiabetic control animals. Depletion of SCF by intravitreous injection of anti-SCF-neutralizing immunoglobulin G significantly prevented vascular hyperpermeability in the retinas of streptozotocin-injected diabetic mice. CONCLUSIONS: Our data reveal that SCF disrupts the endothelial adherens junction and enhances vascular leakage, as well as suggest that anti-SCF/cKit therapy may hold promise as a potential therapy for the treatment of hyperpermeable vascular diseases.

Isolation and characterization of novel, highly proliferative human CD34/CD73-double-positive testis-derived stem cells for cell therapy.

Human adult stem cells are a readily available multipotent cell source that can be used in regenerative medicine. Despite many advantages, including low tumorigenicity, their rapid senescence and limited plasticity have curtailed their use in cell-based therapies. In this study, we isolated CD34/CD73-double-positive (CD34(+)/CD73(+)) testicular stromal cells (HTSCs) and found that the expression of CD34 was closely related to the cells' stemness and proliferation. The CD34(+)/CD73(+) cells grew in vitro for an extended period of time, yielding a multitude of cells (5.6×10(16) cells) without forming tumors in vivo. They also differentiated into all three germ layer lineages both in vitro and in vivo, produced cartilage more efficiently compared to bone marrow stem cells and, importantly, restored erectile function in a cavernous nerve crush injury rat model. Thus, these HTSCs may represent a promising new autologous cell source for clinical use.

A comparison of human cord blood- and embryonic stem cell-derived endothelial progenitor cells in the treatment of chronic wounds.

Endothelial progenitor cells (EPCs) promote new blood vessel formation and increase angiogenesis by secreting growth factors and cytokines in ischemic tissues. Therefore, EPCs have been highlighted as an alternative cell source for wound healing. EPCs can be isolated from various sources, including the bone marrow, cord blood, and adipose tissue. However, several recent studies have reported that isolating EPCs from these sources has limitations, such as the isolation of insufficient cell numbers and the difficulty of expanding these cells in culture. Thus, human embryonic stem cells (hESCs) have generated great interest as an alternative source of EPCs. Previously, we established an efficient preparation method to obtain EPCs from hESCs (hESC-EPCs). These hESC-EPCs secreted growth factors and cytokines, which are known to be important in angiogenesis and wound healing. In this study, we directly compared the capacity of hESC-EPCs and human cord blood-derived EPCs (hCB-EPCs) to benefit wound healing. The number of hESC-EPCs increased during culture and was always higher than the number of hCB-EPCs during the culture period. In addition, the levels of VEGF and Ang-1 secreted by hESC-EPCs were significantly higher than those produced by hCB-EPCs. After transplantation in a mouse dermal excisional wound model, all EPC-transplanted wounds exhibited better regeneration than in the control group. More importantly, we found that the wounds transplanted with hESC-EPCs showed significantly accelerated re-epithelialization. Thus, hESC-EPCs may be a promising cell source for the treatment of chronic wounds.

Pertussis toxin enhances colony organization of enzymatic-dissociated single human embryonic stem cells.

Human embryonic stem cells (hESCs) self-renew indefinitely as highly organized pluripotent colonies. Unlike mouse pluripotent stem cell colonies, human colonies form a uniform, flat, epithelium-like monolayer. Interestingly, it has been reported that colony morphology is closely correlated with the maintenance of pluripotency. However, the molecular mechanisms that underlie human pluripotent colony formation and organization are poorly understood. In this study, we used real-time imaging tools to examine the in vitro colony formation of enzymatically dissociated single hESCs under feeder-free conditions. We demonstrate that colony formation consists of 4 stages: attachment, migration, aggregation, and colony formation, which are facilitated in an intracellular, calcium-dependent manner. Moreover, we found that blocking G(i)-coupled G protein-coupled receptor (GPCR) signaling results in enhanced cell-cell interactions and plays an integral role in promoting the survival of hESCs in culture. From the imaging results, we identified the conditions required for colony formation, and we identified the importance of blocking G(i)-coupled GPCR by pertussis toxin in modulating hESC colony formation and organization. These results will likely be useful for engineering hESCs to further study the mechanisms involved in their function.

Assessment of differentiation aspects by the morphological classification of embryoid bodies derived from human embryonic stem cells.

In general, the formation of embryoid bodies (EBs) is a commonly known method for initial induction of human embryonic stem cells (hESCs) into their derivatives in vitro. Despite the ability of EBs to mimic developmental processing, the specification and classifications of EBs are not yet well known. Because EBs show various differentiation potentials depending on the size and morphology of the aggregated cells, specification is difficult to attain. Here, we sought to classify the differentiation potentials of EBs by morphologies to enable one to control the differentiation of specific lineages from hESCs with high efficiency. To induce the differentiation of EB formation, we established floating cultures of undifferentiated hESCs in Petri dishes with hESC medium lacking basic fibroblast growth factor. Cells first aggregated into balls; ∼10 days after suspension culture, some different types of EB morphology were present, which we classified as cystic-, bright cavity-, and dark cavity-type EBs. Next, we analyzed the characteristics of each type of EB for its capacity to differentiate into the 3 germ layers via multiplex polymerase chain reaction (PCR), real-time PCR, and immunocytochemistry. Our results indicated that most cells within the cystic EBs were composed of endoderm lineage populations, and both of the cavity EB types were well organized with 3 germ-layer cells. However, the differentiation capacity of the bright cavity EBs was faster than that of the dark cavity EBs. Thus, the bright cavity EBs in this study, which showed equal differentiation tendencies compared with other types of EBs, may serve as the standard for in vitro engineering of EBs. These results indicate that the classification of EB morphologies allows the estimation of the differentiation status of the EBs and may allow the delineation of subsets of conditions necessary for EBs to differentiate into specific cell types.

The use of biodegradable PLGA nanoparticles to mediate SOX9 gene delivery in human mesenchymal stem cells (hMSCs) and induce chondrogenesis.

In stem cell therapy, transfection of specific genes into stem cells is an important technique to induce cell differentiation. To perform gene transfection in human mesenchymal stem cells (hMSCs), we designed and fabricated a non-viral vector system for specific stem cell differentiation. Several kinds of gene carriers were evaluated with regard to their transfection efficiency and their ability to enhance hMSCs differentiation. Of these delivery vehicles, biodegradable poly (DL-lactic-co-glycolic acid) (PLGA) nanoparticles yielded the best results, as they complexed with high levels of plasmid DNA (pDNA), allowed robust gene expression in hMSCs, and induced chondrogenesis. Polyplexing with polyethylenimine (PEI) enhanced the cellular uptake of SOX9 DNA complexed with PLGA nanoparticles both in vitro and in vivo. The expression of enhanced green fluorescent protein (EGFP) and SOX9 increased up to 75% in hMSCs transfected with PEI/SOX9 complexed PLGA nanoparticles 2 days after transfection. SOX9 gene expression was evaluated by RT-PCR, real time-qPCR, glycosaminoglycan (GAG)/DNA levels, immunoblotting, histology, and immunofluorescence.

Differentiation of hESCs into Mesodermal Subtypes: Vascular-, Hematopoietic- and Mesenchymal-lineage Cells.

To date, studies on the application of mesodermally derived mesenchymal-, hematopoietic- and vascular-lineage cells for cell therapy have provided either poor or insufficient data. The results are equivocal with regard to therapeutic efficiency and yield. Since the establishment of human embryonic stem cells (hESCs) in 1998, the capacity of hESCs to differentiate into various mesodermal lineages has sparked considerable interest in the regenerative medicine community, a group interested in generating specialized cells to treat patients suffering from degenerative diseases. Even though hESCs are sensitive, effective methods for guiding the differentiation of hESCs into specific mesodermal cell types are still being developed. In addition, to understand the functional properties of hESC derivatives, numerous animal model studies have been performed by many research groups over the last decade. In this review, we describe and summarize the protocols currently used for differentiation of hESCs into multiple mesodermal lineages and their therapeutic efficiency in different animal models. Furthermore, we discuss the technical hurdles associated with each protocol and the safety of hESC derivatives for therapeutic applications. Technical improvement of the methods used to produce hESC derivatives for therapeutic use in patients with degenerative diseases should remain an objective of future studies, as should the development of effective and stable induction systems.

Functional expression of formyl peptide receptor family in human NK cells.

We determined the expression of the formyl peptide receptor (FPR) family and the functional roles of the FPR family in NK cells. All tested human NK cells express two members of the FPR family (FPR1 and FPR2). The expression of FPR3 was noted to occur in a donor-specific manner. The stimulation of NK cells with FPR family-selective agonists (fMLF (N-formyl-Met-Leu-Phe), MMK-1, F2L, and WKYMVm (Trp-Lys-Tyr-Met-Val-d-Met)) elicited cytolytic activity in resting NK cells, but not in IL-2-activated NK cells; the cytolytic activity was not inhibited by pertussis toxin. The FPR family agonists also stimulated chemotactic migration of IL-2-activated NK cells, but not resting NK cells; the chemotactic migration was completely inhibited by pertussis toxin. WKYMVm stimulates ERK, p38 MAPK, and JNK activities in both resting and IL-2-activated NK cells. WKYMVm-induced chemotactic migration was partially inhibited by PD98059 (2'-amino-3'-methoxyflavone); however, the inhibition of JNK by its selective inhibitor (SP600125, anthra[1,9-cd]pyrazol-6(2H)-one) dramatically inhibited the WKYMVm-induced cytolytic activity. Furthermore, WKYMVm-induced chemotactic migration and cytolytic activity were partly inhibited by FPR family-selective antagonists (cyclosporin H and WRWWWW). Taken together, our findings indicate that human NK cells express functional members of the FPR family, and in turn the activation of the three members of the FPR receptor family elicit cytolytic activity in NK cells, thus suggesting that the receptors are potentially important therapeutic targets for the modulation of NK cell-mediated immune responses.

Depletion of nucleophosmin via transglutaminase 2 cross-linking increases drug resistance in cancer cells.

It has been suggested that nucleophosmin has an anti-apoptotic function via Bax binding. We found that nucleophosmin is a substrate of transglutaminase 2 (TGase 2) in cancer cells. Increased expression of TGase 2 expression is highly associated with drug resistance, and polymerization of nucleophosmin by TGase 2 also can be correlated with the drug resistance of cancer cells. In the present study, an accumulation of nucleophosmin in cytosol was detected when doxorubicin was treated to cancer cells, and it was found, moreover, that an increase of cytosolic nucleophosmin can result in drug-induced apoptosis. Nucleophosmin was polymerized by TGase 2, and the polymerization was inhibited with the TGase 2 inhibitor, cystamine, in vitro. The nucleophosmin level in the cytosolic cell fraction was reduced when TGase 2 was expressed, and the reduced nucleophosmin level was rescued by cystamine treatment. Moreover, nucleophosmin cross-linked by TGase 2 was eradicated in MCF7 cells via the ubiquitin-proteasomal pathway. In parallel with this nucleophosmin-level restoration, the pro-apoptotic Bax protein level was increased. Therefore, depletion of cytosolic nucleophosmin by TGase 2 can decrease Bax protein stability and lead to anti-apoptosis. Drug-resistant cancer cells became sensitive to doxorubicin treatment when nucleophosmin was expressed in cytosol. Taking these results together, it can be concluded that TGase 2 inhibits accumulation of cytosolic nucleophosmin through polymerization, which results in drug resistance in cancer cells.

A new regulatory mechanism of NF-kappaB activation by I-kappaBbeta in cancer cells.

Transglutaminase 2 (TGase 2) catalyzes covalent isopeptide bond formation between glutamine and lysine residues. Recently, we reported that TGase 2 activates nuclear factor-kappa B (NF-kappaB) by depleting inhibitor of NF-kappaBalpha (I-kappaBalpha) levels via polymer formation. Furthermore, TGase 2 expression synergistically increases NF-kappaB activity with canonical pathway. The major I-kappaB proteins such as I-kappaBalpha and I-kappaBbeta resemble each other in both primary sequence and tertiary structure. However, I-kappaBbeta does not degrade fully, while I-kappaBalpha degrades immediately in response to most stimuli. We found that I-kappaBbeta does not contain any of the previously identified TGase 2 target sites. In this study, both an in vitro cross-linking assay and a TGase 2 transfection assay revealed that I-kappaBbeta is independent from TGase 2-mediated polymerization. Furthermore, increased I-kappaBbeta expression reversed NF-kappaB activation in cancer cells, compensating for the loss of I-kappaBalpha via TGase 2 polymerization.

Lysophosphatidylglycerol stimulates chemotactic migration in human natural killer cells.

We observed that lysophosphatidylglycerol (LPG) stimulates chemotactic migration in human natural killer (NK) cells. The LPG-induced chemotactic migration of NK cells was completely inhibited by pertussis toxin (PTX). LPG also stimulated the extracellular signal-regulated kinase (ERK) and Akt activities in NK cells. LPG-stimulated ERK activity was inhibited by PTX, indicating the involvement of PTX-sensitive G-proteins. The preincubation of NK cells with an ERK inhibitor (PD98059) or phosphoinositide-3-kinase (PI3K) inhibitors (wortmannin and LY294002) completely inhibited LPG-induced chemotactic migration, suggesting the essential role of ERK and PI3K in the process. Moreover, LPG-induced chemotactic migration in NK cell was inhibited by Ki16425, an LPA(1/3) receptor-selective antagonist, suggesting the involvement of the Ki16425-sensitive G-protein coupled receptor (GPCR) in the process. Taken together, the results indicate that LPG stimulates chemotactic migration in NK cells through GPCR, suggesting a new function of LPG as a modulator of NK cell functioning.

Optimization of lipase pretreatment prior to lipase immobilization to prevent loss of activity.

In our previous work, a method of pretreating lipase was developed to prevent loss of its activity during covalent immobilization. In this study, Rhizopus oryzae lipase was pretreated before immobilization and then immobilized on a silica gel surface. The effects of the various materials and conditions used in the pretreatment stage on the activity of immobilized lipase were investigated. Immobilized lipase pretreated with 0.1% of soybean oil had better activity than those pretreated with other materials. The optimal temperature, agitation speed, and pretreating time for lipase pretreatment were determined to be 40 degrees C, 200 rpm, and 45 min, respectively. The activity of immobilized soybean oil pretreated lipase was 630 U/g matrix, which is 20 times higher than that of immobilized non-pretreated lipase. In addition, immobilized lipase activity was maintained at levels exceeding 90% of its original activity after 10 reuses.

Lysophosphatidylethanolamine stimulates chemotactic migration and cellular invasion in SK-OV3 human ovarian cancer cells: involvement of pertussis toxin-sensitive G-protein coupled receptor.

We investigated whether lysophosphatidylethanolamine (LPE) modulates cellular signaling in different cell types. SK-OV3 ovarian cancer cells and OVCAR-3 ovarian cancer cells were responsive to LPE. LPE-stimulated intracellular calcium concentration ([Ca(2+)](i)) increase was inhibited by U-73122, suggesting that LPE stimulates calcium signaling via phospholipase C activation. Moreover, pertussis toxin (PTX) almost completely inhibited [Ca(2+)](i) increase by LPE, indicating the involvement of PTX-sensitive G-proteins. Furthermore, we found that LPE stimulated chemotactic migration and cellular invasion in SK-OV3 ovarian cancer cells. We examined the role of lysophosphatidic acid receptors on LPE-stimulated cellular responses using HepG2 cells transfected with different LPA receptors, and found that LPE failed to stimulate nuclear factor kappa B-driven luciferase. We suggest that LPE stimulates a membrane bound receptor, different from well known LPA receptors, resulting in chemotactic migration and cellular invasion in SK-OV3 ovarian cancer cells.

F2L, a peptide derived from heme-binding protein, inhibits formyl peptide receptor-mediated signaling.

F2L is an acetylated amino-terminal peptide derived from the cleavage of the human heme-binding protein. Very recently, F2L was identified as an endogenous chemoattractant peptide acting specifically through formyl peptide receptor-like (FPRL)2. In the present study, we report that F2L stimulates chemotactic migration in human neutrophils. However, F2L inhibits formyl peptide receptor (FPR) and FPRL1 activities, resulting in the complete inhibition of intracellular calcium increases, and superoxide generation induced by N-formyl-Met-Leu-Phe, MMK-1, or Trp-Lys-Tyr-Met-Val-d-Met (WKYMVm) in human neutrophils. In terms of the inhibitory role of F2L on FPR- and FPRL-mediated signaling, we found that F2L competitively inhibits the binding of (125)I-WKYMVm to its specific receptors, FPR and FPRL1. F2L is the first endogenous molecule that inhibits FPR- and FPRL1-mediated signaling, and is expected to be useful in the study of FPR and FPRL1 signaling and in the development of drugs to treat diseases involving the FPR family of receptors.