Selective Cell Adhesion Research Articles

Fabrication and characterization of nanopatterns for selective cell adhesion

Nanopatterns with chemical functionalization are of importance in biological sensing and drug delivery systems. Despite recent advances in nanotechnology, producing large-area nanopatterned surfaces for controlling the size of cellular adhesion sites is still a challenge. Unlike for homogeneous substrates, only a few techniques suitable for characterizing the chemical properties of nanopatterned substrates with a sufficient lateral resolution exist. In this research, we fabricated nanopatterned substrates composed of 2D arrays of gold nanoislands by using nanosphere lithography (NSL). Orthogonal chemistry was applied to achieve a selective adsorption of fibronectins to nanoislands. The contact angle and the X-ray photoelectron spectroscopy (XPS) data confirmed the success of the dual functionalization performed on 1/2Au-1/2glass substrates. The chemical properties of nanopatterns were investigated by using Atomic Force Microscopy (AFM). The adhesion maps and the topographic images confirmed nanometer-scale chemical functionalization.

ESAM predicts cardiovascular mortality in diabetic hemodialysis patients

Aim: To assess endothelial cell selective adhesion molecule (ESAM) as predictor of cardiovascular mortality in diabetic dialysis patients (DDPs).Methods: ESAM, clinical and laboratory parameters were assessed in 73 DDP. Cardiovascular mortality was recorded in a 2 years’ prospective observational study.Results: Baseline ESAM was 17.1 (10.05–24.8) ng/ml and was correlated to phosphate (r = −0.42, p = 0.008), parathormone (r = −0.36, p = 0.048), albumin (r = −0.24, p = 0.048). ESAM significantly predicted cardiovascular death in univariate [HR = 1.03, 95% CI (1.006–1.054), p = 0.01] and multivariate [HR = 1.034, 95% CI (1.003–1.066), p = 0.03] Cox analysis. Time to cardiovascular death was shorter for patients with ESAM >12.44 ng/ml, p = 0.0045.Conclusion: ESAM is an independent predictor of cardiovascular mortality in DDP.

Selective Cell Adhesion and Biosensing Applications of Bio-Active Block Copolymers Prepared by CuAAC/Thiol-ene Double Click Reactions.

N-Acetyl-l-cysteine (NAC)-capped poly(methyl methacrylate)-b-polycaprolactone block copolymer (PMMA-b-PCL-NAC) was prepared using the previously described one-pot photoinduced sequential CuAAC/thiol-ene double click procedure. PMMA-b-PCL-NAC had previously shown good applicability as a matrix for cell adhesion of cells from the Vero cell line (African green monkey kidney epithelial). Here, in this work, PMMA-b-PCL-NAC served as an excellent immobilization matrix for biomolecule conjugation. Covalent binding of RGD (R: arginine, G: glycine, and D: aspartic acid) peptide sequence onto the PMMA-b-PCL-NAC-coated surface was performed via EDC chemistry. RGD-modified PMMA-b-PCL-NAC (PMMA-b-PCL-NAC-RGD) as a non-toxic cell proliferation platform was used for selective "integrin αvβ3-mediated cell adhesion and biosensing studies. Both optical and electrochemical techniques were used to monitor the adhesion differences between "integrin αvβ3" receptor positive and negative cell lines on to the designed biofunctional surfaces.

Age-related changes in monocytes exacerbate neointimal hyperplasia after vascular injury.

Neointimal hyperplasia is the leading cause of restenosis after endovascular interventions. It is characterized by the accumulation of myofibroblast-like cells and extracellular matrix in the innermost layer of the wall and is exacerbated by inflammation. Monocytes from either young or aged rats were applied perivascularly to injured vascular walls of young recipient animals. Monocytes from aged rats, but not young donors, increased neointima thickness. Accordingly, the gene expression profiles of CD11b+ monocytes from aged rats showed significant up-regulation of genes involved in cellular adhesion, lipid degradation, cytotoxicity, differentiation, and inflammation. These included cadherin 13 (Cdh13), colony stimulating factor 1 (Csf1), chemokine C-X-C motif ligand 1 (Cxcl1), endothelial cell-selective adhesion molecule (Esam), and interferon gamma (Ifng). In conclusion, our results suggest that the increased inflammatory and adhesive profile of monocytes contributes to pathological wall remodeling in aged-related vascular diseases.

Dendritic cell SIRPα regulates homeostasis of dendritic cells in lymphoid organs

Signal regulatory protein α (SIRPα), an immunoglobulin superfamily protein that is expressed predominantly in myeloid lineage cells such as dendritic cells (DCs) or macrophages, mediates cell-cell signaling. In the immune system, SIRPα is thought to be important for homeostasis of DCs, but it remains unclear whether SIRPα intrinsic to DCs is indeed indispensable for such functional role. Thus, we here generated the mice, in which SIRPα was specifically ablated in CD11c(+) DCs (Sirpa(Δ) (DC) ). Sirpa(Δ) (DC) mice manifested a marked reduction of CD4(+) CD8α(-) conventional DCs (cDCs) in the secondary lymphoid organs, as well as of Langerhans cells in the epidermis. Such reduction of cDCs in Sirpa(Δ) (DC) mice was comparable to that apparent with the mice, in which SIRPα was systemically ablated. Expression of SIRPα in DCs was well correlated with that of either endothelial cell-selective adhesion molecule (ESAM) or Epstein-Barr virus-induced molecule 2 (EBI2), both of which were also implicated in the regulation of DC homeostasis. Indeed, ESAM(+) or EBI2(+) cDCs were markedly reduced in the spleen of Sirpa(Δ) (DC) mice. Thus, our results suggest that SIRPα intrinsic to CD11c(+) DCs is essential for homeostasis of cDCs in the secondary lymphoid organs and skin.

Surface modulation of complex stiffness via layer-by-layer assembly as a facile strategy for selective cell adhesion.

In-stent restenosis and thrombosis are the main severe problems that occur after the percutaneous vascular intervention. The competition between endothelial cells (ECs) and smooth muscle cells (SMCs) plays a key role during these pathological changes. The regulation of this competition offers new opportunities to design biomaterials in the cardiovascular fields. Bioactive molecules have been typically employed to increase EC adhesion and thereafter to enhance EC competitiveness; however, this method is associated with limitations from the point of view of practical and industrial applications. Herein, we present an approach to enhance EC competitiveness over that of SMC through the selective EC adhesion, which is achieved by modulating a complex surface stiffness based on the technique of layer-by-layer (LbL) assembly. This complex stiffness can be achieved by regulating the thickness of multilayer films coordinating with a rigid underlying substrate. The selective cell adhesion is attributed to changes in the complex surface stiffness and a different intrinsic property between ECs and SMCs. This study provides a facile and broadly applicable approach for the purpose of the enhancement of EC competitiveness over that of SMC, which has great potential for the development of cell-based functional biomaterials in the cardiovascular field.

1C33 表面の幾何学的特徴が細胞の選択接着機構におよぼす影響(OS2-3:細胞・分子のバイオメカニクス(3))

1C33 表面の幾何学的特徴が細胞の選択接着機構におよぼす影響(OS2-3:細胞・分子のバイオメカニクス(3))

Controlled surface density of RGD ligands for cell adhesion: evidence for ligand specificity by using QCM-D.

RGD peptides (Arg-Gly-Asp) are known to promote cell adhesion. As a consequence, numerous materials have been functionalized using these peptides for several medical applications. We report herein the controlled functionalization of surfaces to study the influence of RGD density on cell selectivity. For this purpose, we selected a quartz crystal microbalance QCM-D as this technique allows real-time monitoring of cell adhesion to RGD surfaces. We observed that a critical spacing of nearly 40 nm between RGD ligands is required to observe selective cell adhesion whereas a higher density is not specific.

Analysis of biomarkers for risk of acute kidney injury after primary angioplasty for acute ST-segment elevation myocardial infarction: results of the HORIZONS-AMI trial.

Contrast-induced acute kidney injury (CI-AKI) may occur after percutaneous coronary intervention (PCI). We evaluated patients with ST-elevation myocardial infarction (STEMI) undergoing emergency PCI with serial biomarkers. Of the 390 patients enrolled in the HORIZONS-AMI biomarker substudy, 56 (14.3%) developed AKI. In the AKI group, the levels of B-type natriuretic peptide were consistently higher than in the no-AKI group at baseline (P = 0.0327), hospital discharge (P = 0.0002), 30-day follow-up (P = 0.0193), and 1-year follow-up (P = 0.031). At hospital discharge, the AKI group had elevated biomarkers compared to the no-AKI group: D-dimer (P = 0.0066), C-reactive protein (P = 0.0468), endothelial cell-selective adhesion molecule (P = 0.0169), adiponectin (P = 0.0346), and von Willebrand factor (P = 0.0168); there was also a trend toward higher cystatin C (P = 0.0585) in the AKI group. Similar correlations between biomarker panel increase and the development of CI-AKI were consistent at baseline, 30-day, and 1-year follow-up. Chemokine (C-C motif) ligand 23 showed an opposite pattern with an increase at all time points in the no-AKI compared to the AKI group. The risk of CI-AKI after primary PCI for STEMI may be associated with hemostatic imbalances, activation of procoagulants, decreased endogenous anticoagulants, enhanced inflammation, platelet activation, or decreased fibrinolytic activity.

O1-11-6 - Expression of Esam and Vwf as Angiogenesis Marker in Tumor Growth and Metastasis of Carcinoma

Background: The formation of new vascular (angiogenesis) is one of important step in tumor growth and have correlation with the capability of tumor cell to mestastase in distant place. Endothelial cell-Selective Adhesion Molecule (ESAM) is protein which expressed in endothelial cell of blood vessel. Von Willebrand Factor (vWF) is glycoprotein which is found in blood plasma and were produced by both endothelial cell and megakaryocytes. Meanwhile, Ki-67 is one of cell protein which is coded by MKi-67 gene in human cells and have relations with cell proliferation.Methods: A series of 79 tissue samples from carcinoma mammae were stained immunohistochemically with ESAM, vWF and Ki-67 antibodies to evaluate its vascularity and cell proliferation.Results: From these samples, it was found that 31 (39%) samples have low vascularity and 48 (61%) samples have high vascularity according to ESAM expression. Meanwhile, in correlated with vWF expression, there were 34 (43%) samples with low vascularity and 45 (54%) samples with high vascularity. There was a significant correlation of totally number of vascular in tumor samples which have stained with ESAM and vWF with totally number of cells which expressed Ki-67 and tumor size/volume. There was only a significant correlation in between totally number of vascularity which expressed ESAM and the presence of lymphe nodes metastasis. There was also significant correlation between increase of vascularity number and number of cells with Ki-67 expression.Conclusion: It was concluded that angiogenesis was highly required in tumor growth and metastases and both of ESAM and vWF have involved in angiogenesis process of breast cancer. However, ESAM looks more sensitive than vWF to predict the presence of metastases in breast cancer.

Abstract 3303: Engineering of highly controlled in vitro co-culture model to study the mesenchymal stem cells mediated signaling in breast cancer cells

Abstract Mesenchymal stem cells (MSCs) mediated signaling in breast cancer cells (BCCs) are critical for epithelial-mesenchymal transition (EMT) and metastasis. The mutual interactions between tumor cells and MSCs via direct contact or paracrine interaction through the recruitment of growth factors, cytokines and chemokines are thought to modulate tumor growth, invasion, metastasis and angiogenesis. Therefore, the cell-cell interactions that control the behaviour of cancer cells are targets with as much potential for the development of effective therapies as the tumor cells themselves. To this end, co-culture of BCCs with MSCs is critical to understand the role of MSCs on breast cancer progression. However, establishing co-culture of these cells with BCCs is challenging as the variation in culture condition can compromise the viability and functionality of co-cultured cells. In the present study, we have developed an in vitro co-culture method for controlling the interaction of BCCs with MSCs using biocompatible synthetic materials. We used cationic and anionic polymers to create stable and consistent surfaces. After that, these polymers were screened for the selective adhesion of cells, to create the patterned co-culture of BCCs and MSCs. Unlike other synthetic and natural polymers, one of the greatest advantages of screened polymers is that they are biologically inactive material for BCCs and MSCs cells, thus, do not alter the gene or protein expression profile of BCCs and MSCs. Therefore, the results obtained using this method provide true interaction of BCCs with MSCs in co-culture system without interference of interaction of cells with matrix. In addition, the in vitro co-culture model established in this study provides advantage on random co-culture by spatially organise cells in definite patterns, thereby, controlling the number and interaction of BCCs with MSCs. Also, provides an opportunity to study direct (through E-cadherin) and indirect (through cytokines) interaction in a single system which is important to study collective response of interaction BCCs and MSCs on breast cancer biology. After established co-culture model, we investigated MSCs mediated signaling in BCCs. qRT-PCR, western blotting and immunostaining data suggested that MSCs induce the expression of oncogene, increased proliferation of BCCs and up or downregulate the gene related to the drug resistance. Citation Format: Amita Daverey, Allison Drain, Karleen Crone, Srivatsan Kidambi. Engineering of highly controlled in vitro co-culture model to study the mesenchymal stem cells mediated signaling in breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3303. doi:10.1158/1538-7445.AM2014-3303

Peptide REDV-modified polysaccharide hydrogel with endothelial cell selectivity for the promotion of angiogenesis.

Rapid and controlled vascularization of engineered tissues remains one of the key limitations in thick tissue engineering. Although many studies have focused on improving the rapid vascularization through the immobilization of bioactive molecules, the competition in growth between endothelial cells (ECs) and other cell types is to some extent neglected. In this study, we developed a peptide GREDV-modified scaffold for selective adhesion of human umbilical vein endothelial cells (HUVECs) through the specific recognition of the REDV peptide and integrin α4 β1 . In vitro studies showed that GREDV-conjugated alginate (ALG-GREDV) improved HUVEC adhesion, migration and proliferation when compared with a non-modified group. Furthermore, ALG-GREDV exhibited a superior capability for promoting the proliferation and selective adhesion of HUVEC over that of other peptide (RGD and YIGSR) modified groups (ALG-Pep). In vivo angiogenic assays demonstrated that the ALG-GREDV scaffold induced an angiogenic potential by stimulating new vessel formation and showed the highest blood vessel density among all samples after 21 days of implanting (83.7 vessels/mm(2) ). More importantly, the blood vessel density in cambium fibrous tissue of ALG-GREDV was about 1.5 times greater than other ALG-Pep groups, indicating facilitation of ALG-GREDV on selective angiogenesis in vivo. These results demonstrated that REDV-conjugated alginate could be a useful scaffold for stimulating and inducing angiogenesis in tissue-engineered applications.

A combinatorial study of nanocomposite hydrogels: on-chip mechanical/viscoelastic and pre-osteoblast interaction characterization.

Nanocomposite hydrogels were prepared in a combinatorial way with chitosan, bioglass nanoparticles (BG-NPs) and distinct amounts of crosslinker (genipin), in a total of 30 formulations. Such miniaturized hydrogels were prepared by dispensing the precursor solutions in wettable spots previously patterned onto superhydrophobic surfaces. The chips were used as platforms to analyze the biomaterials on-chip both for mechanical/viscoelastic and cell-biomaterial interactions. We adapted a mechanical dynamic analyzer (DMA) in order to perform the in situ totally unconfined solid-state rheological characterization of biomaterials under physiological-like conditions. We concluded that the viscoelastic properties of the hydrogels are dependent on the three factors studied. Besides influencing biomaterials' mechanical properties, bioglass fillers also confer bioactivity. We immersed the chips with 20 distinct biomaterial formulations in a cell suspension of MC3T3-E1 pre-osteoblasts and quantified - using image analysis compatible with the maintenance of the integrity of the chip - selective cell adhesion after 1 day of cell culture, as well as cell proliferation and cell morphology at day 3. Linear regression studies showed that for the range of conditions studied herein, neither cell adhesion nor proliferation depended directly on the biomaterials' mechanical/viscoelastic properties. Rather, cell proliferation was favoured in the presence of an intermediate amount of BGNPs (12.5% w/w) for all chitosan/genipin conditions, especially in softer hydrogels (2% (w/v) chitosan, 2.5% (w/w) genipin). This hit-spotted condition also favoured cell spreading. Interestingly, the elastic modulus measured for this formulation meets the values reported for the granulation tissue occurring during bone regeneration, where fibroblasts produce collagen. We believe that this approach will facilitate the complete on-chip rapid study of miniaturized biomaterials, in order to get more adequate formulations to be used in tissue engineering or other biomedical applications.

JAM-related proteins in mucosal homeostasis and inflammation

Mucosal surfaces are lined by epithelial cells that form a physical barrier protecting the body against external noxious substances and pathogens. At a molecular level, the mucosal barrier is regulated by tight junctions (TJs) that seal the paracellular space between adjacent epithelial cells. Transmembrane proteins within TJs include junctional adhesion molecules (JAMs) that belong to the cortical thymocyte marker for Xenopus family of proteins. JAM family encompasses three classical members (JAM-A, JAM-B, and JAM-C) and related molecules including JAM4, JAM-like protein, Coxsackie and adenovirus receptor (CAR), CAR-like membrane protein and endothelial cell-selective adhesion molecule. JAMs have multiple functions that include regulation of endothelial and epithelial paracellular permeability, leukocyte recruitment during inflammation, angiogenesis, cell migration, and proliferation. In this review, we summarize the current knowledge regarding the roles of the JAM family members in the regulation of mucosal homeostasis and leukocyte trafficking with a particular emphasis on barrier function and its perturbation during pathological inflammation.

Relationship of endothelial cell selective adhesion molecule to markers of oxidative stress in type 2 diabetes

Background. Endothelial dysfunction is an important contributor to micro and macrovascular complications of type 2 diabetes (T2D) and is reflected by increased systemic oxidative stress. Endothelial cell selective adhesion molecule (ESAM) influences endothelial function. We aimed to assess, for the first time to our knowledge, the relationship of soluble ESAM to markers of systemic oxidative stress. Materials and methods. ESAM, malondialdehyde (MDA) level and catalase activity were determined in 54 T2D patients and 43 controls. Results. T2D patients had significantly higher ESAM when compared to controls (16.07 ± 5.77 μg/L versus 8.57 ± 5.28 μg/L, p < 0.0001), they also had higher MDA level (3.88 ± 1.50 μmol/L vs. 1.58 ± 0.72 μmol/L, p < 0.0001) and lower catalase activity (3.07 (2.63–3.44) U/mg vs. 8.72 (4.55–10.46) U/mg, p < 0.0001). In T2D patients ESAM was inversely related to catalase activity (r = −0.27, p = 0.04), relationship to MDA level was direct but not significant (r = 0.16, p = 0.24). MDA concentration correlated inversely to catalase activity (r = −0.28, p = 0.04). In multiple regression catalase activity remained significantly correlated to ESAM (p = 0.02) and MDA level was significantly related to glycated hemoglobin (p = 0.01); there was trend towards a positive correlation of MDA level to ESAM (p = 0.08). When patients were divided according to oxidative stress, those with increased oxidative stress (defined as MDA concentration > 2.98 μmol/L and catalase activity < 3.38 U/mg) had higher ESAM than the rest of the patients (17.99 ± 5.02 μg/L vs. 14.29 ± 5.94 μg/L p = 0.01). Conclusion. ESAM is higher in T2D than in controls and parallels oxidative stress: ESAM is inversely related to catalase activity and higher ESAM is found in T2D patients with increased oxidative stress.

Folic acid modified clay/polymer nanocomposites for selective cell adhesion.

A folic acid (FA) modified poly(epsilon-caprolactone)/clay nanocomposite (PCL/MMT-(CH2CH2OH)2-FA) resulting in selective cell adhesion and proliferation was synthesized and characterized as a cell culture and biosensing platform. For this purpose, first the FA modified clay (MMT-(CH2CH2OH)2-FA) was prepared by treating the organo-modified clay, Cloisite 30B [MMT-(CH2CH2OH)2] with FA in chloroform at 60 °C. Subsequent ring opening polymerization of ε-caprolactone in the presence of tin octoate (Sn(Oct)2) using MMT-(CH2CH2OH)2-FA at 110 °C resulted in the formation of MMT-(CH2CH2OH)2-FA with an exfoliated clay structure. The structures of intermediates and the final nanocomposite were investigated in detail by FT-IR spectral analysis and DSC, TGA, XRD, SEM and AFM measurements. The combination of FA, PCL and clay provides a simple and versatile route to surfaces that allows controlled and selective cell adhesion and proliferation. FA receptor-positive HeLa and negative A549 cells were used to prove the selectivity of the modified surfaces. Both microscopy and electrochemical sensing techniques were applied to show the differences in cell adherence on the modified and pristine clay platforms. This approach is expected to be adapted into various bio-applications such as 'cell culture on chip', biosensors and design of tools for targeted diagnosis or therapy.

Chitosan-modified, collagen-based biomimetic nanofibrous membranes as selective cell adhering wound dressings in the treatment of chemically burned corneas.

Corneal chemical injury is a general but intractable ocular emergency, the sequelae of which are particularly challenging to treat. Human amniotic membrane (HAM) is one of the resources as a wound dressing for damaged corneal reconstruction, but the concerns related to the possible transmission of infectious diseases are the main drawbacks. Here we present a versatile method utilizing electrospinning and surface modification processes to develop optically highly transparent, microstructurally stable (>20 MPa in tensile strength in the wet state) biomimetic nanofibrous membranes. These membrane nanofibers, mainly consisting of a collagen-hyaluronate interior and a chitosan surface coating, showed superior mechanical and biological performances compared to HAM, and were favorable to the selective adhesion of epithelial cells (corneal, conjunctival) and fibroblasts. The alkali-burned corneal damage model in rats demonstrated that the biomimetic membranes could markedly improve re-epithelialization in corneal tissue within one week. Therefore, such bioactive multifunctional membranes may find widespread biomedical applications in wound healing and postoperative anti-adhesion in the near future.

Characterization of the liver-macrophages isolated from a mixed primary culture of neonatal swine hepatocytes

We recently developed a novel procedure to obtain liver-macrophages in sufficient number and purity using a mixed primary culture of rat and bovine hepatocytes. In this study, we aim to apply this method to the neonatal swine liver. Swine parenchymal hepatocytes were isolated by a two-step collagenase perfusion method and cultured in T75 culture flasks. Similar to the rat and bovine cells, the swine hepatocytes retained an epithelial cell morphology for only a few days and progressively changed into fibroblastic cells. After 5–13 days of culture, macrophage-like cells actively proliferated on the mixed fibroblastic cell sheet. Gentle shaking of the culture flask followed by the transfer and brief incubation of the culture supernatant resulted in a quick and selective adhesion of macrophage-like cells to a plastic dish surface. After rinsing dishes with saline, the attached macrophage-like cells were collected at a yield of 106 cells per T75 culture flask at 2–3 day intervals for more than 3 weeks. The isolated cells displayed a typical macrophage morphology and were strongly positive for macrophage markers, such as CD172a, Iba-1 and KT022, but negative for cytokeratin, desmin and α-smooth muscle actin, indicating a highly purified macrophage population. The isolated cells exhibited phagocytosis of polystyrene microbeads and a release of inflammatory cytokines upon lipopolysaccharide stimulation. This shaking and attachment method is applicable to the swine liver and provides a sufficient number of macrophages without any need of complex laboratory equipments.

Establishment of c-myc-immortalized Kupffer cell line from a C57BL/6 mouse strain

We recently demonstrated in several mammalian species, a novel procedure to obtain liver-macrophages (Kupffer cells) in sufficient numbers and purity using a mixed primary culture of hepatocytes. In this study, we applied this method to the C57BL/6 mouse liver and established an immortalized Kupffer cell line from this mouse strain. The hepatocytes from the C57BL/6 adult mouse liver were isolated by a two-step collagenase perfusion method and cultured in T25 culture flasks. Similar to our previous studies, the mouse hepatocytes progressively changed their morphology into a fibroblastic appearance after a few days of culture. After 7–10 days of culture, Kupffer-like cells, which were contaminants in the hepatocyte fraction at the start of the culture, actively proliferated on the mixed fibroblastic cell sheet. At this stage, a retroviral vector containing the human c-myc oncogene and neomycin resistance gene was introduced into the mixed culture. Gentle shaking of the culture flask, followed by the transfer and brief incubation of the culture supernatant, resulted in a quick and selective adhesion of Kupffer cells to a plastic dish surface. After selection with G418 and cloning by limiting dilutions, a clonal cell line (KUP5) was established. KUP5 cells displayed typical macrophage morphology and were stably passaged at 4–5 days intervals for more than 5 months, with a population doubling time of 19h. KUP5 cells are immunocytochemically positive for mouse macrophage markers, such as Mac-1, F4/80. KUP5 cells exhibited substantial phagocytosis of polystyrene microbeads and the release of inflammatory cytokines upon lipopolysaccharide stimulation. Taken together, KUP5 cells provide a useful means to study the function of Kupffer cells in vitro.

Endothelial Cell-Selective Adhesion Molecule Marks Human Hematopoietic Stem Cells Regardless Of The HSC Sources

Identification of novel markers associated with hematopoietic stem cells (HSCs) is important to progress basic and clinical research regarding the HSC biology. We previously reported that endothelial cell-selective adhesion molecule (ESAM) marks HSCs throughout life in mice (Yokota et al. Blood, 2009). We also demonstrated that ESAM can be a useful indicator of activated HSCs after bone marrow (BM) injury and that ESAM is functionally important for recovering hematopoiesis by using ESAM knockout mice (Sudo et al. J Immunol, 2012). However, the discrepancy between species has been a long-standing obstacle to apply findings in mice to human. For example, established murine HSC markers such as Sca-1 or CD150 are not expressed on human HSCs. Thus, it is important to know if ESAM marks HSCs beyond species and serves as a functional molecule for the HSC property, but information regarding ESAM expression in human HSCs has been quite limited. In this study, we have examined the ESAM expression pattern on human HSCs derived from diverse sources. In addition, we have performed functional assessment of the ESAM-expressing cells.Cord blood (CB), aspirated BM, and granulocyte-colony stimulating factor-mobilized peripheral blood (GMPB) were obtained from healthy donors. BM was also obtained from head of femora of patients who received the hip replacement surgery. All of the protocols were approved by the Institutional Review Board of Osaka University School of Medicine, and we obtained the written agreement form with informed consent from all participants. Mononuclear cells were separated using Ficoll centrifugation from CB, aspirated BM and GMPB. For preparation of BM cells adjacent to bone tissues, trabecular tissues of femora were treated with 2 mg/ml collagenase IV and DNase and gently agitated for 1 hour at 37 °C. Collected cells were analyzed using flow cytometry for cell surface expression of ESAM and other markers. Further, the CD34+ CD38−cells were fractionated according to the intensity of ESAM expression and evaluated in vivo and in vitro functional assays.Flow cytometry analyses revealed that the majority of CB CD34+ CD38− cells expressed ESAM. According to the expression level, CB CD34+ CD38− cells could be subdivided into three populations, namely ESAM−/Low, ESAMHigh, and ESAMBright. While all CB contained a robust ESAMHigh population in CD34+ CD38− cells, the percentage of ESAMBright cells varied widely among CB samples. The ESAMHigh CD34+ CD38− cells also expressed CD90 and CD133, which are known as HSC markers. Methylcellulose colony-forming assays and limiting dilution assays revealed that ESAMHigh fraction enriches primitive hematopoietic progenitors. Further, ESAMHigh cells also reconstituted the long-term human hematopoiesis in NOD/Shi-scid, IL-2Rγnull (NOG) mice. Therefore, as in mice, ESAMHighmarks authentic HSCs in human.On the other hand, ESAMBright CD34+ CD38− cells showed low colony-forming activities and no reconstitution of human hematopoiesis in NOG mice. These ESAMBright CD34+ CD38− cells expressed CD118/leukemia inhibitor factor receptor and endothelial markers such as VE-Cadherin, Flk-1, and CD146, but not CD45. These results suggested that ESAMBright cells in the CB CD34+ CD38− fraction are non-hematopoietic cells. With respect to the other HSC sources such as aspirated BM and GMPB, almost all CD34+ CD38− cells were ESAMHigh and ESAMBright cells were not found in this fraction. Interestingly, however, ESAMBright cells were found in the CD34+ CD38− fraction isolated from collagenase-treated femora. These BM-derived ESAMBright CD34+ CD38− cells expressed endothelial markers as did the CB-derived cells. They could generate CD31+endothelial cells, but not hematopoietic cells in coculture with MS5 stromal cells with vascular endothelial growth factor, stromal-cell-derived factor, and interleukin 16.In conclusion, ESAM expression serves as a marker to enrich HSCs in human regardless of the HSC sources. In addition, the very high intensity of this marker might be useful to isolate non-hematopoietic progenitors from CD34+ CD38− cells, which has been conventionally used as human HSCs. The common feature of ESAM expression of murine and human HSCs suggests a possibility that functional significance of ESAM expression obtained from mouse studies could be applicable to human. Disclosures:No relevant conflicts of interest to declare.