Spatiotemporal changes of tissue glycans depending on localization in cardiac aging.

Heart failure is caused by various factors, making the underlying pathogenic mechanisms difficult to identify. Since cardiovascular disease tends to worsen over time, early diagnosis is key for treatment. In addition, understanding the qualitative changes in the heart associated with aging, where information on the direct influences of aging on cardiovascular disease is limited, would also be useful for treatment and diagnosis. To fill these research gaps, the focus of our study was to detect the structural and functional molecular changes associated with the heart over time, with a focus on glycans, which reflect the type and state of cells. METHODS: We investigated glycan localization in the cardiac tissue of normal mice and their alterations during aging, using evanescent-field fluorescence-assisted lectin microarray, a technique based on lectin-glycan interaction, and lectin staining. RESULTS: The glycan profiles in the left ventricle showed differences between the luminal side (medial) and wall side (lateral) regions. The medial region was characterized by the presence of sialic acid residues. Moreover, age-related changes in glycan profiles were observed at a younger age in the medial region. The difference in the age-related decrease in the level of α-galactose stained with Griffonia simplicifolia lectin-IB4 in different regions of the left ventricle suggests spatiotemporal changes in the number of microvessels. CONCLUSIONS: The glycan profile, which retains diverse glycan structures, is supported by many cell populations, and maintains cardiac function. With further research, glycan localization and changes have the potential to be developed as a marker of the signs of heart failure.

PRC2-dependent regulation of ganglioside expression during dedifferentiation contributes to the proliferation and migration of vascular smooth muscle cells.

Phenotypic switching between contractile (differentiated state) and proliferative (dedifferentiated state) vascular smooth muscle cells (VSMCs) is a hallmark of vascular remodeling that contributes to atherosclerotic diseases. Gangliosides, a group of glycosphingolipids, have been detected in atherosclerotic lesions and are suspected to contribute to the disease process. However, the underlying mechanism, specifically with respect to their role in VSMC phenotype switching, is not clear. In this study, we sought to reveal the endogenous expression of gangliosides and their functional significance in VSMCs during atherosclerosis. We found that switching from the contractile to proliferative phenotype was accompanied by upregulation of a- and b-series gangliosides, which in turn, were regulated by polycomb repressor complex 2 (PRC2). Downregulation of ganglioside expression using an siRNA targeting ST3GAL5, which is required for the synthesis of a- and b-series gangliosides, attenuated the proliferation and migration of dedifferentiated VSMCs. Therefore, we concluded that the increased expression of a- and b-series gangliosides via PRC2 activity during dedifferentiation is involved in the proliferation and migration of VSMCs. Gangliosides may be an effective target in VSMCs for atherosclerosis prevention and treatment.

Tissue Glycome Mapping: Lectin Microarray-Based Differential Glycomic Analysis of Formalin-Fixed Paraffin-Embedded Tissue Sections.

Lectin microarray (LMA) is a high-sensitive glycan analysis technology used to obtain global glycomic profiles of both N- and O-glycans attached not only to purified glycoproteins but also to crude glycoprotein samples. Through additional use of laser microdissection (LMD) for tissue collection, we developed an LMA-based glycomic profiling technique for a specific type of cells in a tiny area of formalin-fixed paraffin-embedded (FFPE) tissue sections. This LMD-LMA method makes it possible to obtain reproducible tissue glycomic profiles that can be compared with each other, using a unified protocol for all procedures, including FFPE tissue preparation, tissue staining, protein extraction and labeling, and LMA analysis. Here, we describe the standardized LMD-LMA procedure for a "tissue glycome mapping" approach, which facilitates an in-depth understanding of region- and tissue-specific protein glycosylation. We also describe potential applications of the spatial tissue glycomic profiles, including histochemical analysis for evaluating distribution of lectin ligands and a fluorescence LMD-LMA method for cell type-selective glycomic profiling using a cell type-specific probe, composed of a lectin and an antibody. The protocols presented here will accelerate the effective utilization of FFPE tissue specimens by providing tissue glycome maps for the discovery of the biological roles and disease-related alterations of protein glycosylation.

Cell Surface and Functional Features of Cortical Bone Stem Cells.

The newly established mouse cortical-bone-derived stem cells (mCBSCs) are unique stem cells compared to mouse mesenchymal stem cells (mMSCs). The mCBSC-treated hearts after myocardial infarction have been reported to have greater improvement in myocardial structure and functions. In this study, we examined the stemness features, cell surface glycan profiles, and paracrine functions of mCBSCs compared with mMSCs. The stemness analysis revealed that the self-renewing capacity of mCBSCs was greater than mMSCs; however, the differentiation capacity of mCBSCs was limited to the chondrogenic lineage among three types of cells (adipocyte, osteoblast, chondrocyte). The cell surface glycan profiles by lectin array analysis revealed that α2-6sialic acid is expressed at very low levels on the cell surface of mCBSCs compared with that on mMSCs. In contrast, the lactosamine (Galß1-4GlcNAc) structure, poly lactosamine- or poly N-acetylglucosamine structure, and α2-3sialic acid on both N- and O-glycans were more highly expressed in mCBSCs. Moreover, we found that mCBSCs secrete a greater amount of TGF-ß1 compared to mMSCs, and that the TGF-ß1 contributed to the self-migration of mCBSCs and activation of fibroblasts. Together, these results suggest that unique characteristics in mCBSCs compared to mMSCs may lead to advanced utility of mCBSCs for cardiac and noncardiac repair.

Glycan characteristics of human heart constituent cells maintaining organ function: relatively stable glycan profiles in cellular senescence.

Cell surface glycoproteins, which are good indicators of cellular types and biological function; are suited for cell evaluation. Tissue remodeling using various cells is a key feature of regenerative therapy. For artificial heart remodeling, a mixture of heart constituent cells has been investigated for organ assembly, however, the cellular characteristics remain unclear. In this study, the glycan profiles of human cardiomyocytes (HCMs), human cardiac fibroblasts (HCFs), and human vascular endothelial cells (ECs) were analyzed using evanescent-field lectin microarray analysis, a tool of glycan profiling, to clarify the required cellular characteristics. We found that ECs had more "α1-2fucose" and "core α1-6fucose" residues than other cells, and that "α2-6sialic acid" residue was more abundant in ECs and HCMs than in HCFs. HCFs showed higher abundance of "ß-galactose" and "ß-N-acetylgalactosamine" residues on N-glycan and O-glycan, respectively, compared to other cells. Interestingly, cardiac glycan profiles were insignificantly changed with cellular senescence. The residues identified in this study may participate in organ maintenance by contributing to the preservation of glycan components. Therefore, future studies should investigate the roles of glycans in optimal tissue remodeling since identifying cellular characteristics is important for the development of regenerative therapies.

Rapamycin promotes endothelial-mesenchymal transition during stress-induced premature senescence through the activation of autophagy.

BACKGROUND: Rapamycin is known to be effective in suppressing senescence and the senescence-associated secretory phenotype (SASP). Therefore, it is highly expected to represent an anti-aging drug. Its anti-aging effect has been demonstrated at the mouse individual level. However, there are not many clinical findings with respect to its activity in humans. Here, we aimed to clarify the effect of rapamycin on human endothelial cells (ECs) as an in vitro model of human blood vessels. METHODS: Over the course of oxidative stress-induced senescence using hydrogen peroxide, we examined the effect of rapamycin on human coronary artery ECs (HCAECs). Senescence was evaluated by detecting senescence-associated ß-galactosidase (SA-ß-Gal) activity and the real-time PCR analysis of p16INK4a. Furthermore, expression levels of SASP factors were examined by real-time PCR and the expression of senescence-related antigens, such as intercellular adhesion molecule-1 (ICAM-1) and ganglioside GM1, were examined by fluorescence-activated cell sorting analysis and immunostaining. The inhibitory effect of rapamycin on mTOR signaling was examined by immunoblotting. The adhesion of leukocytes to HCAECs was evaluated by adhesion assays. Endothelial-mesenchymal transition (EndMT) induced by rapamycin treatment was evaluated by real-time PCR analysis and immunostaining for EndMT markers. Finally, we checked the activation of autophagy by immunoblotting and examined its contribution to EndMT by using a specific inhibitor. Furthermore, we examined how the activation of autophagy influences TGF-ß signaling by immunoblotting for Smad2/3 and Smad7. RESULTS: A decrease in SA-ß-Gal activity and the suppression of SASP factors were observed in HCAECs undergoing stress-induced premature senescence (SIPS) after rapamycin treatment. In contrast, ICAM-1 and ganglioside GM1 were upregulated by rapamycin treatment. In addition, leukocyte adhesion to HCAECs was promoted by this treatment. In rapamycin-treated HCAECs, morphological changes and the promotion of EndMT were also observed. Furthermore, we found that autophagy activation induced by rapamycin treatment, which led to activation of the TGF-ß pathway, contributed to EndMT induction. CONCLUSIONS: We revealed that although rapamycin functions to inhibit senescence and suppress SASP in HCAECs undergoing SIPS, EndMT is induced due to the activation of autophagy. Video abstract.

Mutations of histone demethylase genes encoded by X and Y chromosomes, Kdm5c and Kdm5d, lead to noncompaction cardiomyopathy in mice.

Mammalian X and Y chromosomes evolved from a pair of autosomes. Although most ancestral genes have been lost from the Y chromosome, a small number of ancestral X-Y gene pairs are still present on the sex chromosomes. The KDM5C and KDM5D genes, which encode H3K4 histone demethylases, are a surviving ancestral gene pair located on the X and Y chromosomes, respectively. Mutations in KDM5C cause X-linked intellectual disability in human males, suggesting functional divergence between KDM5C and KDM5D in the nervous system. In this study, to explore the functional conservation and divergence between these two genes in other organs, we generated female mice lacking Kdm5c (homozygous X5c- X5c- females) and male mice lacking both Kdm5c and Kdm5d (compound hemizygous X5c- Y5d- males). Both X5c- X5c- females and X5c- Y5d- males showed lower body weights and postnatal lethality. Histological examination of the hearts showed prominent trabecular extension and a thin layer of compacted myocardium in the left and right ventricles, indicating noncompaction cardiomyopathy. However, hemizygous males lacking either Kdm5c or Kdm5d showed no signs of noncompaction cardiomyopathy. These results clearly demonstrate that the function of Kdm5c and Kdm5d in heart development is conserved.

Ganglioside GM2, highly expressed in the MIA PaCa-2 pancreatic ductal adenocarcinoma cell line, is correlated with growth, invasion, and advanced stage.

Gangliosides, a group of glycosphingolipids, are known to be cell surface markers and functional factors in several cancers. However, the association between gangliosides and pancreatic ductal adenocarcinoma (PDAC) has not been well elucidated. In this study, we examined the expression and roles of ganglioside GM2 in PDAC. GM2+ cells showed a higher growth rate than GM2- cells in the adherent condition. When GM2- and GM2+ cells were cultured three-dimensionally, almost all cells in the spheres expressed GM2, including cancer stem cell (CSC)-like cells. A glycolipid synthesis inhibitor reduced GM2 expression and TGF-ß1 signaling in these CSC-like cells, presumably by inhibiting the interaction between GM2 and TGFß RII and suppressing invasion. Furthermore, suppression of GM2 expression by MAPK inhibition also reduced TGF-ß1 signaling and suppressed invasion. GM2+ cells formed larger subcutaneous tumors at a high incidence in nude mice than did GM2- cells. In PDAC cases, GM2 expression was significantly associated with younger age, larger tumor size, advanced stage and higher histological grade. These findings suggest that GM2 could be used as a novel diagnostic and therapeutic target for PDAC.

Gangliosides Contribute to Vascular Insulin Resistance.

Insulin in physiological concentrations is important to maintain vascular function. Moreover, vascular insulin resistance contributes to vascular impairment. In the elderly, other factors including hypertension, dyslipidemia, and chronic inflammation amplify senescence of vascular endothelial and smooth muscle cells. In turn, senescence increases the risk for vascular-related diseases such as arteriosclerosis, diabetes, and Alzheimer's disease. Recently, it was found that GM1 ganglioside, one of the glycolipids localized on the cell membrane, mediates vascular insulin resistance by promoting senescence and/or inflammatory stimulation. First, it was shown that increased GM1 levels associated with aging/senescence contribute to insulin resistance in human aortic endothelial cells (HAECs). Second, the expression levels of gangliosides were monitored in HAECs treated with different concentrations of tumor necrosis factor-alpha (TNFα) for different time intervals to mimic in vivo acute or chronic inflammatory conditions. Third, the levels of insulin signaling-related molecules were monitored in HAECs after TNFα treatment with or without inhibitors of ganglioside synthesis. In this review, we summarize the molecular mechanisms of insulin resistance in aged/senescent and TNFα-stimulated endothelial cells mediated by gangliosides and highlight the possible roles of gangliosides in vascular insulin resistance-related diseases.

Comparison of functional glycans between cancer stem cells and normal stem cells.

Cancer stem cells (CSCs) are a small group of cells within a tumor that preserve stemness and enhance regrowth of cancer cells. CSCs have important implications in resistance to conventional therapies and tumor relapse, although their detailed properties remain unknown. Thus, CSCs represent promising targets to improve cancer treatment. So far, a number of cell surface markers containing glycans have been exploited to identify and isolate CSCs. Cell surface glycans are well-known markers for specific cell types and also play important cellular roles, such as regulation of cell signaling. In normal stem cells, including embryonic and tissue stem cells, glycan markers in an undifferentiated state have been identified. These markers are mostly known to regulate signaling pathways required for maintenance of stemness. In contrast, CSC-specific glycans have not been well characterized yet. In this review, we summarize functional commonalities between CSCs and normal stem cells in glycan-mediated signaling pathways. Identification of CSC-specific glycans may lead to early diagnosis and radical treatment of cancer.

Qualitative and quantitative alterations in intracellular and membrane glycoproteins maintain the balance between cellular senescence and human aging.

Glycans are associated with and serve as biomarkers for various biological functions. We previously reported that cell surface sialylated glycoproteins of dermal fibroblasts decreased with cellular senescence and human aging. There is little information on the changes in glycoprotein expression and subcellular localization during the aging process. Here, we examined intracellular glycan profiles of fibroblasts undergoing cellular senescence and those derived from aging human subjects using lectin microarray analysis. We found a sequential change of the intracellular glycan profiles was little during cellular senescence. The intracellular glycans of cells derived from aged fetus and from elderly subjects showed similar localized patterns while repeating unsteady changes. The ratio of α2-3/2-6sialylated intracellular glycoproteins in total cell extracts increased, except for a part of α2-3sialylated O-glycans. These findings are in contrast to those for membrane glycoprotein, which decreased with aging. Interestingly, the ratio of increasing sialylated glycoproteins in the fetus-derived cells showing cellular senescence was similar to that in cells derived from the elderly. Thus, intracellular glycans may maintain cellular functions such as ubiquitin/proteasome-mediated degradation and/or autophagy during aging by contributing to the accumulation of intracellular glycosylated proteins. Our findings provide novel mechanistic insight into the molecular changes that occur during aging.

Characteristic glycopeptides associated with extreme human longevity identified through plasma glycoproteomics.

BACKGROUND: Glycosylation is highly susceptible to changes of the physiological conditions, and accordingly, is a potential biomarker associated with several diseases and/or longevity. Semi-supercentenarians (SSCs; older than 105 years) are thought to be a model of human longevity. Thus, we performed glycoproteomics using plasma samples of SSCs, and identified proteins and conjugated N-glycans that are characteristic of extreme human longevity. METHODS: Plasma proteins from Japanese semi-supercentenarians (SSCs, 106-109 years), aged controls (70-88 years), and young controls (20-38 years) were analysed by using lectin microarrays and liquid chromatography/mass spectrometry (LC/MS). Peak area ratios of glycopeptides to corresponding normalising peptides were subjected to orthogonal projections to latent structures discriminant analysis (OPLS-DA). Furthermore, plasma levels of clinical biomarkers were measured. RESULTS: We found two lectins such as Phaseolus vulgaris, and Erythrina cristagalli (ECA), of which protein binding were characteristically increased in SSCs. Peak area ratios of ECA-enriched glycopeptides were successfully discriminated between SSCs and controls using OPLS-DA, and indicated that tri-antennary and sialylated N-glycans of haptoglobin at Asn207 and Asn211 sites were characterized in SSCs. Sialylated glycans of haptoglobin are a potential biomarker of several diseases, such as hepatocellular carcinoma, liver cirrhosis, and IgA-nephritis. However, the SSCs analysed here did not suffer from these diseases. CONCLUSIONS: Tri-antennary and sialylated N-glycans on haptoglobin at the Asn207 and Asn211 sites were abundant in SSCs and characteristic of extreme human longevity. GENERAL SIGNIFICANCE: We found abundant glycans in SSCs, which may be associated with human longevity.

Ganglioside GM1 contributes to extracellular/intracellular regulation of insulin resistance, impairment of insulin signaling and down-stream eNOS activation, in human aortic endothelial cells after short- or long-term exposure to TNFα.

Vascular insulin resistance induced by inflammatory cytokines leads to the initiation and development of vascular diseases. In humans, circulating TNFα levels are increased during aging, suggesting a correlation between vascular insulin resistance and plasma TNFα levels. Currently, the precise molecular mechanisms of vascular insulin resistance mediated by TNFα are not well characterized. We aimed at clarifying whether glycosphingolipids contribute to vascular insulin resistance after inflammatory stimulation. In this study, we examined vascular insulin resistance using human aortic endothelial cells after treatment with different concentrations of TNFα for different time intervals for mimicking in vivo acute or chronic inflammatory situations. We show that ganglioside GM1 levels on cell membranes change depending on time of exposure to TNFα and its concentration and that the GM1 expression is associated with specific extracellular/intracellular regulation of the insulin signaling cascade. Furthermore, we provide evidence that factors such as aging and senescence affect the regulation of insulin resistance. Our data suggest that GM1 is a key player in the induction of vascular insulin resistance after short- or long-term exposure to TNFα and is a good extracellular target for prevention and cure of vascular diseases.

Sugar-Binding Profiles of Chitin-Binding Lectins from the Hevein Family: A Comprehensive Study.

Chitin-binding lectins form the hevein family in plants, which are defined by the presence of single or multiple structurally conserved GlcNAc (N-acetylglucosamine)-binding domains. Although they have been used as probes for chito-oligosaccharides, their detailed specificities remain to be investigated. In this study, we analyzed six chitin-binding lectins, DSA, LEL, PWM, STL, UDA, and WGA, by quantitative frontal affinity chromatography. Some novel features were evident: WGA showed almost comparable affinity for pyridylaminated chitotriose and chitotetraose, while LEL and UDA showed much weaker affinity, and DSA, PWM, and STL had no substantial affinity for the former. WGA showed selective affinity for hybrid-type N-glycans harboring a bisecting GlcNAc residue. UDA showed extensive binding to high-mannose type N-glycans, with affinity increasing with the number of Man residues. DSA showed the highest affinity for highly branched N-glycans consisting of type II LacNAc (N-acetyllactosamine). Further, multivalent features of these lectins were investigated by using glycoconjugate and lectin microarrays. The lectins showed substantial binding to immobilized LacNAc as well as chito-oligosaccharides, although the extents to which they bound varied among them. WGA showed strong binding to heavily sialylated glycoproteins. The above observations will help interpret lectin-glycoprotein interactions in histochemical studies and glyco-biomarker investigations.

Sialylation regulates myofibroblast differentiation of human skin fibroblasts.

BACKGROUND: Fibroblasts are key players in maintaining skin homeostasis and in orchestrating physiological tissue repair and skin regeneration. Dysfunctions in fibroblasts that occur with aging and the senescent process lead to the delayed healing observed in elderly people. The molecular mechanisms leading to fibroblast dysfunction during aging and the senescent process have not yet been clarified. Previously, changes in patterns of glycosylation were observed in fibroblasts in aging and the senescent process, but the effect of these changes on the function of fibroblasts has not been well documented. Here, we investigated whether changes in glycosylation during the process to senescence may have functional effects on fibroblasts. METHODS: The changes in cell surface glycans on skin fibroblasts during the process to senescence were examined in early-passage (EP) and late-passage (LP) skin fibroblasts by fluorescence-activated cell sorting analysis using lectins. The contributors to the changes in cell surface glycans were examined by real-time polymerase chain reaction or Western blot analysis. The effects of changes in glycosylation on proliferation, migration, induction of cellular senescence, and myofibroblast differentiation induced by transforming growth factor (TGF)-ß1 stimulation were examined in EP fibroblasts. The changes in glycosylation were performed by GalNAc-α-O-benzyl or sialidase treatment. RESULTS: A decrease in sialylation of glycoproteins and an increase in sialidase NEU1 were observed in LP fibroblasts. The reduction of sialylation did not have any effect on proliferation, migration, or induction of cellular senescence. On the other hand, myofibroblast differentiation was inhibited by the reduction of sialylation, indicating that sialylation is important for myofibroblast differentiation. The localization of CD44 in lipid rafts, which is required for myofibroblast differentiation, was inhibited by the reduction of sialylation. Furthermore, reduced myofibroblast differentiation in LP fibroblasts was restored by a sialidase inhibitor. CONCLUSIONS: Desialylation of CD44 with increased sialidase during the process to senescence reduced the localization of CD44 in lipid rafts after TGF-ß1 stimulation, leading to the inhibition of myofibroblast differentiation. Thus, regulation of sialylation may be an attractive strategy for the prevention and regenerative therapy of age-related skin diseases, cosmetic skin alterations, and chronic wounds caused by delayed healing in elderly people.

A standardized method for lectin microarray-based tissue glycome mapping.

The significance of glycomic profiling has been highlighted by recent findings that structural changes of glycans are observed in many diseases, including cancer. Therefore, glycomic profiling of the whole body (glycome mapping) under different physiopathological states may contribute to the discovery of reliable biomarkers with disease-specific alterations. To achieve this, standardization of high-throughput and in-depth analysis of tissue glycome mapping is needed. However, this is a great challenge due to the lack of analytical methodology for glycans on small amounts of endogenous glycoproteins. Here, we established a standardized method of lectin-assisted tissue glycome mapping. Formalin-fixed, paraffin-embedded tissue sections were prepared from brain, liver, kidney, spleen, and testis of two C57BL/6J mice. In total, 190 size-adjusted fragments with different morphology were serially collected from each tissue by laser microdissection and subjected to lectin microarray analysis. The results and subsequent histochemical analysis with selected lectins were highly consistent with previous reports of mass spectrometry-based N- and/or O-glycome analyses and histochemistry. This is the first report to look at both N- and O-glycome profiles of various regions within tissue sections of five different organs. This simple and reproducible mapping approach is also applicable to various disease model mice to facilitate disease-related biomarker discovery.

N- and O-glycan cell surface protein modifications associated with cellular senescence and human aging.

BACKGROUND: Glycans play essential roles in biological functions such as differentiation and cancer. Recently, glycans have been considered as biomarkers for physiological aging. However, details regarding the specific glycans involved are limited. Here, we investigated cellular senescence- and human aging-dependent glycan changes in human diploid fibroblasts derived from differently aged skin donors using a lectin microarray. RESULTS: We found that α2-6sialylated glycans in particular differed between elderly- and fetus-derived cells at early passage. However, both cell types exhibited sequentially decreasing α2-3sialylated O-glycan structures during the cellular senescence process and showed similar overall glycan profiles. CONCLUSIONS: We observed a senescence-associated decrease in sialylation and increase in galactose exposure. Therefore, glycan profiling using lectin microarrays might be useful for the characterization of biomarkers of aging.

Ganglioside GM1 Contributes to the State of Insulin Resistance in Senescent Human Arterial Endothelial Cells.

Vascular endothelial cells (ECs) play central roles in physiologically important functions of blood vessels and contribute to the maintenance of vascular integrity. Therefore, it is considered that the impairment of EC functions leads to the development of vascular diseases. However, the molecular mechanisms of the EC dysfunctions that accompany senescence and aging have not yet been clarified. The carbohydrate antigens carried by glycoconjugates (e.g. glycoproteins, glycosphingolipids, and proteoglycans) mainly present on the cell surface serve not only as marker molecules but also as functional molecules. In this study, we have investigated the abundance and functional roles of glycosphingolipids in human ECs during senescence and aging. Among glycosphingolipids, ganglioside GM1 was highly expressed in abundance on the surface of replicatively and prematurely senescent ECs and also of ECs derived from an elderly subject. Insulin signaling, which regulates important functions of ECs, is impaired in senescent and aged ECs. Actually, by down-regulating GM1 on senescent ECs and overloading exogenous GM1 onto non-senescent ECs, we showed that an increased abundance of GM1 functionally contributes to the impairment of insulin signaling in ECs. Taken together, these findings provide the first evidence that GM1 increases in abundance on the cell surface of ECs under the conditions of cellular senescence and aging and causes insulin resistance in ECs. GM1 may be an attractive target for the detection, prevention, and therapy of insulin resistance and related vascular diseases, particularly in older people.

Novel detergent for whole organ tissue engineering.

Whole organ tissue engineering for various organs, including the heart, lung, liver, and kidney, has demonstrated promising results for end-stage organ failure. However, the sodium dodecyl sulfate (SDS)-based protocol for standard decellularization has drawbacks such as clot formation in vascularized transplantation and poor cell engraftment in recellularization procedures. Preservation of the surface milieu of extracellular matrices (ECMs) might be crucial for organ generation based on decellularization/recellularization engineering. We examined a novel detergent, sodium lauryl ether sulfate (SLES), to determine whether it could overcome the drawbacks associated with SDS using rat heart and kidney. Both organs were perfused in an antegrade fashion with either SLES or SDS. Although immunohistochemistry for collagen I, IV, laminin, and fibronectin showed similar preservation in both detergents, morphological analysis using scanning electron microscopy and an assay of glycosaminoglycan content on ECMs showed that SLES-treated tissues had better-preserved ECMs than SDS-treated tissues. Mesenteric transplantation revealed SLES did not induce significant inflammation, as opposed to SDS. Platelet adhesion to decellularized tissues was significantly reduced with SLES. Overall, SLES could replace older detergents such as SDS in the decellularization process for generation of transplantable recellularized organs.

Large-scale cell production of stem cells for clinical application using the automated cell processing machine.

BACKGROUND: Cell-based regeneration therapies have great potential for application in new areas in clinical medicine, although some obstacles still remain to be overcome for a wide range of clinical applications. One major impediment is the difficulty in large-scale production of cells of interest with reproducibility. Current protocols of cell therapy require a time-consuming and laborious manual process. To solve this problem, we focused on the robotics of an automated and high-throughput cell culture system. Automated robotic cultivation of stem or progenitor cells in clinical trials has not been reported till date. The system AutoCulture used in this study can automatically replace the culture medium, centrifuge cells, split cells, and take photographs for morphological assessment. We examined the feasibility of this system in a clinical setting. RESULTS: We observed similar characteristics by both the culture methods in terms of the growth rate, gene expression profile, cell surface profile by fluorescence-activated cell sorting, surface glycan profile, and genomic DNA stability. These results indicate that AutoCulture is a feasible method for the cultivation of human cells for regenerative medicine. CONCLUSIONS: An automated cell-processing machine will play important roles in cell therapy and have widespread use from application in multicenter trials to provision of off-the-shelf cell products.