Xenogeneic-Free Human Intestinal Organoids for Assessing Intestinal Nutrient Absorption.

Since many nutrients, including the three major ones of glucose, dipeptides, and cholesterol, are mainly absorbed in the small intestine, the assessment of their effects on intestinal tissue is important for the study of food absorption. However, cultured intestinal cell lines, such as Caco-2 cells, or animal models, which differ from normal human physiological conditions, are generally used for the evaluation of intestinal absorption and digestion. Therefore, it is necessary to develop an alternative in vitro method for more accurate analyses. In this study, we demonstrate inhibitory effects on nutrient absorption through nutrient transporters using three-dimensional xenogeneic-free human intestinal organoids (XF-HIOs), with characteristics of the human intestine, as we previously reported. We first show that the organoids absorbed glucose, dipeptide, and cholesterol in a transporter-dependent manner. Next, we examine the inhibitory effect of natural ingredients on the absorption of glucose and cholesterol. We reveal that glucose absorption was suppressed by epicatechin gallate or nobiletin, normally found in green tea catechin or citrus fruits, respectively. In comparison, cholesterol absorption was not inhibited by luteolin and quercetin, contained in some vegetables. Our findings highlight the usefulness of screening for the absorption of functional food substances using XF-HIOs.

Rapid and sensitive mycoplasma detection system using image-based deep learning.

A major concern in the clinical application of cell therapy is the manufacturing cost of cell products, which mainly depends on quality control. The mycoplasma test, an important biological test in cell therapy, takes several weeks to detect a microorganism and is extremely expensive. Furthermore, the manual detection of mycoplasma from images requires high-level expertise. We hypothesized that a mycoplasma identification program using a convolutional neural network could reduce the test time and improve sensitivity. To this end, we developed a program comprising three parts (mycoplasma detection, prediction, and cell counting) that allows users to evaluate the sample and verify infected/non-infected cells identified by the program. In experiments conducted, stained DNA images of positive and negative control using mycoplasma-infected and non-infected Vero cells, respectively, were used as training data, and the program results were compared with those of conventional methods, such as manual counting based on visual observation. The minimum detectable mycoplasma contaminations for manual counting and the proposed program were 10 and 5 CFU (colony-forming unit), respectively, and the test time for manual counting was 20 times that for the proposed program. These results suggest that the proposed system can realize a low-cost and streamlined manufacturing process for cellular products in cell-based research and clinical applications.

The Fate of Transplanted Periodontal Ligament Stem Cells in Surgically Created Periodontal Defects in Rats.

Periodontal disease is chronic inflammation that leads to the destruction of tooth-supporting periodontal tissues. We devised a novel method ("cell transfer technology") to transfer cells onto a scaffold surface and reported the potential of the technique for regenerative medicine. The aim of this study is to examine the efficacy of this technique in periodontal regeneration and the fate of transplanted cells. Human periodontal ligament stem cells (PDLSCs) were transferred to decellularized amniotic membrane and transplanted into periodontal defects in rats. Regeneration of tissues was examined by microcomputed tomography and histological observation. The fate of transplanted PDLSCs was traced using PKH26 and human Alu sequence detection by PCR. Imaging showed more bone in PDLSC-transplanted defects than those in control (amnion only). Histological examination confirmed the enhanced periodontal tissue formation in PDLSC defects. New formation of cementum, periodontal ligament, and bone were prominently observed in PDLSC defects. PKH26-labeled PDLSCs were found at limited areas in regenerated periodontal tissues. Human Alu sequence detection revealed that the level of Alu sequence was not increased, but rather decreased. This study describes a novel stem cell transplantation strategy for periodontal disease using the cell transfer technology and offers new insight for cell-based periodontal regeneration.

Cell transfer technology for tissue engineering.

We recently developed novel cell transplantation method "cell transfer technology" utilizing photolithography. Using this method, we can transfer ex vivo expanded cells onto scaffold material in desired patterns, like printing of pictures and letters on a paper. We have investigated the possibility of this novel method for cell-based therapy using several disease models. We first transferred endothelial cells in capillary-like patterns on amnion. The transplantation of the endothelial cell-transferred amnion enhanced the reperfusion in mouse ischemic limb model. The fusion of transplanted capillary with host vessel networks was also observed. The osteoblast- and periodontal ligament stem cell-transferred amnion were next transplanted in bone and periodontal defects models. After healing period, both transplantations improved the regeneration of bone and periodontal tissues, respectively. This method was further applicable to transfer of multiple cell types and the transplantation of osteoblasts and periodontal ligament stem cell-transferred amnion resulted in the improved bone regeneration compared with single cell type transplantation. These data suggested the therapeutic potential of the technology in cell-based therapies for reperfusion of ischemic limb and regeneration of bone and periodontal tissues. Cell transfer technology is applicable to wide range of regenerative medicine in the future.

Exosomes of human placenta-derived mesenchymal stem cells stimulate angiogenesis.

BACKGROUND: The therapeutic potential of mesenchymal stem cells (MSCs) may be attributed partly to humoral factors such as growth factors, cytokines, and chemokines. Human term placental tissue-derived MSCs (PlaMSCs), or conditioned medium left over from cultures of these cells, have been reported to enhance angiogenesis. Recently, the exosome, which can transport a diverse suite of macromolecules, has gained attention as a novel intercellular communication tool. However, the potential role of the exosome in PlaMSC therapeutic action is not well understood. The purpose of this study was to evaluate PlaMSC-derived exosome angiogenesis promotion in vitro and in vivo. METHODS: MSCs were isolated from human term placental tissue by enzymatic digestion. Conditioned medium was collected after 48-h incubation in serum-free medium (PlaMSC-CM). Angiogenic factors present in PlaMSC-CM were screened by a growth factor array. Exosomes were prepared by ultracentrifugation of PlaMSC-CM, and confirmed by transmission electron microscopy, dynamic light scattering, and western blot analyses. The proangiogenic activity of PlaMSC-derived exosomes (PlaMSC-exo) was assessed using an endothelial tube formation assay, a cell migration assay, and reverse transcription-PCR analysis. The in-vivo angiogenic activity of PlaMSC-exo was evaluated using a murine auricle ischemic injury model. RESULTS: PlaMSC-CM contained both angiogenic and angiostatic factors, which enhanced endothelial tube formation. PlaMSC-exo were incorporated into endothelial cells; these exosomes stimulated both endothelial tube formation and migration, and enhanced angiogenesis-related gene expression. Laser Doppler blood flow analysis showed that PlaMSC-exo infusion also enhanced angiogenesis in an in-vivo murine auricle ischemic injury model. CONCLUSIONS: PlaMSC-exo enhanced angiogenesis in vitro and in vivo, suggesting that exosomes play a role in the proangiogenic activity of PlaMSCs. PlaMSC-exo may be a novel therapeutic approach for treating ischemic diseases.

Double-layered cell transfer technology for bone regeneration.

For cell-based medicine, to mimic in vivo cellular localization, various tissue engineering approaches have been studied to obtain a desirable arrangement of cells on scaffold materials. We have developed a novel method of cell manipulation called "cell transfer technology", enabling the transfer of cultured cells onto scaffold materials, and controlling cell topology. Here we show that using this technique, two different cell types can be transferred onto a scaffold surface as stable double layers or in patterned arrangements. Various combinations of adherent cells were transferred to a scaffold, amniotic membrane, in overlapping bilayers (double-layered cell transfer), and transferred cells showed stability upon deformations of the material including folding and trimming. Transplantation of mesenchymal stem cells from periodontal ligaments (PDLSC) and osteoblasts, using double-layered cell transfer significantly enhanced bone formation, when compared to single cell type transplantation. Our findings suggest that this double-layer cell transfer is useful to produce a cell transplantation material that can bear two cell layers. Moreover, the transplantation of an amniotic membrane with PDLSCs/osteoblasts by cell transfer technology has therapeutic potential for bone defects. We conclude that cell transfer technology provides a novel and unique cell transplantation method for bone regeneration.

Placenta Mesenchymal Stem Cell Derived Exosomes Confer Plasticity on Fibroblasts.

Mesenchymal stem cell (MSC)-conditioned medium (MSC-CM) has been reported to enhance wound healing. Exosomes contain nucleic acids, proteins, and lipids, and function as an intercellular communication vehicle for mediating some paracrine effects. However, the function of MSC-derived exosomes (MSC-exo) remains elusive. In this study, we isolated human placenta MSC (PlaMSC)-derived exosomes (PlaMSC-exo) and examined their function in vitro. PlaMSCs were isolated from human term placenta using enzymatic digestion. PlaMSC-exo were prepared from the conditioned medium of PlaMSC (PlaMSC-CM) by ultracentrifugation. The expression of stemness-related genes, such as OCT4 and NANOG, in normal adult human dermal fibroblasts (NHDF) after incubation with PlaMSC-exo was measured by real-time reverse transcriptase PCR analysis (real-time PCR). The effect of PlaMSC-exo on OCT4 transcription activity was assessed using Oct4-EGFP reporter mice-derived dermal fibroblasts. The stimulating effects of PlaMSC-exo on osteoblastic and adipocyte-differentiation of NHDF were evaluated by alkaline phosphatase (ALP), and Alizarin red S- and oil red O-staining, respectively. The expression of osteoblast- and adipocyte-related genes was also assessed by real-time PCR. The treatment of NHDF with PlaMSC-exo significantly upregulated OCT4 and NANOG mRNA expression. PlaMSC-exo also enhanced OCT4 transcription. The NHDF treated with PlaMSC-exo exhibited osteoblastic and adipocyte-differentiation in osteogenic and adipogenic induction media. PlaMSC-exo increase the expression of OCT4 and NANOG mRNA in fibroblasts. As a result, PlaMSC-exo influence the differentiation competence of fibroblasts to both osteoblastic and adipocyte-differentiation. It shows a new feature of MSCs and the possibility of clinical application of MSC-exo. J. Cell. Biochem. 117: 1658-1670, 2016. © 2015 Wiley Periodicals, Inc.

An automated multiplex specific IgE assay system using a photoimmobilized microarray.

An automated microarray diagnostic system for specific IgE using photoimmobilized allergen has been developed. Photoimmobilization is useful for preparing microarrays, where various types of biological components are covalently immobilized on a plate. Because the immobilization is based on a photo-induced radical cross-linking reaction, it does not require specific functional groups on the immobilized components. Here, an aqueous solution of a photoreactive poly(ethylene glycol)-based polymer was spin-coated on a plate, and an aqueous solution of each allergen was microspotted on the coated plate and allowed to dry in air. Finally, the plate was irradiated with an ultraviolet lamp for covalent immobilization. An automated machine using these plates was developed for the assay of antigen-specific IgE. Initially, the patient serum was added to the microarray plate, and after reaction of the microspotted allergen with IgE, the adsorbed IgE was detected by a peroxidase-conjugated anti-IgE-antibody. The chemical luminescence intensity of the substrate decomposed by the peroxidase was automatically detected using a sensitive charge-coupled device camera. All the allergens were immobilized stably using this method, which was used to screen for allergen-specific IgE. The results were comparable with those using conventional specific IgE. Using this system, six different allergen-specific IgE were assayed using 10 µL of serum within a period of 20 min.

The use of cationic nanogels to deliver proteins to myeloma cells and primary T lymphocytes that poorly express heparan sulfate.

Fusion proteins containing protein transduction domain (PTD) are widely used for intracellular delivery of exogenous proteins. PTD-mediated delivery requires expression of heparan sulfate on the surface of the target cells. However, some of metastatic tumor cells and primary lymphocytes poorly express heparan sulfate. Here we demonstrate that proteins complexed with nanosize hydrogels formed by cationic cholesteryl group-bearing pullulans (cCHP) are efficiently delivered to myeloma cells and primary CD4(+) T lymphocytes probably by induction of macropinocytosis, although these cells are resistant to PTD-mediated protein delivery as a consequence of poor heparan sulfate expression. The anti-apoptotic protein Bcl-xL delivered by cCHP nanogels efficiently blocked apoptosis of these cells, establishing functional regulation of cells by proteins delivered by cCHP nanogels. Thus, cCHP nanogel is a useful tool to deliver proteins for development of new cancer therapy and immune regulation.

Self-assembled cationic nanogels for intracellular protein delivery.

An effective intracellular protein delivery system with self-assembled cationic nanogels is reported. Interaction of proteins with self-assembled nanogels of cationic cholesteryl group-bearing pullulans (CHPNH 2) was investigated by dynamic light scattering (DLS), transmission electron micrograph (TEM), fluorescence resonance energy transfer (FRET), and fluorescence correlation spectroscopy (FCS). The cationic nanogels strongly interacted with bovine serum albumin (BSA) and formed monodispersed nanoparticels (<50 nm). The complex more effectively internalized into HeLa cells than cationic liposomes and a protein transduction domain (PTD) based carrier even in the presence of serum. The higher efficiency of the nanogel carrier is probably due to the formation of colloidally stable nanoparticles with the protein. The enzymatic activity of beta-galactosidase (beta-Gal) was retained after internalization into cells. The nanogel carrier promoted nuclear delivery of a GFP-conjugated nuclear localization signal and Tat as a PTD (Tat-NLS-GFP). A blocking experiment with chemical inhibitors revealed the possible involvement of macropinocytosis in the uptake of the nanogel complex. After cellular uptake, the complex of the nanogel-protein was dissociated and the protein was released inside the cell. Such a self-assembled cationic nanogel system should create opportunities for novel applications of protein delivery.

Suppression of VEGF transcription in renal cell carcinoma cells by pyrrole-imidazole hairpin polyamides targeting the hypoxia responsive element.

Hypoxia inducible factor (HIF), a master regulator of critical genes for cell survival under hypoxic conditions, is known to be related to tumorigenesis and progression of renal cell carcinoma. N-methylpyrrole (Py)-N-methylimidazole (Im) hairpin polyamides are synthetic organic compounds that recognize and bind to the minor grooves of specific DNA sequences. We synthesized three Py-Im hairpin polyamides targeting the flanking sequences of hypoxia responsive element (HRE; a binding site of HIF) in the promoter region of the vascular endothelial growth factor (VEGF) gene. The effects of the polyamides on HIF-induced transcription were evaluated by a luciferase assay using a reporter plasmid containing a VEGF promoter. Real time reverse-transcriptase polymerase chain reaction and enzyme-linked immunosorbent assay were performed to examine the effects of the polyamides on the transcription and secretion of VEGF in A498 renal cell carcinoma cells, which have a frame-shift mutation in the von Hippel-Lindau gene. A combination of three Py-Im hairpin polyamides suppressed HIF-induced transcription in reporter assays using 293 cells and successfully suppressed transcription and translation of the VEGF gene in A498 cells. Inhibition of the HIF-HRE interaction was confirmed by an electrophoresis mobility shift assay. An approach using Py-Im hairpin polyamides may be a new strategy for the treatment of renal cell carcinoma.

Development of gene silencing pyrrole-imidazole polyamide targeting the TGF-beta1 promoter for treatment of progressive renal diseases.

Pyrrole-imidazole (Py-Im) polyamides are nuclease-resistant novel compounds that inhibit gene expression by binding to the minor groove of DNA. A Py-Im polyamide that targets rat TGF-beta1 was designed as a gene-silencing agent for progressive renal diseases, and the distribution and the effects of this polyamide on renal injury were examined in Dahl-salt sensitive (Dahl-S) rats. For identification of transcription factor binding elements for activation of the rat TGF-beta1 gene, recombinant TGF-beta1 reporter plasmids were transfected into HEK-293 cells, and promoter activity was measured. Py-Im polyamide was designed to the activator protein-1 binding site of the rat TGF-beta1 promoter. This Py-Im polyamide showed strong, fast, and specific binding to the target DNA in gel mobility shift and Biacore assays. Py-Im polyamide significantly inhibited TGF-beta1 promoter activity and expression of TGF-beta1 mRNA and protein in rat mesangial cells. Intravenously administered fluorescein-labeled polyamide distributed to the kidney of rats. Py-Im polyamide significantly inhibited expression of TGF-beta1 mRNA and protein in the renal cortex of Dahl-S rats and reduced the increase in urinary protein and albumin in Dahl-S rats independent of changes in blood pressure. These results indicate that Py-Im polyamide that targets TGF-beta1 will be a novel gene-silencing agent for the TGF-beta1-associated diseases, including progressive renal diseases.

Synthetic pyrrole-imidazole polyamide inhibits expression of the human transforming growth factor-beta1 gene.

Pyrrole-imidazole (Py-Im) polyamides can bind to the predetermined base pairs in the minor groove of double-helical DNA with high affinity. These synthetic small molecules can interfere with transcription factor-DNA interaction and inhibit or activate the transcription of corresponding genes. In the present study, we designed and synthesized a Py-Im polyamide to target -545 to -539 base pairs of human transforming growth factor-beta1 (hTGF-beta1) promoter adjacent to the fat-specific element 2 (FSE2) to inhibit the expression of the gene. Gel mobility shift assay showed that the synthetic Py-Im polyamide binds to its corresponding double-strand oligonucleotides, whereas the mismatch polyamides did not bind. Fluorescein isothiocyanate-labeled Py-Im polyamide was detected in the nuclei of human vascular smooth muscle cells (VSMCs) after 2- to 48-h incubation. Py-Im polyamide significantly decreased the promoter activity of hTGF-beta1 determined by in vitro transcription experiments and luciferase assay. In cultured human VSMCs, Py-Im polyamide targeting hTGF-beta1 promoter significantly inhibited expressions of hTGF-beta1 mRNA and protein. These results indicate that the synthetic Py-Im polyamide designed to bind hTGF-beta1 promoter inhibited hTGF-beta1 gene and protein expression successfully. This novel agent will be used for the TGF-beta-related diseases as a gene therapy.

Binding of distamycin A to UV-damaged DNA.

We have found that distamycin A can bind to DNA duplexes containing the (6-4) photoproduct, one of the major UV lesions in DNA, despite the changes, caused by photoproduct formation, in both the chemical structure of the base moiety and the local tertiary structure of the helix. A 20-mer duplex containing the target site, AATT.AATT, was designed, and then one of the TT sequences was changed to the (6-4) photoproduct. Distamycin binding to the photoproduct-containing duplex was detected by CD spectroscopy, whereas specific binding did not occur when the TT site was changed to a cyclobutane pyrimidine dimer, another type of UV lesion. Distamycin binding was analyzed in detail using 14-mer duplexes. Curve fitting of the CD titration data and induced CD difference spectra revealed that the binding stoichiometry changed from 1:1 to 2:1 with photoproduct formation. Melting curves of the drug-DNA complexes also supported this stoichiometry.

Enantioselective DNA alkylation by a pyrrole-imidazole S-CBI conjugate.

Conjugates 12S and 12R of N-methylpyrrole (Py)-N-methylimidazole (Im) seven-ringed hairpin polyamide with both enantiomers of 1,2,9,9a-tetrahydrocyclopropa[1,2-c]benz[1,2-e]indol-4-one (CBI) were synthesized, and their DNA alkylating activity was examined. High-resolution denaturing gel electrophoresis revealed that 12S selectively and efficiently alkylated at one match sequence, 5'-TGACCA-3', in 450-bp DNA fragments. The selectivity and efficiency of the DNA alkylation by 12S were higher than those of the corresponding cyclopropapyrroloindole (CPI) conjugate, 11. In sharp contrast, another enantiomer, 12R, showed very weak DNA alkylating activity. Product analysis of the synthetic decanucleotide confirmed that the alkylating activity of 12S was comparable with 11 and that 12S had a significantly higher reactivity than 12R. The enantioselective reactivity of 12S and 12R is assumed to be due to the location of the alkylating cyclopropane ring of the CBI unit in the minor groove of the DNA duplex. Since the CBI unit can be synthesized from commercially available 1,3-naphthalenediol, the present results open up the possibility of large-scale synthesis of alkylating Py-Im polyamides for facilitating their use in future animal studies.

Analysis of distamycin A binding to UV-damaged DNA.

We have found that distamycin A can bind to DNA duplexes containing the (6-4) photoproduct, one of the major UV lesions in DNA, in spite of the changes caused by photoproduct formation in the chemical structure of the base moiety and the local tertiary structure of the duplex. Distamycin binding was analyzed in detail using 14-mer duplexes. Curve fitting of the CD titration data and induced CD difference Spectra revealed that the binding stoichiometry changed from 1:1 to 2:1 with the photoproduct formation. Melting curves of the drug-DNA complexes also supported this stoichiometry.

Fmoc solid-phase synthesis and its application to pyrrole-imidazole polyamides.

Improvements to the Fmoc solid-phase synthesis of N-methylpyrrole (Py) and N-methylimidazole (Im) polyamides were examined. It was found that a different combination of coupling agents and activators, and the introduction of a dimer unit significantly enhanced the yield of polyamides. Using this improved method, various types of polyamides were synthesized. The uptake and localization of polyamides in living mammalian cells were investigated using FITC labeled Py-Im polyamides.