A Comparative Immunohistochemical Study of Anal Canal Epithelium in Humans and Swine, Focusing on the Anal Transitional Zone Epithelium and the Anal Glands.

To better understand the cellular origins and differentiation of anal canal epithelial neoplasms, the immunohistochemical profiles of the anal canal epithelium in humans and swine were evaluated. Formalin-fixed tissue sections were immunostained for mucin (MUC: MUC2, MUC5AC, MUC5B), desmoglein 3 (DGS3), p63, CDX2, SOX2, and α-smooth muscle actin (α-SMA). The anal transitional zone (ATZ) epithelium covered the anal sinus and consisted of a stratified epithelium with mucous cells interspersed within the surface lining. Anal glands opened into the anal sinus. Ducts and acini of intraepithelial or periepithelial mucous type were the main structures of human anal glands, whereas those of swine were compound tubuloacinar mixed glands. Distal to the ATZ epithelium, non-keratinized stratified squamous epithelium merged with the keratinized stratified squamous epithelium of the perianal skin. MUC5AC expression predominated over MUC5B expression in the ATZ epithelium, while MUC5B expression was higher in the anal glands. SOX2 was positive in the ATZ epithelium, anal glands, and squamous epithelium except in the perianal skin. In humans, DGS3 was expressed in the ATZ epithelium, anal gland ducts, and squamous epithelium. p63 was detected in the ATZ epithelium, anal glands, and squamous epithelium. Myoepithelial cells positive for α-SMA and p63 were present in the anal glands of swine. Colorectal columnar cells were MUC5B+ /MUC2+ /CDX2+ /MUC5AC- /SOX2- . The ATZ epithelium seems to be a distinctive epithelium, with morphological and functional features allowing smooth defecation. The MUC5AC+ /SOX2+ /MUC2- /CDX2- profile of the ATZ epithelium and anal glands is a useful feature for diagnosing adenocarcinoma arising from these regions. Anat Rec, 301:796-805, 2018. © 2017 Wiley Periodicals, Inc.

Characterization of clinically isolated thymidine-dependent small-colony variants of Escherichia coli producing extended-spectrum ß-lactamase.

PURPOSE: Thymidine-dependent small-colony variants (TD-SCVs) are difficult to detect or test for antimicrobial susceptibility. We investigated the characteristics of clonal TD-SCVs of Escherichia coli, both with and without blaCTX-M-3, isolated from a patient. METHODOLOGY: Mutation in the thyA gene was analysed by sequencing, and morphological abnormalities in the colonies and cells of the isolates were examined. Additionally, conjugational transfer experiments were performed to prove the horizontal transferability of plasmids harbouring resistance genes. RESULTS: The TD-SCVs contained a single nucleotide substitution in the thyA gene, c.62G>A, corresponding to p.Arg21His. Morphologically, their colonies were more translucent and flattened than those of the wild-type strain. In addition, cells of the TD-SCVs were swollen and elongated, sometimes with abnormal and incomplete divisions; a large amount of cell debris was also observed. Changing c.62G>A back to the wild-type sequence reversed these abnormalities. Conjugational transfer experiments showed that the TD-SCV of E. coli with blaCTX-M-3 failed to transfer blaCTX-M-3 to E. coli CSH2. However, the TD-SCV of E. coli without blaCTX-M-3 experimentally received the plasmid encoding blaSHV-18 from Klebsiella pneumoniae ATCC 700603 and transferred it to E. coli CSH2. CONCLUSION: Mutation in the thyA gene causes morphological abnormalities in the colonies and cells of E. coli, as well as inducing thymidine auxotrophy. In addition, TD-SCVs horizontally transmit plasmids encoding resistance genes. It is important to detect TD-SCVs based on their characteristics because they serve as reservoirs of transferable antibiotic resistance plasmids.

The fibrous form of intracellular inclusion bodies in recombinant variant fibrinogen-producing cells is specific to the hepatic fibrinogen storage disease-inducible variant fibrinogen.

Fibrinogen storage disease (FSD) is a rare disorder that is characterized by the accumulation of fibrinogen in hepatocytes and induces liver injury. Six mutations in the γC domain (γG284R, γT314P, γD316N, the deletion of γG346-Q350, γG366S, and γR375W) have been identified for FSD. Our group previously established γ375W fibrinogen-producing Chinese hamster ovary (CHO) cells and observed aberrant large granular and fibrous forms of intracellular inclusion bodies. The aim of this study was to investigate whether fibrous intracellular inclusion bodies are specific to FSD-inducible variant fibrinogen. Thirteen expression vectors encoding the variant γ-chain were stably or transiently transfected into CHO cells expressing normal fibrinogen Aα- and Bß-chains or HuH-7 cells, which were then immunofluorescently stained. Six CHO and HuH-7 cell lines that transiently produced FSD-inducible variant fibrinogen presented the fibrous (3.2-22.7 and 2.1-24.5%, respectively) and large granular (5.4-25.5 and 7.7-23.9%) forms of intracellular inclusion bodies. Seven CHO and HuH-7 cell lines that transiently produced FSD-non-inducible variant fibrinogen only exhibit the large granular form. These results demonstrate that transiently transfected variant fibrinogen-producing CHO cells and inclusion bodies of the fibrous form may be useful in non-invasive screening for FSD risk factors for FSD before its onset.

A novel surfactant protein C L55F mutation associated with interstitial lung disease alters subcellular localization of proSP-C in A549 cells.

BACKGROUND: Heterozygous mutations of SFTPC, the gene-encoding surfactant protein C (SP-C), result in interstitial lung disease (ILD). However, characterization of mutations located in the mature domain of precursor SP-C (proSP-C) is limited. This study examined the molecular pathogenesis of such a mutation of ILD. METHODS: We employed sequencing of SFTPC and established A549 cells stably expressing several proSP-C mutants. Histopathology and transmission electron microscopy (TEM) of lung tissue from a pediatric patient with ILD were assessed. Effects of mutant proSP-C were evaluated by western blotting, immunofluorescence, and TEM. RESULTS: Sequencing of SFTPC revealed a novel heterozygous mutation, c.163C>T (L55F). In lung tissue, abnormal localization of proSP-C was observed by immunohistochemistry, and small and dense lamellar bodies (LBs) in type II alveolar epithelial cells (AECs) were detected by TEM. TEM of A549 cells stably expressing proSP-C(L55F) displayed abnormal cytoplasmic organelles. ProSP-C(L55F) exhibited a band pattern similar to that of proSP-C(WT) for processed intermediates. Immunofluorescence studies demonstrated that proSP-C(L55F) partially colocalized in CD63-positive cytoplasmic vesicles of A549 cells, which was in contrast to proSP-C(WT). CONCLUSION: We detected a novel c.163C>T mutation located in the mature domain of SFTPC associated with ILD that altered the subcellular localization of proSP-C in A549 cells.

Helicobacter heilmannii sensu stricto-related gastric ulcers: a case report.

A spiral bacterium (SH9), morphologically different from Helicobacter pylori (H. pylori), was found in a 62-year-old woman's gastric mucosa. Gastroscopic examination revealed multiple gastric ulcers near the pyloric ring; mapping gastric biopsy showed mild mononuclear infiltration with large lymphoid follicles in the antrum, without corpus atrophy. Urea breath test and H. pylori culture were negative, but Giemsa staining of biopsies revealed tightly coiled bacteria that immunostained with anti-H. pylori antibody. Sequencing of SH9 16S rRNA and the partial urease A and B subunit genes showed that the former sequence had highest similarity (99%; 1302/1315 bp) to Helicobacter heilmannii (H. heilmannii) sensu stricto (H. heilmannii s.s.) BC1 obtained from a bobcat, while the latter sequence confirmed highest similarity (98.3%; 1467/1493 bp) to H. heilmannii s.s. HU2 obtained from a human. The patient was diagnosed with multiple gastric ulcers associated with H. heilmannii s.s. infection. After triple therapy (amoxicillin, clarithromycin, and lansoprazole) with regimen for eradicating H. pylori, gastroscopy showed ulcer improvement and no H. heilmannii s.s. upon biopsy.

γ375W fibrinogen-synthesizing CHO cells indicate the accumulation of variant fibrinogen within endoplasmic reticulum.

BACKGROUND: Hepatic endoplasmic reticulum (ER) storage disease (HERSD) associated with hypofibrinogenemia has been reported in patients with four types of heterozygous γ-chain variant fibrinogen in the C terminal region. Of interest, substitution of γR375W induced hypofibrinogenemia and HERSD, whereas γR375G induced dysfibrinogenemia. OBJECTIVES: To analyze the synthesis of variant fibrinogen and morphological characteristics, we established variant fibrinogen-producing cells and compared them with wild-type fibrinogen-synthesizing cells. METHODS: The fibrinogen γ-chain expression vectors coding γ375W and γ375G were altered by oligonucleotide-directed mutagenesis and transfected into Chinese hamster ovary (CHO) cells. Synthesis of fibrinogen (media and cell lysates) was measured by ELISA for each cloned cell line and morphological characteristics were observed by immunofluorescence and transmission electron microscopy. RESULTS: The medium/cell lysate fibrinogen ratio of γ375W-CHO cells was markedly lower than that of the normal cells and γ375G-CHO cells. Immunostaining with anti-fibrinogen antibody showed only γ375W-CHO cells, but revealed two types of cells containing cytoplasmic inclusion bodies, scattered large-granular bodies and fibrous forms. Observation by confocal microscopy indicated that both inclusion bodies were colocalized with fibrinogen and ER-membrane protein; furthermore, transmission electron microscopic observation demonstrated dilatation of the ER by large-granular inclusion bodies and fibrous forms filled with regularly structured fibular materials within the dilated ER. CONCLUSION: These results demonstrated that assembled and non-secreted γ375W fibrinogen was accumulated in the dilated ER and aggregated variant fibrinogen was seen as regularly structured fibular materials, which was similar to the fingerprint-like pattern observed at inclusion bodies in patients' hepatocytes affected with HERSD.

A case of Helicobacter heilmannii-associated primary gastric mucosa-associated lymphoid tissue lymphoma achieving complete remission after eradication.

A 46-year-old man underwent gastrointestinal endoscopy while visiting the hospital for a general physical check-up. Coarse mucosa in the antrum with superficial erosions was found by endoscopic gastrointestinal examination, but no atrophic changes were seen in the corpus. Histopathological examination of gastric biopsy specimens revealed mucosa-associated lymphoid tissue (MALT) lymphoma. Although Helicobacter pylori was not detected in our patient, H. heilmannii was identified histologically and by polymerase chain reaction analysis, resulting in the diagnosis of H. heilmannii-associated gastric MALT lymphoma. We successfully eradicated H. heilmannii and achieved complete remission of gastric MALT lymphoma by antibiotic therapy. H. heilmannii usually causes milder gastritis than H. pylori, but it has been more closely associated with MALT lymphoma. As such, when H. pylori infection is excluded in patients with gastric MALT lymphoma, physicians should next consider the possibility of H. heilmannii. Furthermore, our research suggests that eradication therapy is effective for treatment of localized H. heilmannii-associated gastric MALT lymphoma.

Biocompatibility and bone tissue compatibility of alumina ceramics reinforced with carbon nanotubes.

AIMS: The addition of carbon nanotubes (CNTs) remarkably improves the mechanical characteristics of base materials. CNT/alumina ceramic composites are expected to be highly functional biomaterials useful in a variety of medical fields. Biocompatibility and bone tissue compatibility were studied for the application of CNT/alumina composites as biomaterials. METHODS & RESULTS: Inflammation reactions in response to the composite were as mild as those of alumina ceramic alone in a subcutaneous implantation study. In bone implantation testing, the composite showed good bone tissue compatibility and connected directly to new bone. An in vitro cell attachment test was performed for osteoblasts, chondrocytes, fibroblasts and smooth muscle cells, and CNT/alumina composite showed cell attachment similar to that of alumina ceramic. DISCUSSION & CONCLUSION: Owing to proven good biocompatibility and bone tissue compatibility, the application of CNT/alumina composites as biomaterials that contact bone, such as prostheses in arthroplasty and devices for bone repair, are expected.

Bactericidal activities of woven cotton and nonwoven polypropylene fabrics coated with hydroxyapatite-binding silver/titanium dioxide ceramic nanocomposite "Earth-plus".

BACKGROUND: Bacteria from the hospital environment, including linens and curtains, are often responsible for hospital-associated infections. The aim of the present study was to evaluate the bactericidal effects of fabrics coated with the hydroxyapatite-binding silver/titanium dioxide ceramic nanocomposite "Earth-plus". METHODS: Bactericidal activities of woven and nonwoven fabrics coated with Earth-plus were investigated by the time-kill curve method using nine bacterial strains, including three Staphylococcus aureus, three Escherichia coli, and three Pseudomonas aeruginosa strains. RESULTS: The numbers of viable S. aureus and E. coli cells on both fabrics coated with Earth-plus decreased to below 2 log(10) colony-forming units/mL in six hours and reached the detection limit in 18 hours. Viable cell counts of P. aeruginosa on both fabrics coated with Earth-plus could not be detected after 3-6 hours. Viable cells on woven fabrics showed a more rapid decline than those on nonwoven fabrics. Bacterial cell counts of the nine strains on fabrics without Earth-plus failed to decrease even after 18 hours. CONCLUSION: Woven cotton and nonwoven polypropylene fabrics were shown to have excellent antibacterial potential. The woven fabric was more bactericidal than the nonwoven fabric.

A thin carbon-fiber web as a scaffold for bone-tissue regeneration.

Due to the rapid progress being made in tissue regeneration therapy, biomaterials used as scaffolds are expected to play an important role in future clinical application. We report the development of a 3D web (sheet) consisting of high-purity carbon fibers in a nanoscale structure. When the thin carbon-fiber web (TCFW) and recombinant human bone morphogenetic protein 2 (rhBMP-2) composite is implanted in the murine back muscle, new ectopic bone is formed, and the values of the bone mineral content and bone mineral density are significantly higher than those obtained with a collagen sheet. Observation of the interface between the carbon fibers and bone matrix reveal that the fibers are directly integrated into the bone matrix, indicating high bone-tissue compatibility. Further, the rhBMP-2/TCFW composite repairs a critical-size bone defect within a short time period. These results suggest that the TCFW functions as an effective scaffold material and will play an important role in tissue regeneration in the future.

Localization of Liv2 as an immature hepatocyte marker in EB outgrowth.

The objective of this study was to establish Liv2, a surface marker of mouse immature hepatocytes (hepatoblasts), as a selection tool for embryonic stem (ES) cell-derived immature hepatocytes by acquiring basic data on Liv2 in normal mouse embryos and by confirming Liv2 expression in mouse ES-derived cells. The estimated molecular weight of Liv2 was 40-45 kDa, and immunoreactivity was definitively detected in the cell membrane of fetal hepatocytes on embryonic day (E) 9.5, declined gradually until E12.5,and subsequently became undetectable. Liv2 was localized on and close to the cell membrane. Embryoid bodies (EB) were formed from mouse ES cells whose undifferentiated state was confirmed with immunostaining of Nanog by the hanging drop method. A few Liv2-positive cells occurred as a cluster in EB outgrowth on day 7, but only some of these were albumin (ALB)-positive on day 13. These cells had the same pattern of immunoreactivity, i.e., localization on the cell membrane, as immature hepatocytes in the developing liver, although there were other types of cells with a different pattern of immunoreactivity that were seen only as a granular pattern in the cytoplasm and without ALB or the neuronal marker nestin. These results suggest thatLiv2 may be useful as a surface marker for immature hepatocytes derived from ES cells.This application would allow for the sole selection of immature hepatocytes and provide a useful tool for regenerative medicine.

Sialic acid moiety of apolipoprotein E and its impact on the formation of lipoprotein particles in human cerebrospinal fluid.

BACKGROUND: Apolipoprotein (apo) E in the cerebrospinal fluid (CSF) is abundant with sialic acid (SA), and sialylation of certain proteins is known to modulate biological function. The aim of the present study was to quantify the SA content in CSF apoE and carry out the more detailed characterization of the CSF apoE-containing lipoproteins. METHODS: The method for the determination of the SA in CSF apoE was based on the conversion of SA into p-aminobenzoic acid ethyl ester-derivatized N-acetylmannosamine, followed by HPLC analysis. RESULTS: The levels of CSF SA and serum SA were 25.9+/-1.5 and 2209+/-196 micromol/l, respectively; however, when the SA values were corrected by the total protein concentrations, CSF SA values were approximately 3.5-fold of those in the serum. The SA levels in the CSF apoE-containing lipoprotein fractions were 5.3+/-1.3% of total CSF SA, and were correlated with the CSF apoE concentrations. However, the ratios of SA to apoE were inversely proportional to the CSF lipid concentrations. The lipoprotein particle sizes were larger when the ratios of SA to CSF apoE were greater. CONCLUSION: The SA moiety of the CSF apoE molecules may affect the formation of the apoE-containing lipoprotein particles and the regulation of lipid delivery in CNS.

Tumor growth suppression by adenovirus-mediated introduction of a cell-growth-suppressing gene tob in a pancreatic cancer model.

TOB (transducer of ErbB-2) is a tumor suppressor that interacts with protein-tyrosine kinase receptors, including ErbB-2. Introduction of the tob gene into NIH3T3 cells results in cell growth suppression. In this study, we evaluated the effect of tob expression in pancreatic cell lines (AsPC-1, BxPC-3, SOJ) and discuss the tumor-suppressing effects of adenoviral vector expressing tob cDNA. We first measured the levels of endogenous tob mRNA being expressed in all pancreatic cancer cell lines. Then, we examined the effect of adenoviral vector containing tob cDNA (Ad-tob vector) on cancer cell lines. The viral vector was expanded with transfection in 293 cells. The titer of the vector was 350x10(6) pfu/ml. These cancer cells were able to be transfected with MOI 20 without adenoviral toxicity. The transfection of Ad-tob vector results in growth suppression of SOJ and AsPC-1 cell lines. The magnitude of the expression of the Ad-tob gene in cancer is correlated to tumor suppressive activity. We prepared pancreatic cancer peritonitis models using a peritoneal injection of AsPC-1 cells. In this model, bloody ascites and multiple tumor nodules were seen at the mesentery after 16 days. AdCAtob (50x10(6) pfu/day) was administered from day 5 to day 9 after 4 days of peritoneal injection of 2x10(6) AsPC-1 cells. Tumor growth suppression occurred 10 days after peritoneal injection of AdCAtob compared with the control group. There were no tumor nodules in the abdomen and no bloody ascites. These results suggest that the peritoneal injection of AdCAtob has potential to suppress the formation of pancreatic cancer peritonitis, and can be applied for chemotherapy-resistant cancer peritonitis.

Critical roles of VEGF-C-VEGF receptor 3 in reconnection of the collecting lymph vessels in mice.

Molecular mechanisms of reconnection of collecting lymph vessels were analyzed by using murine popliteal prenodal lymph vessels. At 1 and 2 weeks after being divided by cutting the lymph vessel, lymphatic reconnection was frequently observed accompanied by mesh-like lymphatic channels. Electron microscopic study also showed a monolayer of endothelial cells in the newly developed lymph vessels. Smooth muscle markers were immunofluorescently demonstrated in the wall of the new vessels. At 1 week after the procedure of cutting, augmented expressions of VEGF receptors 1, 2 and 3 were found immunohistochemically at the site of the reconnected lymph vessels. The expression of mRNA for VEGF receptor 3 was enhanced at 5 days and 1 week in small pieces of the tissues containing the reconnected lymph vessels, compared with that in the corresponding tissues obtained with sham operated ones. The administration of VEGF-C at the cutting site of the collecting lymph vessel significantly increased the rate of the reconnected lymph vessels, whereas additional treatment with Flt4/Fc chimera protein significantly reduced the rate of the reconnected ones. These results suggest that activation of VEGF-C-VEGF receptor 3 has critical roles in reconnection of the collecting lymph vessels in adult mice.

Cryopreservation of mouse embryoid bodies.

Cryopreservation of embryonic stem (ES) cells is essential to establish them as a resource for regenerative therapy. We evaluated survival adhesion rate, cell structure, gene expression, and multipotency of frozen and thawed embryoid bodies (EBs) derived from mouse ES cells. EBs were cryopreserved using the BICELL and the Program Freezer. After one week the EBs were thawed and cultured. EBs prepared in the Program Freezer had the highest survival adhesion (Program Freezer; 55-69%, BICELL; 30-38%). Though many cells in the thawed EBs were damaged, some were not, especially those prepared in the Program Freezer. RT-PCR analysis showed that genes characteristic of the three embryonic germ layers were expressed in thawed EBs cultured for one week. EBs transplanted into mice formed teratomas consisting of cells derived from the three germ layers. In conclusion, EBs frozen in the Program Freezer had higher survival adhesion rates compared to the BICELL and formed differentiated cells characteristic of the three embryonic germ layers.

Structural differentiation, proliferation, and association of human embryonic stem cell-derived cardiomyocytes in vitro and in their extracardiac tissues.

The proliferation, structural differentiation, and capacity of association of human ES cell-derived cardiomyocytes were assessed in culture and in extracardiac graft tissues. Embryoid body (EB) outgrowths having cardiomyocytes, and their transplants in mice retroperitoneum or renal subcapsular region were analyzed mainly by immunochemistry. During the culture of EB outgrowths, colonies of cardiomyocytes grew in size exhibiting synchronized beatings. Subcellular structures of those cardiomyocytes involved in the contraction, hormone production, and intercellular integration differentiated with distinct immunoreactivity for constituent proteins/peptides. Judging from PCNA staining, proliferation potential was maintained in part for more than 70 days. In teratoma tissues on post-transplantation Day 7, cardiomyocytes maintained their integration with connexin 43 and cadherin at their junctions. They partly exhibited strong PCNA reactivity. On Day 28, large part of the cardiomyocytes lost their association, dispersing among non-cardiac cells without discernible cadherin reactivity. Proliferation potential was generally low irrespective of their tissue diversity. From these results, structural differentiation and active proliferation of human ES cell-derived cardiomyocytes occurred in vitro, maintaining their association. When developed in extracardiac tissues, however, the cardiomyocytes showed low proliferation potential and reduced cellular integration. This leads to the proposal that some procedure will be necessary to accelerate or maintain the proliferation of cardiomyocytes in vivo.

Glucagon-like peptide-1 differentiation of primate embryonic stem cells into insulin-producing cells.

The present study was performed to determine whether glucagon-like peptide-1 (GLP-1) stimulates differentiation of nestin-selected embryonic stem cells into insulin-producing cells. Our experimental strategy began with the production of a highly enriched population of nestin-positive cells from embryoid bodies. These cells differentiated into insulin-producing cells after addition of GLP-1. Islet-like cell clusters (ICCs) formed in inducing culture. These nestin-positive cell-derived ICCs expressed numerous beta-cell lineage genes, including insulin; Glut-2; pancreatic duodenal homebox-1 protein (PDX-1); islet amyloid polypeptide (IAPP); neurogenin 3 (ngn3); and alpha, gamma, and delta cell gene markers. Cells of ICCs showed increased insulin protein expression, glucose-dependent insulin release, and coexpression of insulin and C-peptide. In addition, ICCs were characterized by coexpression of nestin/insulin and nestin/PDX-1. The levels of pancreas-related gene and protein expression and insulin secretion in the GLP-1 group were stronger than those in the normal controls. GLP-1 has been shown to be involved in stimulating the signaling pathways downstream of the transcription factor PDX-1, by increasing its protein and messenger RNA levels. In vivo, ICCs displayed the ability to reverse hyperglycemia in diabetic severe combined immunodeficiency (SCID) mice. We concluded that GLP-1 induced differentiation of nestin-positive progenitor embryonic stem cells into insulin-producing cells, which was achieved by upregulation of PDX-1 expression. This method may have future applications in stem cell therapy of diabetes.

Sendai virus-mediated gene delivery into hepatocytes via isolated hepatic perfusion.

The recombinant Sendai virus vector is a promising tool for human gene therapy, capable of inducing high-level expression of therapeutic genes in tissue cells in situ. The target tissues include airway epithelium, blood vessels, skeletal muscle, retina and the central nervous system, but application to hepatic tissues has not yet been achieved, because direct intraportal injection of the vector is not feasible. We report an efficient and harmless procedure of gene delivery by recombinant Sendai virus into rat parenchymal hepatocytes, based on isolated hepatic perfusion with controlled inflow. Critical parameters for successful hepatic gene delivery are a brief preperfusion period (25 degrees C, 5 min); appropriate vector concentration in the perfusate (10(7) pfu/ml); moderate portal vein pressure (12 mmHg) and a brief hyperthermic postperfusion period (42 degrees C, 5 min). Under these optimized conditions, marker genes were expressed in most parenchymal hepatocytes without significant damage to hepatic tissues. Furthermore, expression of the marker genes was undetectable in nonhepatic tissues, including the gonads, indicating that this approach strictly targets hepatic tissues and thus offers good clinical potential for human gene therapy.

Induction of midbrain dopaminergic neurons from primate embryonic stem cells by coculture with sertoli cells.

The aim of this study was to produce dopaminergic neurons from primate embryonic stem (ES) cells following coculture with mouse Sertoli cells. After 3 weeks of induction, immunostaining revealed that 90% +/- 9% of the colonies contained tyrosine hydroxylase-positive (TH(+)) neurons, and 60% +/- 7% of the tubulin beta III-positive (Tuj III(+)) neurons were TH(+). Reverse transcription-polymerase chain reaction analyses showed that Sertoli-induced neurons expressed midbrain dopaminergic neuron markers, including TH, dopamine transporter, aromatic amino acid decarboxylase (AADC), receptors such as TrkB and TrkC, and transcription factors NurrI and Lmx1b. Neurons that had been differentiated on Sertoli cells were positive for Pax2, En1, and AADC, midbrain-related markers, and negative for dopamine-beta-hydroxylase, a marker of noradrenergic neurons. These Sertoli cell-induced dopaminergic cells can release dopamine when depolarized by high K(+). Sertoli cell-conditioned medium contained glial cell line-derived neurotrophic factor (GDNF) and supported neuronal differentiation. After pretreatment with anti-GDNF antibody, the percentage of Tuj III(+) colonies was reduced to 14%. Thus, GDNF contributed significantly to inducing primate ES cells into dopaminergic neurons. When transplanted into a 6-hydroxydopamine-treated Parkinson's disease model, primate-derived dopaminergic neurons integrated into the mouse striatum. Two weeks after transplantation, surviving TH(+) cells were present. These TH(+) cells survived for 2 months. Therefore, the induction method of coculture ES cells with Sertoli cells provides an unlimited source of primate cells for the study of pathogenesis and transplantation in Parkinson's disease.