Cotransplantation of neuroepithelial stem cells with interstitial cells of Cajal improves neuronal differentiation in a rat aganglionic model.

BACKGROUND: Recent advances have raised the possibility of treating Hirschsprung's disease (HSCR) with transplanted neural stem cells. Although these cells have been shown to migrate and differentiate after transplantation, the restoration of intestinal motility by grafted cells is also mediated via the interstitial cells of Cajal (ICCs). We therefore aimed to evaluate the effect of cografting with ICC on neural stem cells after transplanted into aganglionic bowel. METHODS: Neuroepithelial stem cells (NESCs) and ICCs were isolated from neural tube of embryonic rat (embryonic day 11.5) and colon of neonatal rats respectively. After cotransplantation into the benzalkonium chloride-induced rat aganglionic colon, survival and differentiation of the implanted cells were assessed 8weeks posttransplantation using immunofluorescence and Western blotting. Colonic motility was assessed by measuring the changes of intraluminal pressure responding to inflating stimulation and the response of muscle strips to electrical field stimulation. RESULTS: Cotransplantation with ICC accelerated neuronal differentiation compared with transplantation of NESCs alone. Moreover, the contractile response to inflation and EFS-induced reaction of the colon after coimplantation were successfully evoked. CONCLUSIONS: These data indicate that reconstitution of elements in the intestinal environment such as ICC enhances efficacy of NESCs therapy. This may aid development of a stem cell-based treatment for HSCR.

The expression of IL-6 and STAT3 might predict progression and unfavorable prognosis in Wilms' tumor.

PURPOSE: To investigate the expression profiles of IL-6 and STAT3 in Wilms' tumor (WT) and their relationship with disease progression. METHODS: Immunohistochemistry was used to examine IL-6 and STAT3 expression in 58 primary tumors and 18 invasive/metastatic ones. RESULTS: Positive expression rate of IL-6/STAT3 was 39.7% (23/58)/29.3% (17/58) in primary WT tissues, while 61.1% (11/18)/33.3% (6/18) in associated invasive/metastatic tissues. The expression rate of IL-6 and STAT3 was higher in primary WT tumors of invasive/metastatic group than that of non-invasive/metastatic group (P=0.033; P=0.012). There was a positive correlation between IL-6 and STAT3 expression in 76 WT tissues (P<0.001, r=0.444). The expression of IL-6 /STAT3 between primary WT and matched invasive/metastatic tissues was concordance (P=0.727; P=0.99). IL-6 expression status and histopathological type were associated with disease-free survival (DFS) and overall survival (OS) (P<0.05), while STAT3 was only correlated with DFS (P<0.05). CONCLUSIONS: IL-6 and STAT3 expression in WT might be correlated with progression and predict unfavorable prognosis, highlighting a new therapy target for invasive or metastatic WTs.

Resveratrol inhibits ß-amyloid-induced neuronal apoptosis through regulation of SIRT1-ROCK1 signaling pathway.

Alzheimer's disease (AD) is characterized by the accumulation of ß-amyloid peptide (Aß) and loss of neurons. Recently, a growing body of evidences have indicated that as a herbal compound naturally derived from grapes, resveratrol modulates the pathophysiology of AD, however, with a largely unclear mechanism. Therefore, we aimed to investigate the protection of resveratrol against the neurotoxicity of ß-amyloid peptide 25-35 (Aß(25-35)) and further explore its underlying mechanism in the present study. PC12 cells were injuried by Aß(25-35), and resveratrol at different concentrations was added into the culture medium. We observed that resveratrol increased cell viability through the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) colorimetric assays. Flow cytometry indicated the reduction of cell apoptosis by resveratrol. Moreover, resveratrol also stabilized the intercellular Ca(2+) homeostasis and attenuated Aß(25-35) neurotoxicity. Additionally, Aß(25-35)-suppressed silent information regulator 1 (SIRT1) activity was significantly reversed by resveratrol, resulting in the downregulation of Rho-associated kinase 1 (ROCK1). Our results clearly revealed that resveratrol significantly protected PC12 cells and inhibited the ß-amyloid-induced cell apoptosis through the upregulation of SIRT1. Moreover, as a downstream signal molecule, ROCK1 was negatively regulated by SIRT1. Taken together, our study demonstrated that SIRT1-ROCK1 pathway played a critical role in the pathomechanism of AD.

Striatal extracts promote the dopaminergic differentiation of GFP-bone mesenchymal stem cells.

Bone mesenchymal stem cells (BMSCs) are an attractive donor graft source because of the potential of self-renewal and multi-direction differentiation. However, it is a great challenge to induce BMSCs to specifically differentiate to dopamine (DA) neurons for the treatment of Parkinson's disease. Because the striatum is the target tissue for the projection of DA neurons in the midbrain, we investigated whether its extracts could promote the dopaminergic differentiation of BMSCs. BMSCs were isolated from green fluorescent protein (GFP) transgenic mice. Flow cytometry was used to identify the expression of CD29 and CD11b in cultured BMSCs; and immunochemical staining was employed to determine the differentiation of BMSCs. Our results showed that striatal extracts could induce differentiation of BMSCs into both neurons and glia, especially the DA neurons. When transplanted to the rat striatum, GFP-BMSCs could differentiate into tyrosine hydroxylase positive neurons and demonstrate potential migration in the brain. Taking together, our results suggest that striatal extracts can specifically promote the dopaminergic differentiation of GFP-BMSCs, thereby providing a feasible strategy for the treatment of Parkinson's disease.

Hyperthermia induces upregulation of connexin43 in the golden hamster neural tube.

BACKGROUND: During early embryonic development, maternal exposure to hyperthermia induces neural tube defects (NTDs). Connexins are essential for the formation of gap junctions and Connexin43 (Cx43) is crucially involved in neural tube development. This study was designed to explore the potential role of Cx43 in NTDs induced by hyperthermia. METHODS: Using PCR, the Cx43 cDNA was screened from the cDNA library of the neural tube from golden hamsters treated with hyperthermia. By Northern blot, the expression of Cx43 in heat-treated and control groups of the golden hamsters at day 8.5 after mating was detected. Finally, by in situ hybridization and RT-PCR, the expression of Cx43 was examined in the neural tube at different time points after heat treatment. RESULTS: Cx43 was stably expressed in heat-treated and control groups of the golden hamsters, whereas the expression was evidently higher in the heat-treated group. Cx43 expression in the neural tube at different time points after heat treatment was significantly higher than in control groups (p < 0.01). Hyperthermia did not induce any mutations in Cx43 cDNA. CONCLUSIONS: Our data provide the first evidence that hyperthermia induces upregulation of Cx43 in the golden hamster neural tube. NTDs caused by hyperthermia may be intimately related with the overexpression of Cx43.

Survival and differentiation of neuroepithelial stem cells on chitosan bicomponent fibers.

Chitosan is a popular biomaterial used in tissue engineering. Fibers of chitosan could provide a favorable anatomical substrate for cell growth which provides a promising application for axonal regeneration during peripheral injury. Neuroepithelial stem cells (NEPs) are the most primitive neural stem cells with multipotential for neuronal and glia differentiation. To assess the biocompatibility between NEPs and chitosan fibers, and to explore whether the NEPs have the ability to differentiation on chitosan fibers, NEPs were harvested from the neural tube and seeded on chitosan fibers in in vitro culture. The biocompatibility of chitosan fibers was tested by MTT assays. The growth and survival were observed by light and scanning electronic microscope at different times in culture. And, the differentiation of NEPs was examined by immunocytochemical staining. The results indicated that NEPs could grow on the chitosan fibers and attach firmly to the surface of fibers. On chitosan fibers, NEPs could differentiate into neurons and glia. Our study demonstrated that chitasan fibers had a good biocompatibility with NEPs which affords a potential alternative for the repair of peripheral nerve injury.

Rpl30 and Hmgb1 are required for neurulation in golden hamster.

Neural tube defects (NTDs) are a group of severe congenital malformations resulting from the failure of neurulation. Genes influencing neurulation have been investigated for their contribution to NTDs. Ribosomal protein (Rp) is an abundant and belongs to a high conservative gene family, which has the complex task of coordinating protein biosynthesis in order to maintain cell homeostasis and survival. However, the mechanisms of Rp in the NTDs are unknown. Understanding the mechanisms will lead to new insights into NTDs. In this report, we constructed a cDNA library from neural tube of golden hamster and screened the cDNA library by a subsection screening method (SSS). Our results demonstrate a possible essential role of the RPL30 cDNA gene during neurulation and in the risk of NTDs. Our study also suggests that another gene, HMGB1, may be significantly associated with neurulation and the risk of NTDs.

Insulin-producing cells derived from rat bone marrow and their autologous transplantation in the duodenal wall for treating diabetes.

Islet cell transplantation is one of the most promising therapies for diabetes mellitus (DM). However, the limited availability of purified islets for transplantation and the risk of immunological rejection severely limit its use. In vitro transdifferentiation of autologous bone marrow-derived mesenchymal stem cells (BMSCs) into insulin-producing cells (IPCs) could provide an abundant source of cells for this procedure and avoid immunological rejection. Here, we isolated and characterized BMSCs and induced their in vitro differentiation into IPCs. Reverse-transcription polymerase chain reaction analysis revealed that these IPCs could express Ins1, Ins2, glucagon, glucose transporter 2, and pancreatic duodenal homeobox-1. Insulin production by the IPCs was confirmed by immunocytochemistry and Western blot analysis. On this basis, donor rats supplying BMSCs were made diabetic by a single intraperitoneal injection of streptozotocin. The IPCs were then autologously transplanted into the duodenal submucosa of diabetic rats. Grafted cells could be visualized in sections after 2, 4, and 8 weeks by immunohistochemical staining for insulin. Furthermore, in the IPC-implanted group, hyperglycemia was normalized, compared with a persistent increase in glucose levels in the diabetic group and intraperitoneal glucose tolerance test-induced responses were observed in the IPC-implanted group. These results on autologous transplantation of IPCs derived from BMSCs into the duodenal wall could offer a novel potential therapeutical protocol for DM.

Interstitial cells of Cajal reduce in number in recto-sigmoid Hirschsprung's disease and total colonic aganglionosis.

Interstitial cells of Cajal (ICCs) play a key role in regulating gastrointestinal tract motility. The pathophysiological basis of colonic aperistalsis in Hirschsprung's disease (HD) is still not fully understood. Many studies reported that decreased numbers or disrupted networks of ICCs were associated with HD. Little information is available on the distribution of different subtypes of ICCs in HD. The aim of this study was to determine the alterations in density of different subtypes of ICC in colonic specimens of patients with total colonic and recto-sigmoid HD. Full thickness colonic specimens were obtained from five children with total colonic aganglionosis (TCA), sixteen with recto-sigmoid HD and seven controls. ICCs were visualized in frozen sections by c-Kit (CD117) fluorescent staining. In the control colon, c-Kit positive ICCs formed a dense network surrounding the myenteric plexus (IC-MY), along the submucosal surface of the circular muscle layer (IC-SM) and in the circular and longitudinal muscle layer (IC-IM). In the aganglionic region of the colon of the patients affected by HD, the number of ICCs (especially IC-IM and IC-SM) was markedly reduced and IC-MY networks were disrupted. Nearly total lack of three subtypes of ICCs was observed in the TCA specimens. This study demonstrated the altered distribution of different subtypes of ICCs in the resected colon of patients with recto-sigmoid HD and TCA. These findings suggest that the reduction of each subtype of ICCs may play an important role in the etiology of HD.

Survival and differentiation of neuroepithelial stem cells following transplantation into the lateral ventricle of rats.

Neuroepithelial stem cells (NEPs) demonstrate a high potential for self-renewal and differentiation during embryonic development. To explore the survival and differentiation of NEPs in vivo, we isolated NEPs from green fluorescence protein (GFP) transgenic embryos and transplanted into the lateral ventricle of rats. In vitro culture, NEPs proliferated into neurospheres and differentiated into both neurons and glia. When transplanted into the lateral ventricle of rats, these GFP positive NEPs (GFP+ NEPs) survived and attached to the wall of ventricle. Moreover, grafted cells differentiated into neuron-specific enolase (NSE) positive neurons and glial fibrillary acidic protein (GFAP) positive astrocytes and migrated into the host brain. Thus, our results indicate that NEPs can survive and differentiate into neurons and astrocytes in the lateral ventricle following transplantation.

Hematopoietic potential of mouse placenta with the application of placenta flushing.

Several major vascular tissues, such as the aorta-gonad-mesonephros region (AGM), yolk sac, and fetal liver have been confirmed to possess hematopoietic function. Recently, the placenta has been demonstrated as another hematopoietic organ. However, it is not conclusive whether the placenta possesses hematopoietic ability. Therefore, we undertook a series of experiments to study the hematopoietic functions of placenta. Fetal blood circulation in the placenta is difficult to be eliminated and its interference in the study of placental hematopoiesis is inevitable. With the application of placental flushing, fetal blood contained in the placenta was eliminated. We then made the further study of placental hematopoiesis after the E12.5 placenta was flushed. Our studies showed that placental cells expressing Sca-1, CD117 and CD34 were mainly restricted to the embryonic vessels of E12.5 placenta. The results of fluorescence activated cell sorter (FACs) analysis and colony forming cells (CFC) assay demonstrated that both placenta and placental blood contained hematopoietic stem/progenitor cells (HS/PCs), including CFU-GMs, CFU-GEMMs, BFU-Es, and HPP-CFCs. The frequency of HS/PCs in the placenta was 2-3 times that of placental blood. Therefore, it is necessary to clear placental blood out of the placenta in the studies of the hematopoietic potential of placenta. The placenta still possessed the hematopoietic potential after the fetal blood is flushed out. These observations provide further evidences that the placenta is a hematopoietic organ, as has been proposed for other embryonic hematopoietic sites.

Hematopoietic potential of mouse placenta with the application of placenta flushing

Several major vascular tissues, such as the aorta-gonad-mesonephros región (AGM), yolk sac, and fetal liver have been confirmed to possess hematopoietic function. Recently, the placenta has been demonstrated as another hematopoietic organ. However, it is not conclusive whether the placenta possesses hematopoietic ability. Therefore, we undertook a series of experiments to study the hematopoietic functions of placenta. Fetal blood circulation in the placenta is difficult to be eliminated and its interference in the study of placental hematopoiesis is inevitable. With the application of placental flushing, fetal blood contained in the placenta was eliminated. We then made the further study of placental hematopoiesis after the El2.5 placenta was flushed. Our studies showed that placental cells expressing Sca-1, CD117 and CD34 were mainly restricted to the embryonic vessels of E12.5 placenta. The results of fluorescence activated cell sorter (FACs) analysis and colony forming cells (CFC) assay demonstrated that both placenta and placental blood contained hematopoietic stem/progenitor cells (HS/PCs), including CFU-GMs, CFU-GEMMs, BFU-Es, and HPP-CFCs. The frequency of HS/PCs in the placenta was 2-3 times that of placental blood. Therefore, it is necessary to clear placental blood out of the placenta in the studies of the hematopoietic potential of placenta. The placenta still possessed the hematopoietic potential after the fetal blood is flushed out. These observations provide further evidences that the placenta is a hematopoietic organ, as has been proposed for other embryonic hematopoietic sites.

Effects of Buyang Huanwu Decoction on neurite outgrowth and differentiation of neuroepithelial stem cells.

To determine the effects of Buyang Huanwu Decoction (BYHWD), a traditional Chinese medicine, on neurite outgrowth and differentiation of neuroepithelial stem cells (NEPs), NEPs were isolated from embryonic neural tube and cultured in medium with rat serum containing BYHWD, which was prepared from rats administrated orally with BYHWD. The average neurite length of NEPs grew significantly longer in rat serum containing BYHWD than in control serum without BYHWD. More neurofilament (NF) positive cells and glial fibrillary acidic protein (GFAP) positive cells were detected in NEPs cultured in the presence of BYHWD. Besides, when cultured NEPs were loaded with Fluo-3-AM, the fluorescence intensity obtained from NEPs cultured in serum with BYHWD was significantly lower than that from NEPs cultured in control serum without BYHWD. Our results indicate that BYHWD could exert a promotion effect on neurite outgrowth and differentiation of NEPs.

Modulation of Dishevelled and Vangl2 by all-trans-retinoic acid in the developing mouse central nervous system and its relationship to teratogenesis.

The response to exposure to all-trans-retinoic acid (RA) during embryogenesis varies from physiologic to severe teratogenic effects and is dependent upon the dose and the stage of development in all species. Vangl2 and Dishevelled genes play key roles in establishing planar cell polarity and regulating convergent extension movements during the neurula period. The effects of RA-mediated teratogenesis might be due to its misregulation of Vangl2 and Dishevelled genes. The aim of this study is to monitor the modulation of Vangl2 and Dishevelled in Kunming mouse embryos following maternal treatment with a single oral dose of 30 mg/(kg body weight) of RA during the neurula period. Exposure of 7.75 d embryos to RA induced characteristic morphological changes. The most obvious external effect was the failure of neural tube closure in the midbrain and forebrain regions in 10 d embryos, resulting in exencephaly in later embryos. RA treatment also led to a pronounced decrease of Vangl2 mRNA at 4 and 18 h and a pronounced increase at 66 h after maternal treatment, as detected by reverse transcription-polymerase chain reaction. Western blot analysis showed a marked decrease of Vangl2 protein at 18 and 42 h and a marked increase at 66 and 90 h after maternal treatment. Dishevelled1/2/3 mRNA was significantly down-regulated at 4 and 18 h and up-regulated at 42 h in the fetus after RA treatment, except for an up-regulation of Dishevelled3 at 66 h. The Dishevelled2 mRNA and its protein matched each other. These results hinted that Vangl2 and Dishevelled genes might take part in RA teratogenesis of mouse embryos.

Hyperglycemic condition disturbs the proliferation and cell death of neural progenitors in mouse embryonic spinal cord.

Spina bifida, which results from failure of fusion in the spinal region of neural tube, is among the most common birth defects associated with diabetic pregnancy. However, the mechanism underlying maternal diabetes-induced congenital malformations including spina bifida is not fully understood. It was hypothesized that hyperglycemic conditions affect the proliferation and apoptosis of neural progenitor cells in the developing spinal neural tube, leading to abnormal neurodevelopment. In the present study, biological processes such as proliferation and apoptosis were investigated in the neuroepithelial cells of the developing spinal neural tube of embryos from diabetic mice, and in embryonic spinal neural tube derived neural progenitor cell cultures exposed to high glucose in vitro. Maternal diabetes caused decreased proliferation and increased apoptosis of the neuroepithelial cells in the developing spinal cord of embryos from diabetic mouse. Decreased proliferation and increased apoptosis were also found in neural progenitor cells exposed to high glucose. In addition, high glucose-induced apoptosis in neural progenitor cells was associated with activation of caspase-3. Thus, high glucose disturbs both proliferation and cell death of neural progenitors in the developing spinal neural tube. This could provide a cellular mechanism by which maternal hyperglycemia induces spina bifida in embryos from diabetic pregnancy.

Dopaminergic differentiation of grafted GFP transgenic neuroepithelial stem cells in the brain of a rat model of Parkinson's disease.

Neuroepithelial stem cells (NEPs) possess multipotent potential for self-renewal and neuronal differentiation. Using green fluorescent protein (GFP) positive NEPs, we explored, firstly, the survival and differentiation of grafted NEPs in the host rat and, secondly, whether or not transplantation of NEPs is a feasible therapeutic option for treating Parkinson's disease. NEPs were harvested from the neural tube of enhanced GFP transgenic embryos. In culture, GFP(+) NEPs generated abundant neurospheres and differentiated into both neurons and glia. When stereotaxically transplanted into the 6-hydroxydopamine (6-OHDA)-lesioned striatum of rats, NEPs survived and tyrosine hydroxylase (TH)-positive cells were detected in the graft. Furthermore, these grafted GFP(+) NEPs significantly ameliorated Parkinsonian behavioral symptoms compared with controls which were treated only with normal saline. Our results suggest that transplanted NEPs accomplish dopaminergic differentiation may be used for treating Parkinson's disease.

[Cloning and characterization of down-regulated genes in neural tube defects of golden hamster induced by hyperthermia].

OBJECTIVE: To clone down-regulated genes in neural tube defects of golden hamster induced by hyperthermia and to explore the molecular mechanism. METHODS: A reversed subtractive cDNA library was constructed using suppression subtractive hybridization. Clones with inserts were selected by combination of alpha complementary phenomenon and colony PCR. Then sequence and homology analysis of the inserts were made. And mRNA expression conditions were confirmed by Northern blot. RESULTS: The reversed subtractive cDNA library was successfully constructed. Fifteen recombinant clones were sequenced and recognized homologous to known genes. The results of Northern blot showed that all these genes were down-regulated in defected neural tubes induced by hyperthermia compared to normal developed neural tubes. CONCLUSION: Some important genes are identified which might be involved in neural tube defects induced by hyperthermia.

Neural-tube-derived neuroepithelial stem cells: a new transplant resource for Parkinson's disease.

To assess the feasibility of using neuroepithelial stem cells as a transplant source for Parkinson's disease, neuroepithelial cells were harvested from the neural tube, cultured and stereotactically transplanted into the striatum of a rat model of Parkinson's disease. In culture, neuroepithelial cells generated abundant neurospheres and differentiated into both neurons and glia. After transplantation, tyrosine-hydroxylase-positive cells were detected in the graft. Furthermore, an apomorphine-induced rotation test showed that the implanted cells successfully promoted functional recovery in animals that underwent this transplantation procedure. Our results demonstrate that neuroepithelial cells may be a new source of donor material for Parkinson's disease.

[Establishment and characterization of human extrahepatic growing hepatocellular carcinoma cell line EGHC-9901].

OBJECTIVE: To establish a new extrahepatic growing hepatocellular carcinoma cell line. METHODS: A specimen from extrahepatic growing hepatocellular carcinoma was cultured in vitro. Cancer cells were studied morphologically and subjected to karyotype analysis, DNA analysis, and tumor formation evaluation. RESULTS: Morphological observation and functional analysis showed that their features were similar to those of HCC. Chromosomes with a variation of 76 approximately 104 were able to secret AFP in vitro and to form bile canaliculi with microvilli. CONCLUSION: EGHC-9901 cell line has characteristics of the extrahepatic growing hepatocellular carcinoma.