BACKGROUNDAdipose tissue-derived stromal cells (ADSCs) can be greatly expanded in vitro, and induced to differentiate into multiple mesenchymal cell types, including osteogenic, chondrogenic, myogenic, and adipogenic cells. This study was designed to investigate the possibility of ADSCs differentiating into neurons.

METHODSAdipose tissue from rats was digested with collagenase, and adherent stromal cells were cultured. A medium containing a low concentration of fetal bovine serum was adopted to induce the cells to differentiate. ADSCs were identified by immunocytochemistry, and semi-quantitative RT-PCR was applied to detect mRNA expression of neurofilament 1 (NF1), nestin, and neuron-specific enolase (NSE).

RESULTSNestin-positive cells were found occasionally among ADSCs. ADSCs were found to express NSE mRNA and nestin mRNA, but not NF1 mRNA. ADSCs could differentiate into neuron-like cells in a medium composed of a low concentration of fetal bovine serum, and these differentiated cells displayed complicated neuron-like morphologies.

CONCLUSIONSThe data support the hypothesis that adipose tissue contains stem cells capable of differentiating into neurons. These stem cells can overcome their mesenchymal commitment, and may represent an alternative autologous stem cell source for CNS cell transplantation.

Adipose Tissue ; cytology ; Animals ; Cell Differentiation ; physiology ; Cells, Cultured ; Immunohistochemistry ; Intermediate Filament Proteins ; analysis ; Nerve Tissue Proteins ; Nestin ; Neurofilament Proteins ; analysis ; Neurons ; cytology ; Phenotype ; Phosphopyruvate Hydratase ; analysis ; Rats

BACKGROUNDMany researchers suggest that adult mammalian central nervous system (CNS) is incapable of completing self-repair or regeneration. And there are accumulating lines of evidence which suggest that endogenous neural stem cells (NSCs) are activated in many pathological conditions, including stroke in the past decades, which might partly account for rehabilitation afterwards. In this study, we investigated whether there was endogenous neural stem cell activation in intracerebral hemorrhagic (ICH) rat brains.

METHODSAfter ICH induction by stereotactical injection of collagenase type VII into globus pallidus, 5-Bromo-2 Deoxyuridine (BrdU) was administered intraperitoneally to label newborn cells. Immunohistochemical method was used to detect Nestin, a marker for neural stem cells, and BrdU.

RESULTSNestin-positive or BrdU-Labeled cells were predominantly located at 2 sites: basal ganglion around hemotoma, ependyma and nearby subventricular zone (SVZ). No positive cells for the 2 markers were found in the 2 sites of normal control group and sham group, as well as in non-leisioned parenchyma, both hippocampi and olfactory bulbs in the 4 groups. Nestin+ cells presented 4 types of morphology, and BrdU+ nucleus were polymorphologic. Positive cell counting around hemotoma showed that at day 2, Nestin+ cells were seen around hemotoma in model group, the number of which increased at day 4, day 7 (P <0.01), peaked at day 14 (P <0.05), and reduced significantly by day 28 (P <0.01).

CONCLUSIONEndogenous neural stem cells were activated in experimental intracerebral hemorrhagic rat brains.

Animals ; Brain ; pathology ; Bromodeoxyuridine ; metabolism ; Cerebral Hemorrhage ; pathology ; Intermediate Filament Proteins ; analysis ; Male ; Nerve Tissue Proteins ; analysis ; Nestin ; Neurons ; pathology ; Rats ; Rats, Sprague-Dawley ; Stem Cells ; pathology

The expression of two intermediate filaments, nestin and vimentin, was studied in spinal cord injury (SCI) to elucidate their roles in the formation of glial scars. Rats were sacrificed 1, 4, and 7 days after induction of compression injury of the spinal cord using an aneurysm clip. The affected spinal cords were studied using antibodies against nestin and vimentin intermediate filaments. One day after spinal cord injury, some clusters of nestin-positive vessels were detected in the center of the injury, but few were seen in other cell types. Vimentin immunostaining was detected in some glial cells in the center and its level of immunoreactivity was enhanced in the ependymal cells of the central canal. On days 4 and 7 after spinal cord injury, astrocytes and some ependymal cells in the central canal were stained positively for nestin and increased expression of nestin was observed in vessels. Vimentin was detected in some macrophages and astrocytes in the lesions. Nestin was co-localized with glial fibrillary acidic protein in some glial cells in SCI. These findings imply that spinal cord cells in adult animals have embryonic capacity, and these cells are activated after injury, which in turn contributes to repair of spinal cord injury through formation of a glial scar.
Animals ; Cicatrix/pathology ; Glial Fibrillary Acidic Protein/analysis ; Immunohistochemistry ; Intermediate Filament Proteins/analysis ; Intermediate Filaments/*physiology ; *Nerve Tissue Proteins ; Neuroglia/*pathology ; Rats ; Rats, Sprague-Dawley ; Spinal Cord Injuries/*pathology ; Vimentin/analysis

Merkel cells are thought to function as slowly adapting mechanoreceptors and are known as targets for sensory nerves. However, the nerve-dependency of Merkel cells remains controversial. In this respect, some investigators have found interregional differences between hairy and glabrous skin and others have shown intraregional differences within denervated rat touch domes. Differences between species have also been reported. This study was performed to determine whether Merkel cells proliferate in vitro in the absence of the systemic factors, blood vessels and the intact nerves in human skin. Suspension organ culture was performed using fetal digits to investigate their in vitro proliferation. Merkel cells and cutaneous nerves were identified using antibodies to cytokeratin 20 and protein gene product 9.5 (PGP 9.5), respectively. Fetal digits of 56-82 day gestational age were cultured in serum free medium in a high O2 (45%) environment. Tissues were harvested before starting culture (D0) and 1,4,7,14, 28d after culture. Merkel cells were observed in the volar pads and dorsal nail matrices at D0. After 28d of suspension organ culture, digits looked healthy structurally and the number of Merkel cells had increased. However, PGP 9.5-immunoreactive nerves were markedly diminished after 1 day of culture and almost disappeared after 4 days. Merkel cell proliferation in vitro suggested that Merkel cell development is probably nerve-independent in human fetal glabrous skin.
Cell Division ; Female ; Human ; Intermediate Filament Proteins/analysis ; Merkel Cells/*physiology ; Organ Culture ; Pregnancy ; Skin/cytology/*embryology/*innervation ; Thiolester Hydrolases/analysis

Animals ; Desmin ; analysis ; genetics ; Female ; Immunohistochemistry ; Intermediate Filament Proteins ; analysis ; genetics ; Kidney Glomerulus ; chemistry ; Nephrosis ; metabolism ; Nerve Tissue Proteins ; analysis ; genetics ; Nestin ; Podocytes ; chemistry ; RNA, Messenger ; analysis ; Rats ; Rats, Inbred WKY ; Vimentin ; analysis ; genetics

OBJECTIVE: To explore the methods of isolation, culture and identification of brain tumor stem cells (BTSCs) in neuroepithelial tumor tissues in vitro, and to study the correlation between BTSCs and the patholorical grades of neuroepithelial tumors. METHODS: Tumor cells from patients undergoing neuroepithelial tumors excision were acutely dissociated, triturated into single cells, and then seeded into serum-free medium. After the primary brain tumor spheres (BTSs) were generated, they were triturated again and passaged in fresh medium. The expression of Nestin and CD133 of BTSs was detected by immunocytochemistry staining, and the expression of CD133 of tumor specimen sections was detected by immunohistochemistry staining . The expression of CD133 of 46 brain tumors and 5 normal brain tissues were analysed by SABC immunohistochemical staining, and the correlation between the expression and pathological grade of the tumors was analysed. RESULTS: BTSCs from neuroepithelial tumors could be isolated and cultured, and could be generated and passaged in vitro. The expression of Nestin and CD133 could be detected in BTSCs. CD133 could be detected in neuroepithelial tumor tissues, but not in normal brain tissues. There was significant difference between the expression of CD133 and the different grades of tumors (P < 0.01), and there was a positive correlation between the expression of CD133 and the histologic grading of tumors (P < 0.01). CONCLUSION A small proportion of stem cells have the ability to self-renew in human neuroepithelial tumors, and there is a positive correlation between the expression of CD133 and histologic grading of tumors.
AC133 Antigen ; Antigens, CD ; analysis ; Brain Neoplasms ; pathology ; Glycoproteins ; analysis ; Humans ; Intermediate Filament Proteins ; analysis ; Neoplasms, Neuroepithelial ; pathology ; Neoplastic Stem Cells ; pathology ; Nerve Tissue Proteins ; analysis ; Nestin ; Peptides ; analysis ; Tumor Cells, Cultured

OBJECTIVETo investigate the clinicopathological features and the immunohistochemical phenotype of perianal Paget's disease (PPD) associated with internal anorectal adenocarcinoma, with emphasis on the histogenesis of Paget's cells.

METHODSThe clinical and pathologic features of three cases of PPD with rectal adenocarcinoma were investigated. Periodic-acid-Schiff (PAS), alcian-blue and mucicarmine staining with and without diastase digestion were performed. The immunohistochemical study was performed on selected sections by a panel of antibodies including carcinoembryonic antigen (CEA), CK7, CK8, CK10/13, CK20 and gross cystic disease fluid protein 15 (GCDFP15).

RESULTSAll three cases occurred in middle to old age male patients complaining of anal bleeding. Digital physical examination revealed ulcerated or cauliflower-like masses in the anus just distal to the dentate line. Perianal skin erythematous patches were found in two cases, and small discrete granules in one case. Histologically, the anorectal neoplasm was either a moderately or poorly differentiated adenocarcinoma. Two types of Paget's cells were noted, namely the classical type characterized by a polygonal shape with vesicular nuclei and abundant pale cytoplasm, and the signet ring type characterized by eccentrically displaced nucleus. Both the rectal adenocarcinoma cells and Paget's cells showed strong positivity for PAS, AB and mucicarmine, which were resistant to the diastase digestion. Immunohistochemically, they were both positive for CEA, CK7, CK8 and CK20, but negative for CK10/13 and GCDFP15.

CONCLUSIONSThe CK20(+)-GCDFP15(-) type Paget's cells in PPD were derived from the direct intraepithelial Pagetoid spread of anorectal adenocarcinomas. PPD was more frequently associated with internal carcinomas than any other type of extramammary Paget's disease. It is recommended that clinicians should carefully examine the anus or rectum in the presence of PPD to ascertain if it is associated with an internal carcinoma.

Aged ; Aged, 80 and over ; Apolipoproteins ; Apolipoproteins D ; Carrier Proteins ; analysis ; Diagnosis, Differential ; Glycoproteins ; analysis ; Humans ; Immunohistochemistry ; Intermediate Filament Proteins ; analysis ; Keratin-20 ; Male ; Membrane Transport Proteins ; Middle Aged ; Paget Disease, Extramammary ; chemistry ; diagnosis ; pathology ; Rectal Neoplasms ; chemistry ; diagnosis ; pathology

OBJECTIVEThe purpose of this study was to culture and identify neural stem cells from mouse embryos in vitro using a modified method and provide a basis for further study of the biology of neural stem cells under hypoxia.

METHODSThe cells were isolated mechanically from the front cortex of fetal Institute of Cancer Research (ICR) mice on embryonic day 14. They were passaged by mechanical dissociation and enzymatic digestion. The neurospheres were identified by immunofluorescent staining of nestin. Cell differentiation was induced by 1% fetal bovine serum and then the cells were identified by immunohistochemistry of β-tubulin III and GFAP.

RESULTSThe cells obtained from the front cortex of fetal ICR mice had the capacity of forming neurospheres which showed nestin immunoreactive positivity. After being induced by 1% fetal bovine serum, the cells were differentiated into β-tubulin III-positive cells and GFAP-positive cells.

CONCLUSIONSUsing mechanical dissociation of primary cells and mechanical dissociation with enzymatic digestion of primary cells, the NSCs from the front cortex of mouse embryos can be obtained.

Animals ; Cell Differentiation ; Cells, Cultured ; Embryo, Mammalian ; cytology ; Female ; Glial Fibrillary Acidic Protein ; analysis ; Intermediate Filament Proteins ; analysis ; Mice ; Mice, Inbred ICR ; Nerve Tissue Proteins ; analysis ; Nestin ; Neural Stem Cells ; chemistry ; cytology ; Tubulin ; analysis

OBJECTIVEThis study investigated the effect of hyperbaric oxygenation (HBO) on neural stem cells (NSCs) and myelin in neonatal rats following hypoxic-ischemic brain damage (HIBD) and aimed to explore the possible mechanism of the protective effect of HBO on HIBD.

METHODSSeven-day-old Sprague-Dawley rat pups were randomly assigned into 4 groups: Normal control, HIBD, hyperbaric air (HBA), and HBO groups (n=30 each). The HIBD model was produced by permanent occlusion of the left common carotid artery and 2 hrs hypoxemia exposure (8% O2 at 37 degrees C). HBA and HBO treatment was administered (2 ATA, once daily for 7 days) in the HBA and HBO groups respectively 1 hr after HIBD. BrdU immunohistochemistry was used to detect the NSCs in the sub-ventricle zone (SVZ) of the lateral ventricle and the dentate gyrus (DG) of the hippocampus. The myelin damage was assessed by myelin basic protein (MBP) immunostaining.

RESULTSThe BrdU-positive cells in the SVZ and the DG of the ischemic hemisphere in the HIBD group were dramatically decreased compared with those of the Normal control group at 3 weeks post-HIBD (P < 0.01). The HBO treatment resulted in an increase of BrdU-positive cells in the DG from 153.7 +/- 37.0 to 193.7 +/- 38.8 (P < 0.05). The nestin expression in the HIBD and HBA groups was reduced compared with that in the Normal control group. There was no difference in the nestin expression between the HBO and the Normal control groups. Hypoxia-ischemia (HI) led to marked myelin damage at 1 week post-HIBD. HBO or HBA treatment alleviated the damage.

CONCLUSIONSThe HBO treatment can result in the proliferation of BrdU-positive cells and alleviate the myelin damage following HIBD in neonatal rats, thereby offering neuroprotectivity against HI insults.

Animals ; Animals, Newborn ; Bromodeoxyuridine ; metabolism ; Female ; Hyperbaric Oxygenation ; Hypoxia-Ischemia, Brain ; metabolism ; pathology ; therapy ; Immunohistochemistry ; Intermediate Filament Proteins ; analysis ; Male ; Mice ; Myelin Basic Protein ; analysis ; Nerve Tissue Proteins ; analysis ; Nestin ; Neurons ; cytology ; Rats ; Rats, Sprague-Dawley ; Stem Cells ; cytology

OBJECTIVEBasic fibral growth factor (bFGF) might have a role in the restoration and regeneration of injured neurons following hypoxic-ischemic brain damage (HIBD), but its mechanism has not been fully elucidated. Nestin is an intermediate filament protein expressed in dividing cells during the early stages of CNS development, but it can be reinduced in adults during regeneration of injured neurons after CNS injury. This study investigated the effect of exogenous bFGF on nestin expression in neonatal rats following hypoxia-ischemia (HI) and to explore the possible mechanism.

METHODSEighty-four 7-day-old SD rats were randomly assigned into a Sham-operation group, a HIBD group and a bFGF intervention group (n=28 each). HIBD was induced by ligation of the left carotid artery along with 8% oxygen exposure in neonatal rats from the latter two groups. The Sham-operation group was not subjected to HI. The bFGF intervention group received an intraperitoneal injection of bFGF daily (4000 U/kg). Each group was randomly subdivided into groups sacrificed immediately, at 3, 12 and 24 hrs and 3, 7 and 14 days after HI (n=4). The expression of nestin in the cerebral cortex, hippocampus and extraventricular zone was examined with immunohistochemical staining and image quantitative analysis.

RESULTSNestin was weakly expressed in the hippocampus and extraventricular zone and not expressed in the cortex in the Sham-operation group. The nestin in the cortex, hippocampus and extraventricular zone was significantly increased after HIBD, peaking at 7 days. bFGF treatment increased the nestin expression in the cortex, hippocampus and extraventricular zone and statistical differences were observed from 1 to 14 days after HI when compared with the untreated HIBD group.

CONCLUSIONSExogenous bFGF can up-regulate the nestin expression in neonatal rats following HIBD. The effects of restoration and regeneration of bFGF on injured neurons may be associated with increased nestin expression in neonatal rats.

Animals ; Animals, Newborn ; Female ; Fibroblast Growth Factor 2 ; pharmacology ; therapeutic use ; Hypoxia-Ischemia, Brain ; drug therapy ; metabolism ; pathology ; Immunohistochemistry ; Intermediate Filament Proteins ; analysis ; Male ; Nerve Tissue Proteins ; analysis ; Nestin ; Rats ; Rats, Sprague-Dawley