OBJECTIVETo obtain recombinant nestin and prepare anti-nestin polyclonal antibody (mAb) to explore the biological roles of nestin in the central nervous system development.

METHODSThe nestin cDNA was cloned from human neural stem cells by RT-PCR and ligated to prokaryotic expression plasmid pQE30 for construction of the recombinant vector pQE30-nestin. After sequencing, the recombinant vector was transformed into E.coli M15 and His-tagged nestin was induced by IPTG. The nestin was purified by Ni-NTA affinity chromatography column and characterized by SDS-PAGE and Western blotting. BALB/c mice were immunized with the purified recombinant protein to prepare the antiserum, which was analyzed by Western blotting, enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry.

RESULTSThe nestin gene was successfully cloned from human neural stem cells, which was identical to that reported in GenBank. After IPTG induction, the E.coli transformed with pQE30-nestin plasmid expressed a 25,000 His-tagged protein, which was successfully purified and identified as nestin by Western blotting. Western blotting, ELISA and immunohistochemistry demonstrated that the antiserum could specifically bind to the recombinant nestin as well as to nestin in fetal human and rat brains.

CONCLUSIONWe successfully cloned the nestin gene and expressed the nestin, and nestin mAb prepared can specifically recognize not only the recombinant nestin, but also nestin from human and rats brain tissues.

Adult Stem Cells ; cytology ; metabolism ; Animals ; Antibodies, Monoclonal ; immunology ; isolation & purification ; Blotting, Western ; Cloning, Molecular ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Gene Expression ; Humans ; Immune Sera ; immunology ; Immunohistochemistry ; Intermediate Filament Proteins ; biosynthesis ; genetics ; immunology ; Mice ; Mice, Inbred BALB C ; Nerve Tissue Proteins ; biosynthesis ; genetics ; immunology ; Nervous System ; cytology ; metabolism ; Nestin ; Recombinant Proteins ; biosynthesis ; immunology ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo study the expression and significance of nestin (a type of cytoskeletal protein) in normal and diseased human kidneys.

METHODSDiseased kidney tissues were obtained from needle biopsies in 32 patients with glomerulonephritis (including 8 cases of membranous glomerulopathy, 3 cases of focal segmental glomerulosclerosis, 17 cases of IgA nephropathy with proteinuria and 4 cases of IgA nephropathy without proteinuria). Control kidney tissues were obtained from nephrectomy specimens for renal tumors. The expression of nestin in the control kidney tissues was studied using immunoelectronic microscopy and immunohistochemistry. The expression of nestin in the diseased kidney tissues was detected by immunohistochemistry and real-time reverse transcription-polymerase chain reaction.

RESULTSIn normal kidney tissues, nestin was detected at the periphery of glomerular capillary loops. Semi-quantitative morphometric analysis showed that the glomerular nestin expression level in cases of IgA nephropathy without proteinuria did not differ from that in the normal controls. However, the glomerular nestin expression levels in cases of IgA nephropathy with proteinuria, membranous glomerulopathy and focal segmental glomerulosclerosis were significantly lower than those in the normal kidneys and IgA nephropathy without proteinuria. The glomerular nestin expression levels inversely correlated with the 24-hour urine protein results.

CONCLUSIONNestin may play an important role in maintaining the normal function of podocytes in human kidney.

Adult ; Gene Expression Regulation ; Glomerulonephritis, IGA ; immunology ; Humans ; Immunohistochemistry ; Intermediate Filament Proteins ; genetics ; metabolism ; Kidney ; Kidney Diseases ; metabolism ; pathology ; Kidney Glomerulus ; metabolism ; pathology ; Middle Aged ; Nephrectomy ; adverse effects ; Nerve Tissue Proteins ; genetics ; metabolism ; Nestin ; Proteinuria ; metabolism

There have been many attempts for regeneration of peripheral nerve injury. In this study, we examined the in vivo effects of non-differentiated and neuronal differentiated adipose-derived stem cells (ADSCs) in inducing the neuronal regeneration in the Sprague-Dawley (SD) rats undergoing nerve defect bridged with the PCL nanotubes. Then, we performed immunohistochemical and histopathologic examinations, as well as the electromyography, in three groups: the control group (14 sciatic nerves transplanted with the PCL nanotube scaffold), the experimental group I (14 sciatic nerves with the non-differentiated ADSCs at a density of 7x105 cells/0.1 mL) and the experimental group II (14 sciatic nerves with the neuronal differentiated ADSCs at 7x105 cells/0.1 mL). Six weeks postoperatively, the degree of the neuronal induction and that of immunoreactivity to nestin, MAP-2 and GFAP was significantly higher in the experimental group I and II as compared with the control group. In addition, the nerve conduction velocity (NCV) was significantly higher in the experimental group I and II as compared with the control group (P=0.021 and P=0.020, respectively). On the other hand, there was no significant difference in the NCV between the two experimental groups (P>0.05). Thus, our results will contribute to treating patients with peripheral nerve defects using PCL nanotubes with ADSCs.
Adipose Tissue/cytology ; Animals ; Cell Differentiation ; Electromyography ; Male ; Nanotubes ; *Nerve Regeneration ; Nerve Tissue Proteins/immunology ; Nestin/immunology ; Neural Conduction/physiology ; Peripheral Nerve Injuries/*surgery ; Phosphoprotein Phosphatases/immunology ; Polyesters/*therapeutic use ; Rats ; Rats, Sprague-Dawley ; Sciatic Nerve/injuries/surgery ; Stem Cell Transplantation/*methods ; Stem Cells/*cytology ; Tissue Engineering/methods

OBJECTIVETo investigate the isolation and expansion of mesenchymal stem cells (MSCs) from human umbilical cord Wharton's jelly and their biological identities, and explore the possibility of inducing human umbilical cord-derived MSCs to differentiate into neurocyte-like cells.

METHODSThe growth and proliferative abilities of human umbilical cord-derived MSCs were observed, and their immunophenotypes were determined by flow cytometry. Salvia miltiorrhiza and beta-sulfhydryl alcohol were adopted to induce the cells to differentiate. The differentiated and undifferentiated cells were identified with immunocytochemistry. The pleiotrophin and nestin genes were measured by RT-PCR.

RESULTSA population of human umbilical cord-derived MSCs were isolated from human umbilical Wharton's jelly; they were processed to obtain a fibroblast-like population of cells and could be maintained in vitro for extended periods with stable population doubling, and they were expanded as undifferentiated cells in culture for more than 10 passages, indicating their proliferative capacity. The human umbilical cord-derived MSCs were positive for CD(29), CD(44), CD(59), CD(105), but negative or weakly expressed the markers of hematopoietic cells such as CD(14), CD(33), CD(34), CD(28), CD(45) and CD(117). The important GVHD correlation markers were negative or weakly expressed, including CD(80) (B7-1), CD(86) (B7-2), CD(40) and CD(40L). Salvia miltiorrhiza beta-sulfhydryl alcohol could induce the MSCs to express nestin, a marker of neuronal precursor stem cells at early stage of differentiation. Later, they exhibited neural phenotypes, expressing beta-tubulin III and neurofilament (NF) and glial fibrillary acidic protein (GFAP). It was confirmed by RT-PCR that the MSCs could express pleiotrophin either before or after the induction of salvia miltiorrhiza, furthermore, after the induction the expression was markedly enhanced and the nestin gene was also expressed.

CONCLUSIONThe human MSCs could be isolated from human umbilical cord Wharton's jelly, and it was easy to propagate these MSCs. The negative GVHD correlated markers might result from the fact that MSCs had no HLA barrier, which may suggest potential clinical significance. The MSCs are capable of differentiating into neurocyte-like cells and they may represent an alternative stem cell source for CNS cells transplantation.

Antigens, CD ; immunology ; Carrier Proteins ; genetics ; Cell Differentiation ; physiology ; Cells, Cultured ; Cytokines ; genetics ; Female ; Flow Cytometry ; Glial Fibrillary Acidic Protein ; metabolism ; Humans ; Immunohistochemistry ; Infant, Newborn ; Intermediate Filament Proteins ; genetics ; Male ; Mesenchymal Stromal Cells ; immunology ; metabolism ; physiology ; Nerve Tissue Proteins ; genetics ; Nestin ; Neurofilament Proteins ; metabolism ; Neurons ; metabolism ; physiology ; Pregnancy ; Reverse Transcriptase Polymerase Chain Reaction ; Tubulin ; metabolism ; Umbilical Cord ; cytology