OBJECTIVETo study proteins correlated with the mechanical properties of engineered cartilage by screening significantly changed proteins during cartilage formation by comparative proteomic analysis.

METHODSHuman chondrocyte, cultured and expanded, were seeded onto a polyglycolic acid/polylactic acid (PGA/PLA) scaffolds. After 4 weeks of culture in vitro, the constructs were divided into three groups. There were 6 specimens in each group. For the regular in vitro culture group (A), the constructs were kept in culture at the original condition for an additional 6 weeks. For in vivo groups, the constructs were implanted subcutaneously into nude mice for either 6 weeks (B) or 12 weeks (C). All specimens were harvested for gross observation, average wet weight and volume measurement, histology, immunohistochemistry and biomechanics to evaluate the results. Meanwhile, comparative proteomic analysis was performed for each group, and those proteins involved in extracellular matrix with at least 2 folds up-regulation were chosen for further exploration. The correlations between Young's modulus and the relative content of the selected proteins were analyzed by Pearson correlation coefficient.

RESULTSAll these samples in the three groups eventually formed hyaline-like cartilage structure. Specimens in C and B groups were similar with adult articular cartilage in appearance, and had multiple mature lacuna in histology. However, those specimens in A group had loose texture with irregular hypertrophy lacuna. Specimens implanted for 12 weeks in vivo had better wet weight (372.5 +/- 35.4) mg and Young's modulus (8.68 +/- 2.65) MPa than those cultured in vivo for 6 weeks (346 +/- 34.5) mg, (3.25 +/- 1.24) MPa (P < 0.01). In group A, they were (184.4 +/- 12.28) mg and (0.7 +/- 0.23) MPa. This study had detected 44 proteins in ECM by comparative proteomic analysis, then chosing the greatest ratio of 6 up-regulation proteins compared between C and A groups. The correlation results indicated the content of Decorin, Chondroadherin and Fibromodulin were linear correlation with the mechanical properties of engineered cartilage (P < 0.05).

CONCLUSIONSComparative proteomic analysis could provide large scale information of associated proteins, making it profit for advanced research on the relationship between extracellular matrix and mechanical properties of engineered cartilage by combination with tissue reconstruction techniques.

Animals ; Cartilage ; cytology ; metabolism ; physiology ; Cells, Cultured ; Chondrocytes ; cytology ; metabolism ; Fetus ; cytology ; Humans ; Mice, Nude ; Proteome ; metabolism ; Proteomics ; Tissue Engineering ; methods ; Tissue Scaffolds

Alzheimer's disease (AD) is the most common cause of age-related dementia. The neuropathological hallmarks of AD include extracellular deposition of amyloid-beta peptides and neurofibrillary tangles that lead to intracellular hyperphosphorylated tau in the brain. Soluble amyloid-beta oligomers are the primary pathogenic factor leading to cognitive impairment in AD. Neural stem cells (NSCs) are able to self-renew and give rise to multiple neural cell lineages in both developing and adult central nervous systems. To explore the relationship between AD-related pathology and the behaviors of NSCs that enable neuroregeneration, a number of studies have used animal and in vitro models to investigate the role of amyloid-beta on NSCs derived from various brain regions at different developmental stages. However, the Abeta effects on NSCs remain poorly understood because of conflicting results. To investigate the effects of amyloid-beta oligomers on human NSCs, we established amyloid precursor protein Swedish mutant-expressing cells and identified cell-derived amyloid-beta oligomers in the culture media. Human NSCs were isolated from an aborted fetal telencephalon at 13 weeks of gestation and expanded in culture as neurospheres. Human NSCs exposure to cell-derived amyloid-beta oligomers decreased dividing potential resulting from senescence through telomere attrition, impaired neurogenesis and promoted gliogenesis, and attenuated mobility. These amyloid-beta oligomers modulated the proliferation, differentiation and migration patterns of human NSCs via a glycogen synthase kinase-3beta-mediated signaling pathway. These findings contribute to the development of human NSC-based therapy for AD by elucidating the effects of Abeta oligomers on human NSCs.
Amyloid beta-Peptides/*pharmacology ; Animals ; Apoptosis ; Cell Aging ; Cell Movement ; Cell Proliferation ; Culture Media, Conditioned/chemistry/pharmacology ; Fetus/cytology ; Glycogen Synthase Kinase 3/*metabolism ; HEK293 Cells ; Humans ; Mice ; Mice, Inbred C57BL ; Neural Stem Cells/*drug effects/metabolism/physiology ; Signal Transduction ; Telomere Shortening

OBJECTIVETo establish a method for organotypic slice culture of neonatal rat cortex in a modified condition and investigate the effect of spatial signals on neural stem cell (NSC) differentiation.

METHODSThe brain slices (200 µm in thickness) of neonatal SD rats (3 to 5 days old) were prepared and cultured in modified serum-free DMEM/F12 medium at 37 degrees celsius; with 95% O(2) and 5% CO(2). The organotypic slice cultures were observed regularly. NSCs isolated from the cortex of rat embryos (14-15 embryonic days) were cultured in serum-free DMEM/F12 supplemented with B27 and N2, and the passage 3 NSCs were labeled by CM-DiI before transplanted onto the organotypic slices cultured for 2 weeks. The survival of transplanted NSCs was assessed, and the cell differentiation was identified by immunofluorescence staining.

RESULTSThe organotypic slice cultures were well maintained for at least 4 weeks in the modified medium. The thickness of the organotypic slices reduced from 200 µm to 130 µm after 2-week culture in vitro due to the migration of the cells on the edge of the slices. CM-DiI-labeled NSCs survived well and differentiated into GFAP(+) glia and β-tubullin III(+) neurons.

CONCLUSIONNeonatal rat organotypic brain slice can be successfully cultured in a modified condition to serve as a model for studying NSC differentiation induced by spatial signals.

Animals ; Animals, Newborn ; Cell Differentiation ; physiology ; Cerebral Cortex ; cytology ; Coculture Techniques ; methods ; Fetus ; Neural Stem Cells ; cytology ; transplantation ; Organ Culture Techniques ; methods ; Rats

OBJECTIVETo explore the effect of Ruiqi tablet on mitochondrion activities in the cerebral cortex neurons of fetal mice.

METHODThe cerebral cortex of fetal Wistar rats after 16- 17 gestation days were collected and randomized into Ruiqi tablet group and blank control group after 4-5 days' culture. Laser scanning confocal microscope was adopted to determine the changes in the mitochondrion activities of the primary cultured cerebral cortex neurons of fetal rats after addition of Ruiqi tablet solution.

RESULTRuiqi tablet can increase the mitochondrion activities of the cerebral cortex neurons. No significant change in the mitochondrion activities of the cerebral cortex neurons was found in the blank control group.

CONCLUSIONRuiqi tablet can increase the mitochondrion activities of the neurons and it has certain application prospects in treatment of some neurodegenerative diseases such as Alzheimer's disease, vascular dementia, Parkinson's disease and so on.

Animals ; Cerebral Cortex ; cytology ; drug effects ; physiopathology ; Drugs, Chinese Herbal ; pharmacology ; Female ; Fetus ; chemistry ; drug effects ; physiopathology ; Humans ; Microscopy, Confocal ; Mitochondria ; drug effects ; physiology ; Models, Animal ; Neurons ; drug effects ; physiology ; Random Allocation ; Rats ; Rats, Wistar ; Tablets

OBJECTIVETo investigate the roles of human fetal liver stromal cells (hFLSCs) and human fetal bone marrow stromal cells (hFBMSCs) in the hematopoietic differentiation of human embryonic stem cells and analyze their gene expression profile changes.

METHODSThe embryonic bodies on day 4 were cocultured with hFLSCs or hFBMSC in the presence of cytokines. Flow cytometry was performed after 8 days of induction to detect the expressions of the hemangioblast markers KDR and CD34, and the differential gene expression profiles between hFBMSC and hFLSCs were examined by cDNA microarray analysis.

RESULTSEight days after the induction, (1.06-/+0.20)% of the hFLSCs and (8.8-/+1.49)% of the hFBMSCs were positive for KDR, with the positivity rates for CD34 of (1.25-/+0.16)% and (9.17-/+2.10)%, respectively. In hFLSCs and hFBMSCs cultures, 0.9-/+0.36 and 10.6-/+0.63 hemagioblast-like cell colonies were found, respectively. cDNA microarray analysis showed that 240 genes were highly expressed in hFBMSCs, and 21 genes related to secreted cytokines, cell adhesion molecules and extracellular matrix proteins were highly expressed.

CONCLUSIONThe microenvironment including the cell matrix protein and cytokines secreted by the hFBMSCs might play an important role in hemangioblastic differentiation of human bone marrow stromal cells in vitro.

Antigens, CD34 ; genetics ; metabolism ; Bone Marrow Cells ; cytology ; Cell Differentiation ; physiology ; Coculture Techniques ; Embryonic Stem Cells ; cytology ; Fetus ; Gene Expression Profiling ; Hematopoietic Stem Cells ; cytology ; Humans ; Liver ; cytology ; Oligonucleotide Array Sequence Analysis ; Stromal Cells ; physiology ; Vascular Endothelial Growth Factor Receptor-2 ; genetics ; metabolism

OBJECTIVETo investigate the effect of fetal bovine serum (FBS) on expression of pigment epithelium-derived factor (PEDF) in normal epidermal keratinocytes and dermal fibroblasts.

METHODSKeratinocytes and fibroblasts were incubated with 10% FBS. PEDF protein level in the cells was determined by immunofluorescence and Western blot.

RESULTSPEDF was localized mostly in the cytoplasm,while some in the nuclei. The distribution of PEDF in cytoplasm was in a granular pattern. 10% FBS increased the expression of PEDF both in keratinocytes and fibroblasts,but histamine and Phorbol 12-myristate 13-acetate (PMA) did not interfere the distribution of PEDF in cells.

CONCLUSION10% FBS can upregulate expression of PEDF in epidermal keratinocytes and dermal fibroblasts.

Animals ; Cattle ; Cells, Cultured ; Epidermis ; cytology ; metabolism ; Eye Proteins ; genetics ; metabolism ; Fetus ; Fibroblasts ; cytology ; metabolism ; Keratinocytes ; cytology ; metabolism ; Nerve Growth Factors ; genetics ; metabolism ; Serpins ; genetics ; metabolism ; Serum ; physiology ; Skin ; cytology ; metabolism ; Up-Regulation

OBJECTIVETo investigate the growth and development of nitric oxide synthase (NOS)-positive neurons in the cerebellum of human fetus in the midanaphase.

METHODThe positive expression of the NOS-positive neurons in the cerebellum of midanaphase human fetus was observed by immunohistochemistry.

RESULTSBy the sixth to seventh month of gestation, NOS-positive neurons were seen in the ependymal layer of the cerebellum. The nucleus was oval-shaped and the neurons had short and small processes. By the eighth to ninth month, NOS-positive neurons were found in the central layer of the cerebellum and the nucleus was round-, oval-, or fusiform-shaped; meanwhile, the neurons grew larger in size with richer cytoplast and heavier staining. The beaded nerve fibers reached the marginal layer and the layer became thickened on the tenth month, which generally was composed of 5 to 6 layers of NOS-positive neurons that were tightly aligned. Some NOS-positive neurons were in smaller size with the cell body and the nerve fibers grew well.

CONCLUSIONNitric oxide generated by NOS of the NOS-positive neurons in the cerebellum plays an important role in the differentiation, proliferation, and migration of neurons and gliacytes.

Cerebellar Cortex ; Fetus ; physiology ; Humans ; Immunohistochemistry ; Nerve Fibers ; Neurons ; cytology ; metabolism ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase ; metabolism ; Nitric Oxide Synthase Type I

OBJECTIVE: To investigate the role of Notch signaling in differentiation of Sprague-Dawley (SD) rat retinal progenitor cells (RPCs). METHODS: RPCs were isolated from 16-day embryonic SD rats and cultured in suspension. RPCs were cultured respectively in media with (treatment group) or without (control group) gamma-secretase inhibitor X which was used to block Notch signaling. Morphological observation and immunocytochemistry were applied at day 14 to determine the cell types and analyze the expression of Notch pathway genes in both groups. RESULTS: Most RPCs expressed Notch1 intracellular domains or its downstream transcriptional factor Hes1. A few expressed bHLH transcriptional factors NeuroD and Mash1. Most auto-differentiated RPCs expressed NeuroD or Mash1, while a few of them expressed Notch1 intracellular domains or Hes1. In the group treated with gamma-secretase inhibitor X, the positive rate of Nestin or GFAP was much lower than that in the control group while the positive rate of beta-tubulin was much higher than that in the control group. The difference in the positive rate of recovering between the two groups was not significant. CONCLUSION In vitro Notch signaling may inhibit retinal stem cells differentiation. Inhibiting Notch signaling in vitro may promote differentiation to neurons and partially inhibit glial differentiation.
Animals ; Basic Helix-Loop-Helix Transcription Factors ; metabolism ; Cell Differentiation ; physiology ; Cells, Cultured ; Female ; Fetus ; Homeodomain Proteins ; metabolism ; Neurons ; cytology ; Rats ; Rats, Sprague-Dawley ; Receptor, Notch1 ; genetics ; metabolism ; Retina ; cytology ; Signal Transduction ; drug effects ; physiology ; Stem Cells ; cytology ; Transcription Factor HES-1

This study was aimed to investigate the biological characteristics of osteoblasts and their hematopoietic supportive function by using human fetal osteoblastic cell line 1.19 (hFOBs) as a model. The pluripotency markers (Oct-4, Rex-1, hTERT) of hFOBs were analyzed by RT-PCR, the multilineage differentiation experiments were conducted in vitro. Flow cytometry (FCM) was used to identify the surface markers of hFOBs, and RT-PCR was used to analyze their hematopoietic cytokine expression in comparison with bone marrow mesenchymal stem cell (BM-MSC). The results showed that hFOBs expressed several ESC pluripotency markers including Oct-4 and Rex-1, except hTERT. Moreover, hFOBs could also undergo multilineage differentiation into the mesodermal lineages of adipocytic cell types in addition to its predetermined pathway, the mature osteoblast. Both hFOBs and BM-MSC expressed CD44, CD73 (SH3), CD105 (SH2) and CD90 (Thy1), and lack expression of CD34, CD45, or HLA-DR surface molecules. In addition, both hFOBs and BM-MSC expressed SCF, IL-6, and SDF-1alpha mRNA, but only hFOBs could express GM-CSF and G-CSF. It is concluded that human fetal osteoblastic cell line 1.19 may provide a good model to study the osteoblastic regulation role in hematopoiesis in vitro.
Cell Differentiation ; physiology ; Cell Line ; Fetus ; Hematopoiesis ; physiology ; Humans ; Mesenchymal Stromal Cells ; cytology ; physiology ; Models, Biological ; Osteoblasts ; cytology ; physiology

Transplanting fetal kidney cells (FKCs) can regenerate kidney. This requires in vitro expansion in cell number to acquire enough cells for transplantation. However, FKCs may change their cellular characteristics during expansion and, thus, may not regenerate kidney tissue upon transplantation. We investigated how cell culture period affects cellular characteristics and in vivo regenerative potential of FKCs. As the passage number increased, cell growth rate and colony forming ability decreased while senescence and apoptosis increased. To examine in vivo regenerative potential, FKCs cultured through different numbers of passages were implanted into the parenchyma of kidneys of immunodeficient mice using fibrin gel for 4 wk. Histological analyses showed passage-dependent kidney tissue regeneration, and the regeneration was better when cells from lower number of passages were implanted. This result shows that in vitro culture of FKCs significantly affects the cell characteristics and in vivo tissue regenerative potential.
Animals ; Apoptosis/physiology ; Cell Aging/physiology ; Cell Culture Techniques ; Cell Proliferation ; Cells, Cultured ; Colony-Forming Units Assay ; Female ; Fetal Tissue Transplantation/methods/physiology ; Fetus/cytology/*physiology ; Kidney/embryology/*physiology ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Rats ; Rats, Sprague-Dawley ; Regeneration/*physiology