Natural lactucopicrin alleviates importin-α3-mediated NF-κB activation in inflammated endothelial cells and improves sepsis in mice.

Lactucopicrin, a bitter sesquiterpene lactone of leafy vegetables, such as chicory, curly escarole, and lettuce, possesses anti-malarial, anti-cancer and analgesic properties. However, it remains unknown whether lactucopicrin could inhibit vascular endothelial nuclear factor-κB (NF-κB) activation, a hallmark of vascular inflammatory diseases including sepsis. In tumor necrosis factor-α-stimulated human or mouse aortic endothelial cells, lactucopicrin dose-dependently inhibited NF-κB activation, and concomitantly repressed both vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1)-mediated monocyte adhesion. The lactucopicrin effect was not due to modulation of inhibitor of NF-κB kinases (IKK) α/ß/γ, inhibitor of NF-κB alpha (IκBα), and NF-κB/p65 DNA binding activity. Instead, lactucopicrin inhibited importin-α3 expression by destabilization of its mRNA, an effect mediating the lactucopicrin effect on NF-κB activity. More importantly, in lipopolysaccharide (LPS)-elicited septic mice, oral gavage with lactucopicrin decreased mortality by 30.5% as compared with the control treatment. This effect was associated with inhibited importin-α3 expression, suppressed NF-κB activation and VCAM-1/ICAM-1 expression, and inhibited leukocyte influx in the vascular endothelium of both lung and aorta. Collectively, our novel data suggest that dietary supplementation with lactucopicrin inhibits endothelial NF-κB activation by down-regulation of importin-α3 and thereby improves sepsis.

Lactucopicrin Inhibits Cytoplasmic Dynein-Mediated NF-κB Activation in Inflammated Macrophages and Alleviates Atherogenesis in Apolipoprotein E-Deficient Mice.

SCOPE: Nuclear factor-κB (NF-κB) activation in macrophages aggravates atherosclerosis. Dietary plant secondary metabolites including sesquiterpene lactone lactucopicrin target multiple organs. This study is focused on the impact of lactucopicrin on NF-κB activation in inflammed macrophages and atherogenesis in a mouse model of atherosclerosis. METHODS AND RESULTS: In LPS-stimulated mouse bone marrow-derived macrophages, lactucopicrin inhibits NF-κB activation, and concomitantly represses the expression of IL-1ß, IL-6, and tumor necrosis factor-alpha. This effect is not due to modulation of the inhibitor of NF-κB kinases (IKK) α/ß/γ and NF-κB inhibitor α, and NF-κB/p65 DNA binding activity. Instead, the lactucopicrin effect is reliant on the inhibition of cytoplasmic dynein-mediated p65 transportation, a prerequisite step for p65 nuclear translocation. In high-fat diet-fed apolipoprotein E-deficient mice, lactucopicrin consumption dose-dependently reduces plaque area, inhibits plaque macrophage accumulation, attenuates plaque macrophage NF-κB activation, and reduces both plaque and serum inflammatory burden. However, lactucopicrin consumption does not affect the levels of serum lipids and anti-inflammatory cytokines (IL-4, IL-10, and transforming growth factor beta). CONCLUSION: Dietary lactucopicrin inhibits atherogenesis in mice likely by its anti-inflammatory property. These findings suggest that dietary supplementation with lactucopicrin is a promising strategy to inhibit atherosclerotic cardiovascular disease.

Protocatechuic Acid Inhibits Vulnerable Atherosclerotic Lesion Progression in Older Apoe-/- Mice.

BACKGROUND: Normalization of arterial inflammation inhibits atherosclerosis. The preventive role for protocatechuic acid (PCA) in early-stage atherosclerosis is well recognized; however, its therapeutic role in late-stage atherosclerosis remains unexplored. OBJECTIVE: We investigated whether PCA inhibits vulnerable atherosclerosis progression by normalizing arterial inflammation. METHODS: Thirty-wk-old male apolipoprotein E-deficient (Apoe-/-) mice with vulnerable atherosclerotic lesions in the brachiocephalic artery were fed the AIN-93G diet alone (control) or supplemented with 0.003% PCA (wt:wt) for 20 wk. Lesion size and composition, IL-1ß, and NF-κB in the brachiocephalic arteries, and serum lipid profiles, oxidative status, and proinflammatory cytokines (e.g., IL-1ß, monocyte chemoattractant protein-1, and serum amyloid A) were measured. Moreover, the effect of PCA on the inflammation response was evaluated in efferocytic macrophages from C57BL/6J mice. RESULTS: Compared with the control treatment, dietary PCA supplementation significantly reduced lesion size (27.5%; P < 0.05) and also improved lesion stability (P < 0.05) as evidenced by increased thin fibrous cap thickness (31.7%) and collagen accumulation (58.3%), reduced necrotic core size (37.6%) and cellular apoptosis (73.9%), reduced macrophage accumulation (45.1%), and increased vascular smooth muscle cell accumulation (51.5%). Moreover, PCA supplementation inhibited IL-1ß expression (53.7%) and NF-κB activation (64.4%) in lesions. However, PCA supplementation did not change serum lipid profiles, total antioxidant capacity, and inflammatory cytokines. In efferocytic macrophages, PCA at 0.5 and 1 µmol/L inhibited Il1b/IL-1ß mRNA (27.2-46.5%) and protein (29.2-49.6%) expression and NF-κB activation (67.0-80.3%) by upregulation of MER proto-oncogene tyrosine kinase (MERTK) and inhibition of mitogen-activated protein kinase 3/1 (MAPK3/1). Strikingly, the similar pattern of the MERTK and MAPK3/1 changes in lesional macrophages of mice after PCA intervention in vivo was recapitulated. CONCLUSION: PCA inhibits vulnerable lesion progression in mice, which might partially be caused by normalization of arterial inflammation by upregulation of MERTK and inhibition of MAPK3/1 in lesional macrophages.

Protocatechuic acid from chicory is bioavailable and undergoes partial glucuronidation and sulfation in healthy humans.

Protocatechuic acid exerts multiple health-promoting effects such as anticancer, anti-atherosclerosis, and neuroprotection in animal models. While protocatechuic acid produced in the lower gastrointestinal tract by microbial catabolism of several flavonoids is bioavailable, the pharmacokinetics of protocatechuic acid has not been evaluated so far in humans following its oral consumption. In this open-label and single-dose pharmacokinetic trial, 16 healthy adults followed a low-phytochemical diet for three days. Next, after overnight fasting, participants consumed 150 g of chicory containing 248 µmol of protocatechuic acid. Blood, urine, and fecal samples were collected before and up to 24 hr after chicory consumption. Protocatechuic acid in the free and glucuronide/sulfate-conjugated forms was almost undetectable in serum, urine, and fecal samples before chicory consumption. Chicory consumption increased the levels of protocatechuic acid and its glucuronide/sulfate conjugates in biological samples. The maximum serum concentrations of protocatechuic acid in the free-, glucuronide-, and sulfate-conjugated forms were 3,273, 519, and 340 nmol/L, respectively. The recovery of total protocatechuic acid in blood circulation, urine, and feces was 23.79%, 12.17%, and 12.79% of the ingested dose, respectively. Moreover, glucuronide and sulfate conjugates of protocatechuic acid made up 34.79%, 60.15%, and 72.70% of its total recovery in blood circulation, urine, and feces, respectively. Collectively, protocatechuic acid from chicory is bioavailable and undergoes partial glucuronidation and sulfation in human adults, and its regular consumption may exert health-promoting effects.

Apoptotic cell induction of miR-10b in macrophages contributes to advanced atherosclerosis progression in ApoE-/- mice.

Aims: We previously found that miR-10b inhibits cholesterol efflux from thioglycollate-elicited mouse peritoneal macrophages through repressing ATP binding cassette transporter (ABCA1). Herein, we deciphered the mechanism underlying macrophage miR-10b expression and the role of miR-10b in atherosclerosis. Methods and results: MiR-10b expression was increased in the arteries with advanced but not early atherosclerotic plaques of ApoE-/- mice. Free cholesterol-induced macrophage apoptotic cells (FC-AM) promoted miR-10b expression in mouse resident peritoneal macrophages (RPM) by up-regulation of Twist1/2 in a Mer receptor tyrosine kinase dependent manner. Surprisingly, antagomiR-10b de-repressed ABCA1 in RPM engulfing FC-AM but not in RPM alone or RPM-derived foam cells; systemic delivery of antagomiR-10b enhanced reverse cholesterol transport from RPM engulfing FC-AM but not from RPM or the foam cells in ApoE-/- mice. Mechanistically, RPM engulfing FC-AM possessed sufficient miR-10b functionally repressing ABCA1 expression, whereas RPM and foam cells had little miR-10b incompetently repressing ABCA1 expression. Notably, antagomiR-10b administration reduced advanced plaque size and also enhanced plaque stability in ApoE-/-mice, which were associated with increased plaque macrophage ABCA1 expression and reduced plaque apoptosis and inflammation. However, antagomiR-10b administration did not affect early atherosclerotic plaque formation in ApoE-/- mice. Conclusion: These data suggest that apoptotic cell induction of miR-10b in macrophages is important in advanced atherosclerosis progression.

Prevalence and genotype distribution of human papillomavirus infections in women attending hospitals in Chaozhou of Guangdong province.

BACKGROUND: Human papillomavirus (HPV) infection is the main cause of cervical cancer. Limited epidemiologic data of HPV prevalence are available for women attending hospitals in southern China. This study aimed to evaluate the profiles of HPV infection and cytology status in gynecological outpatients in Chaozhou City. METHODS: A total of 2833 eligible women were enrolled. The HPV GenoArray test was used for HPV detection and genotyping. Nearly one half of the HPV positive women received liquid-based cytology test. Logistic regression analysis was performed to assess the predictable effects of age and genotype for categories of abnormal cytology. RESULTS: The prevalence of overall, high-risk, and low-risk HPV infection were 24.5%, 19.5% and 8.4%, respectively. A U-shaped age-specific prevalence curve was observed in overall HPV and high- risk HPV, but not in low-risk HPV, which declined with age increasing. The 6 most common high-risk HPV type in descending order, were types 52, 16, 58, 18, 68, and 33. Age and HPV genotype were both important determinants of abnormal cytology incidence, the older women (>45 years) and those infected with HPV type 16 and/or 18 having the highest risk for abnormal cytology. CONCLUSION: Our findings support the hypothesis that second-generation HPV prophylactic vaccines including HPV-52 and -58 may offer higher protection for women residing in Chaozhou and neighboring cities in Guangdong.

Epidemiologic characterization of human papillomavirus infection in rural Chaozhou, eastern Guangdong Province of China.

BACKGROUND: Human papilloma virus (HPV) infection was the main cause of cervical cancer. There were only a few reports and detailed data about epidemiological research of HPV infection in rural population of China. MATERIALS AND METHODS: The cervical cells of rural Chaozhou women were collected, and multiplex real time PCR was firstly performed to detect high-risk HPV (HR-HPV) infection, which could detect 13 types of HR-HPV (types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68). Then, HPV-positive samples were typed by HPV GenoArray test. RESULTS: HR-HPV DNA was detected by multiplex real time-PCR in 3830 of 48559 cases (7.89%). There was a peak incidence in age of 55-60 years group, and a lower incidence in who lived in plain group compared with suburban, mountain and seashore group. 3380 cases of HPV positive sample were genotyped, 11.01% (372/3380) cases could not be classified, among the typed 3008 cases, 101 cases were identified without HR-HPV type infection, 2907 cases were infected with one HR-HPV type at least, the 6 most common HR-HPV types in descending order of infection, were type 52 (33.4%, 16 (20.95%), 58 (15.93%), 33 (9.94%), 68 (9.22%) and 18 (8.36%). The combined prevalence of HPV types 16 and 18 accounted for 28.52% of total infection. However, type 52 plus 58 presented 48.23% of total infection. 2209/2907 cases were infected with a single HPV type and 698/2907 cases were infected with multiple types, and multiple infection constituent ratio increased with age, with a peak incidence in age 55-60 years group. CONCLUSIONS: Our findings showed low prevalence of HPV vaccine types (16 and 18) and relatively high prevalence of HPV-52 and -58, support the hypothesis that the second-generation HPV vaccines including HPV-52 and -58 may offer higher protection for women in rural Guangdong Province.

The biological characteristics of glioma stem cells in human glioma cell line SHG44.

Gliomas are the most common tumors of the central nervous system (CNS) and a frequent cause of death. The treatment of malignant gliomas is often palliative due to their high recurrence rate. A growing body of evidence suggests that glioma may arise from cancer stem cells (CSC) correlated with neural stem cells (NSC), with the capacity for self-renewal and multipotency. CSCs have been isolated from human gliomas and numerous other solid tumors. It is assumed that a number of established malignant cell lines also contain a rare subpopulation of stem cells. This study was designed to investigate the proportion of CSCs in the human glioma cell line SHG44 and to study the limitations of CD133 immunophenotyping in glioma stem cell research. SHG44 cells were cultured in both serum-containing and serum-free medium. The similar shape in growth curves (in the exponential growth phase) revealed that most cells participated in the population amplification. Time gradient BrdU labeling and monoclonal assay revealed that almost every single cell participated in the division growth (98.82%) and possessed the ability to form clones (96.19%). No significant difference was found in the proportions of CD133+ cells in the serum-containing and serum-free groups (38.25%/37.92%). In addition, no significant difference was noted in the proportions of CD133+ cells among monoclones selected randomly in the serum-containing group. These results suggested that CD133- cells generate CD133+ cells and have the ability to form clones. Thus, we concluded that most SHG44 cells were CSCs and serum-free medium was not necessary for the generation of CSCs. In this line, CD133- cells also possessed clonogenic, self-renewal capacities and were also CSCs.

Tumor suppressor gene alterations of spontaneously malignant transformed cells from human embryonic muscle in vitro.

Recent research has shown that mesenchymal stem cells (MSCs) which were cultured for long time could transform malignantly, the transformation mechanism is not clear yet, it might be associated with the activation of oncogenes and inactivation of tumor suppressor genes. In our initial investigation, we found that the cells arising from human embryonic muscle could spontaneously transform into malignancy in vitro and we obtained 6 immortalized cell lines. In this study, polymerase chain reaction (PCR) was used to assay several tumor suppressor genes of these cell lines, and homozygous deletions within chromosomal band 9p2l including MTAP (methylthioadenosine phosphorylase), p16 and p15 were detected. PCR products of p53 exons 7 and 8 of these novel tumor cell lines were assayed by sequencing, and the results showed high prevalence of mutations in these regions, the mutation rate reached as high as 8% in exon 7 and 14% in exon 8, and all of them were point mutations, the intron 7 changed more significantly, including piece deletion, insertion, frameshift and point mutation, it showed almost no similarity to that of the wt p53 sequence, that was totally different from other p53 mutation data published. All the mutation sequences were identical in 6 cell lines, this suggest that there may be a common mutation mechanism and strong selective advantage in these novel tumor cell lines over long-term culture. In conclusion, our research shows that the inactivation of tumor suppressor genes may play an important role in the process of malignant transformation of embryonic muscle cells in vitro.

Several types of soft tissue sarcomas originate from the malignant transformation of adipose tissue-derived stem cells.

The cellular origin of soft tissue sarcomas (STSs) is not fully understood. The cancer stem cell hypothesis presumes that tumors originate from the malignant transformation of stem cells. As a type of multipotent stem cell, adipose tissue-derived stromal/stem cells (ADSCs), which possess an unexpected degree of plasticity and often reside in other tissues, may represent a potential source of soft tissue sarcoma. To ascertain whether ADSCs are responsible for the formation of STSs, ADSCs from mice were cultured and treated with 3-methycholanthrene to derive transformed cells. These transformed ADSCs were then injected subcutaneously into immunodeficient mice to test their tumorigenic potential. We found that they generated several types of STSs, including synovial sarcoma, malignant fibrous histiocytoma and fibrosarcoma. This is the first study to report that ADSCs may be the potential initiating cells for synovial sarcoma. Our findings indicate that STSs might originate from malignantly transformed ADSCs.

Inhibition of the replication of hepatitis B virus in vitro by oxymatrine.

This study investigated the inhibitory capacity of oxymatrine on in vitro hepatitis B virus (HBV) replication. HepG2.2.15 cells were treated with oxymatrine 50, 100, 200, 400, 800 or 1000 mg/l, or with human interferon-alpha2b (IFN-alpha2b 1000 U/l) as a positive control. Levels of hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg) and HBV-DNA in cell supernatants were determined by enzyme-linked immunosorbent assay and fluorescent quantitative-polymerase chain reaction, respectively. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometric analysis and terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labelling were used to evaluate the cytotoxicity of oxymatrine. The inhibitory effects of oxymatrine gradually increased as the concentration increased from 200 to 1000 mg/l for HBsAg and HBeAg, and from 200 to 400 mg/l for HBV-DNA. There was no inhibitory effect of oxymatrine at concentrations < 200 mg/l. No significant difference was seen between human IFN-alpha2b (positive control) and oxymatrine >or= 200 mg/l. It is concluded that oxymatrine can inhibit in vitro HBV replication and antigen expression at concentrations >or= 200 mg/l.

A novel mutated cell line with characteristics of dedifferentiated chondrosarcoma.

Dedifferentiated chondrosarcoma (CS) is a rare, highly malignant variant of CS in which a high-grade sarcoma coexists with a low-grade chondroid tumor. In this study, a novel dedifferentiated CS cell line, MS0812, was spontaneously established from mutated human embryonic muscle cells. Several features of the cell line were investigated, including growth characteristics, cytogenetics, electron microscopic features, expression of various antigenic markers and tumor formation. MS0812 has been cultured continuously for more than 3 years. The growth characteristics of MS0812 are similar to the immortalized cell lines as reported. The cell line exhibited complex karyotypes and hyperploidy, the chromosome number ranged from 50 to 158. MS0812 was positive for vimentin, desmin and muscle actin, indicating their muscle origin. With specific inductive condition, MS0812 differentiates into neural cells and adipocytes. Deletion of the p16 gene, which seemed to play a major role in the malignant phenotype of this cell line, was confirmed by PCR and immunocytochemistry. MS0812 formed tumors in nude mice, and the tumor revealed a fibrosarcoma with chondroid components, which were consistent with dedifferentiated CS as reported. Chondroid components showed metachromasia by Alcian blue and toluidine blue and were S100 and collagen-II positive. To our knowledge, this is the first report of the establishment of a human dedifferentiated chondrosarcoma from mutated human embryonic muscle cells, and it is a useful model for the study of the molecular pathogenesis of dedifferentiated CS.

[Human amnion cells express the phenotypes of neural cells and adipocytes in vitro].

OBJECTIVE: To search for culture conditions in which the cells from human amnion could diferentiate into neural cells and hence to explore a new cell source for neural transplantaion. METHODS: Amnion cells from human were cultured with tissue piece method, passaged by trypsin digestion and identified with immunocytochemistry. RESULTS: Amnion cells migrated from explants and primary culture was established; they could multiply and expand steadily in a short time, and they could be passaged by trypsin digestion. When cultured in serum-free neural stem cell media, these cells could form the same spheroid shape as the neurospheres of neural stem cells. They could express alpha smooth muscle actin and differentiate into smooth muscle cells spontaneously, and could express nestin and vimentin, the markers for neural progenitors. Moreover, they could he stained by anti-beta III-tubulin, anti-neurofilament 200 and anti-NSE; the majorities could he stained by antityrosine hydroxylase, the marker for dopaminergic neurons. Lower than 0.1% of the total cells were stained by GFAP, indicating the existence of astrocytes. The amnion cells could also differentiate into adipocytes under specific induction. CONCLUSION: Amnion cells could differentiate into adipocytes and smooth muscle cells; they could express the protein for neural cells; thus may represent an alternative stem cell source for CNS cell transplantation.

[The in vitro study of the human adipose tissue-derived stromal cells differentiating into the neuron-like cells].

OBJECTIVE: To investigate the possibility of the adipose tissue derived stromal cells (ADSCs) to differentiate into the neuron-like cells and to explore a new cell source for the transplantation related to the central nervous system. METHODS: Adipose was digested by collagenase, cultured in the fetal bovine serum containing a medium. Trypse was used to digest the cells and the cell passage was performed. The 3rd to the 9th passage ADSCs were used to make an induction. Isobutyl-methylxanthine, indomethacin, insulin, and dexamethasone were used to induce the ADSCs to differentiate into the neuronlike cells and adipocytes. Sudan black B and immunocytochemistry were used to identify the cells. RESULTS: A population of the ADSCs could be isolated from the adult human adipose tissue, they were processed to obtain a fibroblast-like population of the cells and could be maintained in vitro for an extended period with the stable population doubling, and they were expanded as the undifferentiated cells in culture for more than 20 passages, which indicated their proliferative capacity. They expressed vimentin and nestin, and characteristics of the neuron precursor stem cells at an early stage of differentiation. And the majority of the ADSCs also expressed the neuron-specific enolase and beta III-tubulin, characteristics of the neurons. Isobutyl-methylxanthine, indomethacin, insulin, and dexamethasone induced 40%-50% of ADSCs to differentiate into adipocytes and 0. 1%-0.2% of ADSCs into neuron-like cells. The neuron-like cells had a complicated morphology of the neurons, and they exhibited a neuron phenotype, expressed nestin, vimentin, neuron-specific enolase and beta III-tubulin, but some neuron-like cells also expressed the smooth muscle actin (SMA), and the characteristics of the smooth muscle cells; however, the neurons from the central nervous system were never reported to express this kind of protein. Therefore, the neuron-like cells from the ADSCs could be regarded as functional neurons. CONCLUSION: Our results support the hypothesis that the adult adipose tissue contains the stem cells capable of differentiating into the neuron-like cells, and they can overcome their mesenchymal commitment, which represents an alternative autologous stem cell source for transplantation related to the central nervous system.

[Long-term culture and differentiation of human glioma stem cells ].

OBJECTIVE: To explore the culture and subculture conditions for glioma stem cells(GSCs) and to investigate the differentiation potential of GSCs. METHODS: The cells from human glioma were mechanically dissociated. Cells were cultured in N2 or B27 medium with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF), and they were identified by immunocytochemistry. RESULTS: Glioma stem cells from human glioma have been successfully cultured. They formed typical neurospheres in suspension, and they could be cultured and passaged steadily in vitro. The majorities of the cells expressed vimentin and nestin, which were the markers for neural stem cells. They could differentiate into neurons and astrocytes, and express glial fibrillary acidic protein and beta III-tubulin respectively. CONCLUSION: Human glioma stem cells could be cultured from gliomas in vitro, and they could differentiate into neurons and astrocytes, thus providing a basis for further studies.

[Intraventricular transplantation of human neural stem cells into embryonic rats].

OBJECTIVE: To explore the migration and distribution of human neural stem cells (NSCs) after they were transplanted into lateral cerebroventricles of embryonic rats. METHODS: This study was conducted with the proxy consent and the permission from the Hospitals Ethical Review Committee. The cells from the embryonic human cortices were mechanically dissociated. N2 medium was used to culture and expand the cells. And the cells were identified by immunocytochemistry; then after being labelled with BrdU or adenovirus carrying LacZ gene, they were implanted into the lateral cerebroventricles of embryonic rats. The rats were sacrificed after they were born and the brain sections were examined by immunocytochemistry at different time points. RESULTS: NSCs from embryonic humans were successfully cultured; they formed typical neurospheres in suspension, and the majorities of the cells expressed nestin, which was the marker for neural progenitor cells, and the cells could differentiate into neurons, astrocytes and oligodendrocytes. NSCs could steadily expressed exogenous gene in vivo and in vitro; after transplantation into embryonic rat brains, the fetal human NSCs incorporated individually into all major compartments of the brains, generating widespread CNS chimerism and having a wide distribution in the host fore-, mid-, and hindbrain. CONCLUSION: NSCs could express foreign gene steadily and were the ideal cell sources for cell and gene therapy. These cells could incorporate into developing brains and generate widespread chimerism. These chimeras provide a unique model for studying human neural cell migration and differentiation in a functional nervous system.

Differentiation of adult human bone marrow mesenchymal stem cells into Schwann-like cells in vitro.

OBJECTIVE: To investigate the differentiative capability of adult human bone marrow mesenchymal stem cells (BMSCs) into Schwann-like cells. METHODS: Bone marrows were aspirated from healthy donors and mononuclear cells were separated by Percoll lymphocytes separation liquid (1.073 g/ml) with centrifugation, cells were cultured in DMEM/F12 (1:1) medium containing 10% fetal bovine serum (FBS), 20 ng/ml epidermal growth factor (EGF) and 20 ng/ml basic fibroblast growth factor (bFGF). Cells of passage 1 were identified with immunocytochemistry. RESULTS: Mononuclear cells separated by Percoll's were passaged 10 times by trypsin/ethylenediaminetetraacetic acid (EDTA) digestion in 40 days, and BMSCs increased about 6x10(7) times in this short period. Immunohistochemistry identified that BMSCs were CD34- and CD31-, but they expressed neuron specific enolase; 0.01%-0.02% of total cells expressed nestin, the marker for neural progenitor cells; 40%-50% cells stained heavily by neurofilament 200; and no glial fibrillary acidic protein (GFAP) positive cells were identified; S100 expression was detected among 0.1%-0.2% cells. CONCLUSIONS: Bone marrow contains the stem cells with the ability of differentiating into Schwann-like cells, which may represent an alternative stem cell sources for neural transplantation.

[Stromal cells from human Wharton's jelly differentiate into neural cells].

OBJECTIVE: To investigate the neural differentiating capability of the umbilical cord tissue-derived stromal cells (UCSCs) in the attempt to find a new cell source for neural transplantation. METHODS: UCSCs from umbilical cord of human were cultured with tissue piece method, passaged by trypsin digestion. And Salvia miltiorrhiza was used to induce the cells to differentiate. Cells were identified with immunocytochemistry. RESULTS: Stromal cells that migrated from explants and primary culture were obtained. These cells could differentiate into smooth muscle cells spontaneously and expressed smooth muscle actin; they could be passaged by trypsin digestion. Salvia miltiorrhiza could induce these cells to differentiate into the neuron-like cells, which displayed typical neuron morphology, expressed nestin, beta III-tubulin and NSE at the early stage of differentiation, and were stained by anti-neurofilament 200 at the late stage of differentiation. With optimal conditions, about 90% of UCSCs expressed neuronal phenotypes, lower than 1% of the differentiated cells expressed GFAP, and no myelin basic protein expression was detected in the differentiated cells, indicating the absence of oligodendrocyte differentiation from stromal cells. CONCLUSION: The data supported the hypothesis that umbilical cord contains the stem cells with the ability of differentiating into neurons, which may provide an alternative stem cell source for CNS cell transplantation.

[Research progress of adipose tissue-derived stromal cells].

OBJECTIVE: To review research progress of adipose tissue-derived stromal cells (ADSCs). METHODS: The recent articles on ADSCs were extensively reviewed, and the culture and differentiation ability of ADSCs were investigated. RESULTS: A population of stem cells could be isolated from adult adipose tissue, they were processed to obtain a fibroblast-like population of cells and could be maintained in vitro for extended periods with stable population doubling. The majority of the isolated cells were mesenchymal origin, with a few pericytes, endothelial cells and smooth muscle cells. ADSCs could be induced to differentiate into multiple mesenchymal cell types, including osteogenic, chondrogenic, myogenic and adipogenic cells, they could also differentiate into nerve cells. CONCLUSION: ADSCs can substitute mesenchymal stem cells and become an alternative stem cells source for tissue engineering.

Culture of skin-derived precursors and their differentiation into neurons.

OBJECTIVE: To investigate the culture method of skin-derived precursors (SKPs) and to explore a new cell source for cell transplantation of central nervous system. METHODS: Cells from skins of juvenile and adult mice were isolated and cultured in serum-free medium. A mechanical method was chosen to passage these cells and they were identified by the immunocytochemistry assay. RESULTS: SKPs could be isolated from adult and neonatal skins. They could be maintained in vitro for long periods with stable proliferation, and expanded as undifferentiated cells in culture for more than 12 passages. About 50% of SKPs expressed nestin and majority of these cells expressed fibronectin when they were plated on polyornithine and laminin coated plates. About 5% cells showed neuronal differentiation and expressed neurofilament-M (NF-M) and NSE when SKPs were plated in serum-containing medium, and these cells could also differentiate into adipocytes and fibroblast-like cells. CONCLUSIONS: The data support the hypothesis that adult skin contains stem cells capable of differentiating into neurons, adipocytes, and fibroblast-like cells. They may represent an alternative autologous stem cell source for CNS cell transplantation.