A novel ethanol-based method to induce differentiation of adipose-derived stromal cells into astrocytes

The quantity and survival time of astrocytes,which were differentiated from adult adipose-derived stromal cells after exposure to an inducer containing 3-isobutyl-1-methylxanthine,have thus far been unsatisfactory.The present study investigated the growth and differentiation characteristics of induced astrocytes by observing their growth curves.After induction for 48 hours with an inducer containing 0.5% ethanol,some adult adult adipose-derived stromal cells displayed typical astrocytic morphology.The cell quantity gradually decreased with prolonged induction time.Nestin,glial fibrillary acidic protein,and S-100 expression reached peak levels at 14 days,but neuron-specific enolase was not expressed.These results suggest that the induced astrocytes reached their peak at 14 days.Further optimization of the culture environment may yield mature astrocytes with normal functions,in greater quantity,and prolonged survival time.

Photobiomodulation:a novel approach to promote trans-differentiation of adipose-derived stem cells into neuronal-like cells

Photobiomodulation,originally used red and near-infrared lasers,can alter cellular metabolism.It has been demonstrated that the visible spectrum at 451-540 nm does not necessarily increase cell proliferation,near-infrared light promotes adipose stem cell proliferation and affects adipose stem cell migration,which is necessary for the cells homing to the site of injury.In this in vitro study,we explored the potential of adipose-derived stem cells to differentiate into neurons for future translational regenerative treatments in neurodegenerative disorders and brain injuries.We investigated the effects of various biological and chemical inducers on trans-differentiation and evaluated the impact of photobiomodulation using 825 nm near-infrared and 525 nm green laser light at 5 J/cm2.As adipose-derived stem cells can be used in autologous grafting and photobiomodulation has been shown to have biostimulatory effects.Our findings reveal that adipose-derived stem cells can indeed trans-differentiate into neuronal cells when exposed to inducers,with pre-induced cells exhibiting higher rates of proliferation and trans-differentiation compared with the control group.Interestingly,green laser light stimulation led to notable morphological changes indicative of enhanced trans-differentiation,while near-infrared photobiomodulation notably increased the expression of neuronal markers.Through biochemical analysis and enzyme-linked immunosorbent assays,we observed marked improvements in viability,proliferation,membrane permeability,and mitochondrial membrane potential,as well as increased protein levels of neuron-specific enolase and ciliary neurotrophic factor.Overall,our results demonstrate the efficacy of photobiomodulation in enhancing the trans-differentiation ability of adipose-derived stem cells,offering promising prospects for their use in regenerative medicine for neurodegenerative disorders and brain injuries.

Apoptosis during β-mercaptoethanol-induced differentiation of adult adipose-derived stromal cells into neurons

β-mercaptoethanol can induce adipose-derived stromal cells to rapidly and efficiently differentiate into neurons in vitro.However,because of the short survival time of the differentiated cells,clinical applications for this technique are limited.As such,we examined apoptosis of neurons differentiated from adipose-derived stromal cells induced with β-mercaptoethanol in vitro using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and transmission electron microscopy.The results revealed that the number of surviving cells decreased and apoptosis rate increased as induction time extended.Taken together,these results suggest that apoptosis occurring in the process of adipose-derived stromal cells differentiating into neurons is the main cause of cell death.However,the mechanism underlying cellular apoptosis should be researched further to develop methods of controlling apoptosis for clinical applications.

Isolation, Culture and Induced Differentiation of Fetal Porcine Islet Derived Pancreatic Stem Cell

To isolate and culture the porcine pancreatic stem cells and investigate their function, the fetal porcine pancreatic stem cells were isolated by the method of suspending plus adhering culture. The isolated cells were then identified by immunohistochemical staining, and their culture viability measured through the MTT method in vitro. This induced them to differentiate into endocrine cells and detect their function. The isolated IPSCS did not express nestin, but expressed CK-19, a marker of ductal epithelia cells and ct-actin, a smooth muscle marker, demonstrating the growth characteristics of ES-like cells, and strong proliferative ability, after 18 passages. They could excrete insulin, and showed ultrastructure changes after being induced. Porcine pancreatic stem cells can be isolated by this method, induced to form islet-like clusters, and can secret insulin.

Wnt7b can replace Ihh to induce hypertrophic cartilage vascularization but not osteoblast differentiation during endochondral bone development

Indian hedgehog （Ihh） is an essential signal that regulates endochondral bone development. We have previously shown that Wnt7b promotes osteoblast differentiation during mouse embryogenesis, and that its expression in the perichondrium is dependent on Ihh signaling. To test the hypothesis that Wnt7b may mediate some aspects of Ihh function during endochondral bone development, we activated Wnt7b expression from the R26-Wnt7b allele with Col2-Cre in the Ihh-/- mouse. Artificial expression of Wnt7b rescued vascularization of the hypertrophic cartilage in the Ihh-/- mouse, but failed to restore orthotopic osteoblast differentiation in the perichondrium. Similarly, Wnt7b did not recover Ihh-dependent perichondral bone formation in the Ihh-/-; Gli3-/- embryo. Interestingly, Wnt7b induced bone formation at the diaphyseal region of long bones in the absence of Ihh, possibly due to increased vascularization in the area. Thus, Ihh-dependent expression of Wnt7b in the perichondrium may contribute to vascularization of the hypertrophic cartilage during endochondral bone development.

Bone morphogenetic protein 4 induces cholinergic differentiation of brain-derived neural stem cells in vivo and in vitro

In vitro studies have demonstrated that many factors of bone morphogenetic proteins （BMPs） induce cholinergic differentiation of neural stem cells. However, BMP retains the potential to induce increased numbers of cholinergic neurons in central nervous system regions that are rich in cholinergic cells, which is an important determinant of BMP. Therefore, BMP-4 was added to neural stem cell culture medium or the adult rat hippocampal dentate gyrus. Results demonstrated that BMP-4 induced cholinergic differentiation of neural stem cells in vitro and increased the number of cholinergic neurons in the adult rat hippocampal dentate gyrus.

Effects of brain-derived neurotrophic factor on induced differentiation of SH-SY5Y cells in vitro

BACKGROUND： Previous studies have demonstrated that brain-derived neurotrophic factor （BDNF） promotes neural differentiation. However, the mechanisms involved in cell cycle-related protein regulation, which highly correlates to neural proliferation and apoptosis, remain poorly understood. OBJECTIVE： To investigate the effects of various concentrations of BDNF on cycle-related protein mRNA expression in induce-differentiated SH-SY5Y cells in vitro prior to and following G2 phase, and to analyze the neuroprotective effects of BDNF. DESIGN, TIME AND SETTING： A comparison, observational study, based on cell biology, was performed at the Department of Biochemistry, Medical College of Tongji University, from March 2005 to October 2006. MATERIALS： SH-SY5Y cells were provided by Shanghai Institute of Cytology, Chinese Academy of Science; BDNF by Alomone Labs, Israel; all-trans retinoic acid （ATRA） by Sigma-Aldrich, USA. METHODS： SH-SY5Y cells were randomly divided into three groups： blank control [cells were treated in Insulin-Transferrin-Selenium （ITS） solution for 7 days], ATRA （cells were treated with ITS solution containing 10 μmol/L ATRA for 7 days）, and BDNF （cells were treated identical to the ATRA group for 5 days, and then respectively treated in ITS solution containing 1, 10, and 100 μg/L BDNF for 2 days）. The experiment was repeated three times for each group. MAIN OUTCOME MEASURES： mRNA expression levels of cyclin A1, B1, B2, cyclin-dependent kinase 1, and 5 were detected using quantitative real-time RT-PCR; percentage of cells in G1, S, and G2 phases were detected using fluorescence-activated cell sorting. RESULTS： mRNA expression levels of cyclin A1 in the high-dose BDNF group was significantly less than the ATRA group （P 〈 0.05）.mRNA expression levels of cyclin B1 was significantly less in the different BDNF concentration groups compared with the control and ATRA groups （P 〈 0.05 or P 〈 0.01）. mRNA expression levels of cyclin B2 and cyclin-dependent kinase 1 were significantly decreased in the high-dose BDNF group （P 〈 0.05 or P 〈 0.01）. Cyclin-dependent kinase 5 mRNA expression was significantly greater in the low-dose and moderate-dose BDNF groups compared with the ATRA group （P 〈 0.05）. The percentage of cells in G1 phase was significantly greater in the different BDNF concentration groups compared with the ATRA and control groups （P 〈 0.01）. Moreover, the percentage of cells in S phase was significantly less in the three BDNF groups compared with the ATRA group （P 〈 0.01）. However, the percentage of cells in S phase was significantly less in the low-dose and high-dose BDNF groups compared with the control group （P 〈 0.01）. CONCLUSION： BDNF enhanced the percentage of cells in G1 phase, but did not alter mRNA expression of cell cycle-related proteins prior to or following G2 phase. These results suggested that BDNF was not a risk factor for inducing apoptosis.

EXPRESSION OF ONCOGENES DURING INDUCED DIFFERENTIATION OF HUMAN HEPATOCARCINOMA CELL LINE

There was no detectable expression of c-fos,but a little c-myc,high c-fms and mederate high IGF-ⅡmRNA in the untreated human hepatocarcinoma cell SMMC- 7721.After treatment with 10 μmol/L retinoic acid or 0.5 mmol/L dibutyryl cyclic-3',5'adenosine monophosphate(db-cAMP),the c-fos was transiently expressed within 20- 60mins.If the treatment of RA or db-cAMP prolonged to 1-5 days, the transcriptions of c- myc were increased,reaching the highest level on the 2nd and 4th day.Simultaneously the transcriptions of c- fms and IGF- Ⅱwere gradually decreased.On the 5th day of the treatment,c-fms and IGF-ⅡmRNA were decreased to 32% and 14%respectively of the control (untreated cell) value by RA,and 35% and 22%respectively by db-cAMP.The biological significance of the above mentioned results was discussed.

Differentiation and tumorigenicity of neural stem cells from human cord blood mesenchymal stem cells

BACKGROUND： Mesenchymal stem cells （MSCs） are capable of differentiating into a variety of tissues and exhibit low immunogenicity. OBJECTIVE： To investigate isolation and in vitro cultivation methods of human cord blood MSCs, to observe expression of neural stem cell （NSC） marker mRNA under induction, and to detect tumorigenicity in animals. DESIGN, TIME AND SETTING： A cell biological, in vitro trial and a randomized, controlled, in vivo experiment were performed at the Department of Neurology, Daping Hospital at the Third Military Medical University of Chinese PLA from August 2006 to May 2008. MATERIALS： Umbilical cord blood was collected from full-term-delivery fetus at the Department of Gynecology and Obstetrics of Daping Hospital, China. Eighteen BALB/C nu/nu nude mice were randomly assigned to three groups： back subcutaneous, cervical subcutaneous, and control, with 6 mice in each group. METHODS： Monocytes were isolated from heparinized human cord blood samples by density gradient centrifugation and then adherent cultivated in vitro to obtain MSC clones. After the cord blood MSCs were cultured for 7 days with nerve growth factor and retinoic acid to induce differentiation into NSCs, the cells （adjusted density of 1 × 10^7/mL） were prepared into cell suspension. In the back subcutaneous and cervical subcutaneous groups, nude mice were hypodermically injected with a 0.5-mL cell suspension into the back and cervical regions, respectively. In the control group, nude mice received a subcutaneous injection of 0.5 mL physiological saline into the back or cervical regions, respectively. MAIN OUTCOME MEASURES： Cellular morphology was observed by inverted microscopy, cultured cord blood MSCs were examined by flow cytometry, expression of nestin and musashi-1 mRNA was detected by reverse-transcriptase polymerase chain reaction prior to and after induction, and tumorigenicity following cord blood MSC transplantation was assayed by hematoxylin-eosin staining. RESULTS： Following adherent cultivation, the majority of cord blood monocytes became rhombic and strongly expressed CD29, but not CD34, CD1 la, or CD11 b. These results supported previously known characteristics of cord blood MSCs. Following differentiation induction, nestin and musashi-1 were expressed on the surface of NSCs, exhibiting strongest expression at 48 hours, and subsequently reducing expression. Cultured cord blood MSCs were not tumorigenic in the nude mice. Cellular morphology displayed no malignant changes between the control and subcutaneous groups. CONCLUSION： MSCs can be isolated from human cord blood, efficiently expanded under culture conditions, differentiated into NSCs following induction, and display no tumorigenicity in nude mice.

Biochemical properties of norepinephrine as a kind of neurotransmitter secreted by bone marrow-derived neural stem cells induced and differentiated in vitro

BACKGROUND： It has been proved by many experimental studies from the aspects of morphology and immunocytochemistry in recent years that bone marrow stromal cells （BMSCs） can in vitro induce and differentiate into the cells possessing the properties of nerve cells. But the functions of BMSCs-derived neural stem cells（NSCs） and the differentiated neuron-like cells are still unclear. OBJECTIVE： To observe whether bone marrow-derived NSCs can secrete norepinephrine （NE） under the condition of in vitro culture, induce and differentiation, and analyze the biochemical properties of BMSCs-derived NSCs. DESIGN： A non-randomized and controlled experimental observation SETTING ： Institute of Neuromedicine of Chinese PLA, Zhujiang Hospital, Southern Medical University MATERIALS： This experiment was carried out in the Institute of Neuromedicine of Chinese PLA, Zhujiang Hospital, Southern Medical University. The bone marrow used in the experiment was collected from 1.5- month-old healthy New Zealand white rabbits. METHODS： This experiment was carried out in the Institute of Neuromedicine of Chinese PLA, Zhujiang Hospital, Southern Medical University. The bone marrow used in the experiment was collected from 1.5 month-old healthy New Zealand white rabbits. BMSCs of rabbits were isolated and performed in vitro culture, induce and differentiation with culture medium of NSCs and differentiation-inducing factor, then identified with immunocytochemical method. Experimental grouping： ①Negative control group： L-02 hepatic cell and RPMI1640 culture medium were used. ② Background culture group： Only culture medium of NSCs as culture solution was added into BMSCs to perform culture, and 0.1 volume fraction of imported fetal bovine serum was supplemented 72 hours later. ③Differentiation inducing factor group： After culture for 72 hours, retinoic acid and glial cell line-derived neurotrophic factors were added in the culture medium of BMSCs and NSCs as corresponding inducing factors. The level of NE in each group was detected on the day of culture and 5, 7, 14 and 20 days after culture with high performance liquid chromatography （HPLC）. The procedure was conducted 3 times in each group.Standard working curve was made according to the corresponding relationship of NE concentration and peak area. The concentration of NE every 1×10^7 cells was calculated according to standard curve and cell counting. MAIN OUTCOME MEASURES ： The level of NE of cultured cells was detected with HPLC; immunocytochemistrical identification of Nestin and neuron specific nuclear protein was performed. RESULTS： ① On the 14^th day after cell culture, BMSCs turned into magnus and round cells which presented Nestin-positive antigen, then changed into neuron-like cells with long processus and presented neuron specific nuclear protein -positive antigen at the 20^th day following culture. ② The ratio of NE concentration and peak area has good linear relationship, and regression equation was Y=1.168 36＋0.000 272 8X,r=-0.998 4. Coefficient variation （CV） was 〈 5% and the recovery rate was 92.39%（ Y referred to concentration and X was peak area）.③NE was well detached within 10 minutes under the condition of this experiment. ④ NE was detected in NSCs and their culture mediums, which were cultured for 7, 14 and 20 days respectively, but no NE in BMSCs, NSCs-free culture medium and L-02 hepatic cell which were as negative control under the HPLC examination. Analysis of variance showed that the level of NE gradually increased following the elongation of culture time （P 〈 0.01 ）. No significant difference in the level of NE existed at the same time between differentiation inducing factor group and basic culture group（P 〉 0.05）. CONCLUSION ： BMSCs of rabbits can proliferate in vitro and express Nestin antigen; They can differentiate into neuron-like cells, express specific neucleoprotein of mature neurons, synthesize and secrete NE as a kind of neurotransmitter.

Proteome changes during bone mesenchymal stem cell differentiation into photoreceptor-like cells in vitro

Human bone marrow stem cell (BMSC) may be directed to differentiate into multiple cell types, including adipocyte, chondrocyte, osteocyte and photoreceptor, among others. At present, little is known about the features of the BMSC and the protein control mechanism underlying their differentiation into photoreceptor-like cells. In the present study, BMSCs are induced to differentiate into photoreceptor-like cells in an in vitro model simulating the in vivo microenvironment. Up to 32 proteins are identified and differentially expressed through two-dimensional difference gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to establish a differential protein database for photoreceptor-like cells from BMSC-induced differentiation. Western blot analysis further confirms the expression of some of the identified proteins. The present study proposes the total protein expression and possible molecular mechanism during the differentiation of BMSCs into photoreceptor cells.

Optimal time point for the transplantation of neural stem cells induced to differentiate with retinoic acid

Previous studies have demonstrated that differentiated neural stem cells （NSCs） are more suitable for transplantation than non-differentiated NSCs. In this study, NSCs were expanded in vitro for two passages, induced with retinoic acid to differentiate, and harvested between 1 6 days later. They were subsequently cultured in artificial cerebrospinal fluid for an additional 3 days, dudng which their growth and morphology was monitored. NSCs induced for 4 days exhibited a peak rate of cells differentiating into neurons and robust growth. Our results indicate that the optimal time point for transplanting NSCs is following a 4-day period of induced differentiation.

Differential Proteomics Reveals the Potential Injury Mechanism Induced by Heavy Ion Radiation in Mice Ovaries

In the present study, we used a proteomics approach based on a two-dimensional electrophoresis （2-DE） reference map to investigate protein expression in the ovarian tissues of pubertal Swiss-Webster mice subjected to carbon ion radiation （CIR）. Among the identified proteins, ubiquitin carboxy-terminal hydrolase L1 （UCH-L1） is associated with the cell cycle[1] and that it influences proliferation in ovarian tissues. We analyzed the expression of UCH-L1 and the proliferation marker proliferation cell nuclear antigen （PCNA） following CIR using immunoblotting and immunofluorescence. The proteomics and biochemical results provide insight into the underlying mechanisms of CIR toxicity in ovarian tissues.

Synergistic effects of brain-derived neurotrophic factor and retinoic acid on inducing the differentiation of bone marrow stromal cells into neuron-like cells in adult rats in vitro

BACKGROUND： Under induction of retinoic acid （RA）, bone marrow stromal cells （BMSCs） can differentiate into nerve cells or neuron-like cells, which do not survive for a long time, so those are restricted to an application. Other neurotrophic factors can also differentiate into neuronal cells through inducing BMSCs; especially, brain-derived neurotrophic factor （BDNF） can delay natural death of neurons and play a key role in survival and growth of neurons. The combination of them is beneficial for differentiation of BMSCs. OBJECTIVE： To investigate the effects of BDNF combining with RA on inducing differentiation of BMSCs to nerve cells of adult rats and compare the results between common medium group and single BDNF group. DESIGN： Randomized controlled animal study SETTING： Department of Neurology, Affiliated Hospital of Xuzhou Medical College MATERIALS： The experiment was carried out in the Clinical Neurological Laboratory of Xuzhou Medical College from September 2003 to April 2005. A total of 24 SD rats, of either gender, 2 months old, weighing 130-150 g, were provided by Experimental Animal Center of Xuzhou Medical College [certification： SYXK （su） 2002-0038]. Materials and reagents： low-glucose DMEM medium, bovine serum, BDNF, RA, trypsin, separating medium of lymphocyte, monoclonal antibody of mouse-anti-nestin, neuro-specific enolase, glial fibrillary acidic protein （GFAP） antibody, SABC kit, and diaminobenzidine （DAB） color agent. All these mentioned above were mainly provided by SIGMA Company, GIBCO Company and Boshide Company. METHODS： Bone marrow of SD rats was selected for density gradient centrifugation. BMSCs were undertaken primary culture and subculture; and then, those cells were induced respectively in various mediums in total of 3 groups, including control group （primary culture）, BDNF group （20 μg/L BDNF） and BDNF＋RA group （20 μg/L BDNF plus 20 μg/L RA）. On the 3^rd and the 7^th days after induction, BMSCs were stained immunocytochemically with nestin （sign of nerve stem cells）, neuron-specific enolase （NSE, sign of diagnosing neurons） and GFAP （diagnosing astrocyte）, and evaluated cellular property. MAIN OUTCOME MEASURES ： Induction and differentiation in vitro of BMSCs in 3 groups RESULTS： （1） Induction and differentiation of BMSCs： Seven days after induction, cells having 2 or more apophyses were observed. Soma shaped like angle or erose form, which were similar to neurons and glial cells having strong refraction. （2） Results of immunocytochemical detection： Three days after induction, rate of positive cells in BDNF＋RA group was higher than that in BDNF group and control group [（86.15±4.58）%, （65.43±4.23）%, （4.18±1.09）%, P 〈 0.01]. Seven days after induction, rate of positive cells was lower in BDNF group and BDNF＋RA group than that in both groups at 3 days after induction [（31.12±3.18）%, （29.35±2.69）%, P 〈 0.01]; however, amounts of positive cells of NSE and GFAP were higher than those at 3 days after induction （P 〈 0.01）; meanwhile, the amount in BDNF＋RA group was remarkably higher than that in BDNF group （P 〈 0.01）. CONCLUSION： Combination of BDNF and RA can cooperate differentiation of BMSCs into neurons and astrocyte, and the effect is superior to single usage of BDNF.

Effects of GW5074 in the process of imDCs inducing differentiation of naive CD4^+ T cells into Treg cells in vitro

Objective To establish a stable and efficient method of culturing imDCs in vitro,and to explore the effect of GW5074,which blocks ERK1 /2 signal pathway in the process of immature dentritic cells ( imDCs) on inducing differentiation of the naive allogeneic CD4 + T

Isolating cDNA clones from rice induced by Magnaporthe grisea using PCR based differential screening method

The differential hybridization technique hasbeen widely used to identify genes that are dif-ferentially expressed.However,this approachhas several drawbacks.First,the screeningprocedures are rather labor-intensive and time-consuming.Second,the amount of phageDNAs transferred onto the two filters may notbe equivalent,which leads to an inaccurate se-lection of a positive clone.Third,isolation ofphage DNA is slow and cumbersome.Here,aPCR based differential screening method that

Transferrin receptor and ferritin-H are developmentally regulated in oligodendrocyte lineage cells

Iron is an essential trophic element that is required for cell viability and differentiation, especially in oligodendrocytes, which consume relatively high rates of energy to produce myelin. Multiple iron metabolism proteins are expressed in the brain including transferrin receptor and ferritin-H. However, it is still unknown whether they are developmentally regulated in oligodendrocyte lineage cells for myelination. Here, using an in vitro cultured differentiation model of oligodendrocytes, we found that both transferrin receptor and ferritin-H are significantly upregulated during oligodendrocyte maturation, implying the essential role of iron in the development of oligodendrocytes. Additional different doses of Fe3＋ in the cultured medium did not affect oligodendrocyte precursor cell maturation or ferritin-H expression but decreased the expression of the transferrin receptor. These results indicate that upregulation of both transferrin receptor and ferritin-H contributes to maturation and myelination of oligodendrocyte precursor cells.

Engineering an enthesis-like graft for rotator cuff repair:An approach to fabricate highly biomimetic scaffold capable of zone-specifically releasing stem cell differentiation inducers

Rotator cuff(RC)attaches to humerus across a triphasic yet continuous tissue zones(bone-fibrocartilage-tendon),termed“enthesis”.Regrettably,rapid and functional enthesis regeneration is challenging after RC tear.The existing grafts bioengineered for RC repair are insufficient,as they were engineered by a scaffold that did not mimic normal enthesis in morphology,composition,and tensile property,meanwhile cannot simultaneously stimulate the formation of bone-fibrocartilage-tendon tissues.Herein,an optimized decellularization approach based on a vacuum aspiration device(VAD)was developed to fabricate a book-shaped decellularized enthesis matrix(O-BDEM).Then,three recombinant growth factors(CBP-GFs)capable of binding collagen were synthesized by fusing a collagen-binding peptide(CBP)into the N-terminal of BMP-2,TGF-β3,or GDF-7,and zone-specifically tethered to the collagen of O-BDEM to fabricate a novel scaffold(CBP-GFs/O-BDEM)satisfying the above-mentioned requirements.After ensuring the low immunogenicity of CBP-GFs/O-BDEM by a novel single-cell mass cytometry in a mouse model,we interleaved urine-derived stem cell-sheets into this CBP-GFs/O-BDEM to bioengineer an enthesis-like graft.Its high-performance on regenerating enthesis was determined in a canine model.These findings indicate this CBP-GFs/O-BDEM may be an excellent scaffold for constructing enthesis-like graft to patch large/massive RC tears,and provide breakthroughs in fabricating graded interfacial tissue.

Tyrosine hydroxylase-positive cells and dopaminergic neuronal function in human embryonic stem cells: An electrophysiological validation

BACKGROUND： Induced differentiation strategies and cytochemical properties of human embryonic stem ceils （hESCs） have been investigated. However, the electrophysiological functions of tyrosine hydroxylase （TH）-positive cells dedved from hESCs remain unclear. OBJECTIVE： To investigate the differentiation efficiency of TH-positive cells from hESCs in vitro using modified four-step culture methods, including embryoid body formation, and to examine the functional characteristics of the differentiated TH-positive cells using electrophysiological techniques. DESIGN, TIME AND SETTING： Neuroelectrophysiology was performed at the Reproductive Medicine Center and Stem Cell Research Center, Peking University Third Hospital, and the Neuroscience Research Institute and Department of Neurobiology, Peking University, from September 2004 to August 2008. MATERIALS： The hESC line, PKU-1.1, a monoclonal cell line derived from a pre-implantation human blastocyst in the Reproductive Medical Center of Peking University Third Hospital. The patch clamp recording system was provided by the Neuroscience Research Institute and Department of Neurobiology, Peking University. METHODS： The hESC line was induced to differentiate into TH-positive cells in vitro using a modified four-step culture method, including the formation of embryoid body, as well as the presence of sonic hedgehog and fibroblast growth factor 8. The cell karyotype was assessed by G-banding karyotype analysis techniques and specific markers were detected immunocytochemically. Whole-cell configuration was obtained after obtaining a tight seal of over 1 GΩ. Ionic currents were detected by holding the cells at -70 mV and stepping to test voltages between -80 and 40 mV in 10-mV increments in voltage-clamp configuration. MAIN OUTCOME MEASURES： We measured the cell karyotype, specific cell markers, and the electrophysiological properties of the voltage-gated ion channels on the cell membrane of TH-positive dopaminergic cells differentiated from our hESCs line in vitro. RESULTS： The differentiated cells had a consistent appearance, and the majority of cells （〉 90%） expressed TH and β-tubulion, as well as the neural progenitor marker, nestino Cell karyotype analysis demonstrated that all of the hESCs had a stable and normal karyotype （46, XX） after differentiation. In addition, patch clamp recording showed that the 10 recorded TH-positive cells exhibited a fast inward current when the test voltage depolarized to -30 mV, and a delayed outward current when the test voltage depolarized to -10 mV. The peak of inward current was obtained at voltage between 10 mV and 0 mV, while the peak of outward current was obtained at 40 mV. The average peak of inward current density was （ -50.05 ± 15.50） pA/pF, and the average peak of outward current density was （41.98 ± 13.55） pA/pE CONCLUSION： More than 90% of the differentiated hESC-derived cells induced by the modified four-step culture method exhibit dopaminergic neuronal properties, including general electrophysiological functional properties, such as functional potassium and sodium channels.

Therapeutic Effects of All Trans-retinoitc Acid Combined with Transarterial Chemoembolization on Walker-256 Hepatoma in Rats

In order to investigate the inhibitory effects of all trans-retinoitc acid(ATRA)on differentiation and apoptosis of Walker-256 hepatocellular carcinoma cells and the therapeutic effects of ATRA combined with transarterial chemoembolization(TACE)on rat Walker-256 transplanted hepatocarcinoma,Walker-256 hepatocarcinoma cell lines were treated with ATRA at different concentrations.After culture for 48h,the inhibitory rate of cell proliferation was determined by MTT assay;the changes of Fas and Bcl-2mRNA expres...