In vitro differentiation of human adipose-derived adult stromal cells into neuron-like cells in hippocampal astrocyte conditioned medium

BACKGROUND: At present, researches on differentiating from human adipose-derived adult stromal cells (hADASC) to neuron-like cells are focus on inducing by artificial-synthetic compound solution; however, hippocampal astrocyte conditioned medium (HCAM) can induce in vitro differentiation from hADASC to neuron-like cells is still unclear. OBJECTIVE: To observe whether HCAM can induce in vitro differentiation from hADASC to neuron-like cells. DESIGN: Randomized control study. SETTING: Department of Neurology, Taixing People's Hospital; Central Laboratory, North China Coal Medical College. MATERIALS: Donor of adipose tissue was donated by female volunteers suffering from caesarean section in the department of obstetrics & gynecology in our hospital and aged 20-35 years. Adipose tissue was collected from subcutaneous tissue of abdomen during the operation. In addition, 8 male newborn Wistar rats within 24 hours with average body mass of 20 g were provided by Animal Institute of Chinese Academy of Medical Sciences. Rabbit-anti-human Nestin polyclonal antibody, rabbit-anti-human glial fibriliary acidic protein (GFAP) polyclonal antibody, rabbit-anti-human neuro-specific enolase polyclonal antibody and mouse-anti-human microtubal associated protein 2 (MAP-2) polyclonal antibody were provided by Wuhan Boster Company. METHODS: The experiment was carried out in the Central Laboratory of North China Coal Medical College from October 2004 to June 2005. hADASC was cultured with HCAM and its growth and morphological changes were observed under inverted phase contrast microscope. Immunocytochemistry, immunofluorescence and Western blotting were used to evaluate the expressions of Nestin, which was a specific sign of nerve precursor, neuro-specific enolase and MAP-2, which was a specific sign of nerve cell, and GFAP, which was a specific sign of neuroglial cells. MAIN OUTCOME MEASURES: Nestin, which was a specific sign of nerve precursor, neuro-specific enolase and MAP-2, which was a specific sign of nerve cell, and GFAP, which was a specific sign of neuroglial cells. RESULTS: On the 3rd day of culture, partial hADASC started deformation from slender shuttle-shape cells to neuron-like cells. It suggested that cells stretched out apophysis, which were mainly double-pole or multiple-pole cells. Five days later, immunohistochemical detection suggested that expression of Nestin (10.5±0.037) was found out in cells; meanwhile, expressions of GFAP (38.4±0.052) and neuro-specific enolase (NSE) (15.7±0.023) were also found out in cells; however, expression of MAP-2 was not observed. Western blot indicated that, 5 days after effect of HCAM, Nestin was found out in hADASC; meanwhile, expressions of GFAP and neuro-specific enolase were also found out; however, expression of MAP-2 was not observed. CONCLUSION: HCAM can induce the differentiation from hADASC to neuron-like cells in vitro.

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.

Lethal effect of mononuclear cells derived from human umbilical cord blood differentiating into dendritic cells after in vitro induction of cytokines on neuroblastoma cells

BACKGROUND： Dendritic cell is the most major antigen presenting cell of organism. It is proved in recent studies that human umbilical cord blood mononuclear cells induced and cultured in vitro by recombinant human granulocyte-macrophage colony stimulating factor （rhG-MCSF） and recombinant human interleukin-4 （rhlL-4） can generate a great many dendritic cells and promote the lethal effect of T cells on human neuroblastoma, but it is unclear that whether the lethal effect is associated with the most proper concentration of dendritic cells. OBJEETIVE： To investigate the lethal effect of human umbilical cord blood mononuclear cells induced in vitro by cytokines differentiating into dendritic cells on human neuroblastoma, and its best concentration range. DESIGN ： Open experiment SEI-FING： Department of Pediatrics, the Medical School Hospital of Qingdao University MATERIALS ： The study was carried out in the Shandong Provincial Key Laboratory （Laboratory for the Department of Pediatrics of the Medical School Hospital of Qingdao University） during September 2005 to May 2006. Human umbilical cord blood samples were taken from the healthy newborn infants of full-term normal delivery during October to November 2005 in the Medical School Hospital of Qingdao University, and were voluntarily donated by the puerperas. Main instruments： type 3111 CO2 incubator （Forma Scientific, USA）, type 550 ELISA Reader （Bio-Rad, USA）. Main reagents： neuroblastoma cell line SK-N-SH （Shanghai Institute of Life Science, Chinese Academy of Sciences）, RPMI-1640 culture fluid and fetal bovine serum （Hyclone）, rhlL-4 （Promega, USA）, rhG-MCSF （Harbin Pharmaceutic Group Bioengineering Co.Ltd）, rat anti-human CDla monoclonal antibody and FITC-labeled rabbit anti-rat IgG （Xiehe Stem cell Gene Engineering Co.Ltd）. METHODS： ① Human umbilical cord blood mononuclear cells obtained with attachment methods differentiated into human umbilical cord blood dendritic cells, presenting typical morphology of dendritic cells after in vitro induction by rhG-MCSF and rhlL-4. ② Different concentrations of dendritic cells[ dendritic cells： neuroblastoma cells=20：1,50：1,100：1 （2×10^8 L^-1,5×10^8 L^-1,1×10^9 L^-1）], 1×10^9 L^-1 T cells and 1×10^7 L^-1 neuroblastoma cells were added in the experimental group. 1 ×10^9 L^-1 T cells and 1 ×10^7 L^-1 neuroblastoma cells were added in the control group. ③ Main surface marker CDla molecules of dendritic cells were detected with indirect immunofluorescence, and the percent rate of dendritic cells was counted with ultraviolet light and expressed as the expression rate of CD1a^＋ cells. ④Single effector cells and target cells were respectively set in the experimental group and control group to obtain the lethal effect. The lethal effect of dendritic cells on neuroblastoma cells was indirectly evaluated by detecting cellular survival with MTT assay. The lethal effect（%）= （1-A experimentat well-A effector cell /A target cell well）×100%.⑤The expenmental data were presented as Mean ±SD, and paired t test was used. MAIN OUTCOME MEASURES： ① Morphological characters of dendritic cells in the process of induction and differentiation. ②CD1a^＋ cellular expression rate. ③Lethal effect of dendntic cells on neuroblastoma cells. RESULTS： ①Morphological characters of dendritic cells in the process of induction and differentiation： On the 15^th day after human umbilical cord blood mononuclear cells were induced by rhG-MCSF and rhlL-4, typical morphology of dendritic cells could be seen under an inverted microscope. ②Expression rate of CD1a^＋ cells was （43.12±5.83）%. ③Lethal effect of dendritic cells on neuroblastoma cells： Lethal effect of dendritic cells stimulated T cells in each experimental group （ dendritic cells： neuroblastoma cells=100：1,50：1, 20：1 respectively） on neuroblastoma cells was significantly higher than that in control group[（31.00 ±4.41 ）%, （30.92±5.27）%,（33.57±5.35）%,（26.23±5.20）%, t=3.51,2.98,4.24, P〈 0.01 ）; But the lethal effect of dendntic cells on neuroblastoma was significantly lower when their ratio was 100：1 and 50：1 in comparison with 20：1 （t=2.01,2.36, P 〈 0.05）, and no significant difference in lethal effect existed between the ratio at 100：1 and 50：1 （t=0.06,P 〉 0.05）. CONCLUSION： Dendritic cells differentiated from human umbilical cord blood mononuclear cells after in vitro induction of cytokines can promote the lethal effect of T cells on neuroblastoma cells. The lethal effect is associated with the concentration of dendritic cells within some range.

Prostaglandin E2 promotes hematopoietic development from human embryonic stem cells

Recent studies have suggested that prostaglan-din(PG)E2(PGE2)and the prostaglandin pathway are essential for hematopoietic stem cell growth and develop-ment.However,similar studies on hematopoietic commit-ment from human embryonic stem cells(hESCs)are still limited.Here we report that the addition of PGE2 promotes hematopoietic differentiation of hESCs.The induced cells from hESCs/OP9 co-culture and in the presence of PGE2 were characterized by reverse transcription-PCR(RT-PCR),flow cytometry,colony-forming assays and Wright-Giemsa staining.Our results demonstrated that PGE2 exposure could alter the gene expression pattern and morphology of co-cultured hESCs and resulted in a robust hematopoietic differentiation with higher frequencies of CD34+and CD45+cells.Furthermore,the Smad signaling pathway may be involved in PGE2 and OP9 induced hematopoietic differentiation of hESCs.This research may improve our knowledge of stem cell regulation and hopefully lead to better stem cell-based therapeutic options.

Regulation of RDN on Th1/ILC1 cell imbalance in HFMD patients caused by EV71 infection

Enterovirus 71(EV71)infection is more likely to cause hand,foot and mouth disease(HFMD)in children,which can lead to neurogenic complications and higher mortality.As a commonly used clinical medicine,Reduning injection(RDN)helps to shorten the symptoms of patients with HFMD and facilitate the early recovery of children.However,the regulatory mechanism of RDN on the HFMD immune system disorder caused by EV71 remains to be discussed.This study collected detailed treatment data of56 children with HFMD who entered the affiliated Children’s Hospital of Nanjing Medical University during 2019.Retrospective analysis of clinical data showed that the symptoms of the RDN treatment group were improved compared with the untreated group.To explore its mechanism,the relevant detection indicators were detected by flow cytometry,enzyme-linked immunosorbent assay and realtime quantitative PCR.It was found that the number and function of innate immune(ILCs)and adaptive immunity(Th1,Th2 and secreted cytokines)were reduced,suggesting that RDN plays a role by regulating cellular immunity.The in vitro differentiation inhibition test further confirmed that RDN affected Th1 differentiation by inhibiting the expression of transcription factors on the basis of Th1 cell differentiation in vitro.