Effect Of Cerebral Cortex Microenvironment On Differentiation Of Umbilical Cord-mesenchymal Stem Cells

Objective:As the coming of aging society, the incidence rate of nervousdegenerative diseases is increasing rapidly. The most commonneurodegenerative diseases, such as presenile dementia and Parkinson, aregetting more attention all over the world. Stem cells are on the behalf of thedevelopment of neural regenerating medicine research which provides a newthinking and theraputic strategy for neural system diseases. Umbilicalcord-mesenchymal stem cell is a multipotential stem cell strain which isderived from mesoderm and has a potential of self renew and differentiation.Compared with somatic stem cell, umbilical cord-mesenchymal stem cellshave stronger potency of proliferation and differentiation. In special internal orexternal environment, umbilical cord-mesenchymal stem cells candifferentiate into any kinds of tissue cells and are used in stem cell therapyrecently. Study on directional differentiation is the focus of stem cells research.In the present study, extracts from rats' cerebral cortex tissue were used toinduce the differentiation of human umbilical cord-mesenchymal stem cells(hUC-MSCs). We observed whether the induced cells are characterized byneuron markers in morphology and molecular aspects. Immunocytochemistrymethod was used to investigate the expression of neuron specific enolase(NSE) and glutamic acid decarboxylase (GAD). LC-MS-MS was used todetermin the level of intracellular GABA. The role of cerebral cortexmicroenvironment in umbilical cord-mesenchymal stem cell differentiation isexpected to be demonstrated.Method:1The preparation of rats' cerebral cortex tissue extractsCerebral cortexs were taken out from rats to put on the ice before10maleSprague Dawley (SD) rats weighing200²30g were killed. Cerebral cortex tissue extracts are made of150mg/mL DMEM/F12. Cerebral cortex tissueextracts were centrifugated at the speed of3000r/min at room temperature for15min. the supernatant is filtrated by membrane filtration of0.22um asinducing culture medium.2Observing the shape of normally culture MSCs and cells induced byrat'scerebral cortex tissue extractsHUC-MSCs we used were from prassege4to6recognized a by flowcytometry. Single cell suspension was consisted of hUC-MSCs with thedensity of5×104/ml.24well culture clusters which was put by cover slip werefilled with0.5mL single cell suspension. Then put the culture clusters intosaturated humidity incubator with37℃,5%CO2.24hours later, normallyculture medium in to culture clusters were changed by rats' cerebral cortextissue extracts. Compared with control group of hUC-MSCs, closely observethe change of cell shape.3Taking use of immunocytochemistry stainingAccording to instruction of SABC immunocytochemistry, the inducedcells were stainned and identified whether to be gamma-aminobutyric acidneurons and infered whether to have the character of gamma-aminobutyricacid neurons.4Collectting and handling cell samplesCulture medium was removed from cell culture clusters on the ice. thecells were rinsed to be cleaned and centrifugated after the adherent cells oncell culture cluster were scraped by cell scraper. Collect the cells to clearagethe cells by cells ultrasonic fragmentator. then collect supernatant to preservein-70℃for measuring5Measuring the quantity of GABA in cell samples by LC-MS-MS.ZIC-HILIC column (3.5μm,2.1mm×50mm,100A PEEK HPLCColumn Merck Sequant company) was used as analytical column; mobilephase is consisted of80%Methyl Cyanides and20%water (0.5%formic acid)with flow rate of200μL·min^-1. GABA was detected using mode in thepositive electrospray ionisation (ESI) mode. The ion spray voltagewas set at 4.0kV and Sheath Gas Pressure(N2) at45psi.SRM: m/z104→m/z87(GABA),m/z285→m/z200(morphine), collection energy:10V(GABA)å’Œ24V(morphine), scan time600ms. A sensitive, accurate and specificity testmethod was set up to measure GABA in cells.Results:1Morphology characters of stem cells in normally cultured groupThe normally cultured cells were observed under microscope, most of thecells are in shuttle shape or polygon; the cells reflect well and nucleoli areeasily seen. A small amounts of cell bodies are large and thin and flat.2Morphology alterations of the hUC-MSCs after induction by rats' cerebralcortex tissue extractsDifferentiation state and morphology alterations of the hUC-MSCs afterrats' cerebral cortex tissue extracts induction were observed. Morphology ofmost cells has an obviously change, it shows cell body shrinks and change tobe round the apophysis arise and become longer with cell body muchbrightness and strong reflected light;48hours later, most cells change fromround to clostridial form and polygons and irregular form.;72hours later,most of cells do not have obvious change in shape.3The expression of NSE and GAD-67of cells after inductionImmunocytochemistry were used to measure the expression of NSE andGAD-67after hUC-MSCs are induced to neuron for72hours. HUC-MSCsdidn't express NSE and GAD-67; most cells of induction express NSE andGAD-67.4Methodology corroboration of detecting GABAA sensitive, facilitated, and reliable HPLC-MS-MS assay was developedto measure GABA in cells. GABA of cells has a satisfactory linear relationshipin limit of5ng⁵00ng·mL^-1. The lower limit of quantization (LLOQ) is5ng·mL^-1. Good linearity, reproducibility and accuracy were observed formeasurement of GABA in cells. Quantification of GABA in hUC-MSCs islower than lower limit of quantization, while the quantity of GABA in cellsafter induction can be measured and quantitated. 5An effect rats'cerebral cortex tissue extracts induction has for the quantity ofGABA in cells.The quantity of GABA in hUC-MSCs is lower than lower limit ofquantization. The quantity of GABA in cells of0d group is0ng; The quantityof GABA in cells of3d group is (48.6±9.6) ng; The quantity of GABA in cellsof5d group is (59.4±4.9) ng; The quantity of GABA in cells of5d group is(28.6±6.0) ng. Compared with3d group, the quantity of GABA in cells of7dgroup decreased51.8%(P<0.05), while the quantity of GABA in cells of5dgroup has no difference(P>0.05).Conclusion:1The shape of MSCs induced by the rat's cerebral cortex tissue extracts has amarked change. The result of immunocytochemistry shows that the inducedcell expresses NSE and GAD-67and indicates the induced cells partly havethe character of aminobutyric acid neuron in shape and molecule marker.2Compared with normally cultured MSCs, quantity of GABA in inducedgroup is obviously different. The result indicates cells induced by rat'scerebral cortex tissue extracts may be cable to synthesis GABA.