Neuronal Differentiation And Protective Effects On Iron-overload Primary Cortical Neurons Of Adipose Derived Stem Cells In Vitro

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Isolation,culture and characterization of rat adipose derived stem cells and primary cortical neuronsBackgrounds Adipose derived stem cells are an important kind of mesenchymal stem cells from adipose tissue.They play an important role in many central nervous system diseases such as degenerative disease,inflammatory demyelination and stroke.Aims To isolate,cultivate primary cortical neurons and adipose derived stem cells which could be passaged stablely in vitro.Methods Inguinal adipose tissue were removed from 4-6 weeks old,weighing 160 g female SD rat under sterile conditions to isolate adipose-derived stem cells.Adipose tissues were chopped,and then digested with Collagenase I for 45 minutes.Add equal volume of complete medium to terminate digestion and ADSCs were incubated in complete culture mediu.Refresh culture medium every 3 days.According to the growth conditions,7-8 days primary adipose-derived stem cells could be treated with passage processing.Primary rat cortical neurons were derived from fetal rats(gestational age of 16-17 days).Stripped of meninges and the cortex tissues were chopped.Debris was digested in trypsin for 15 minutes.Add three times the volume of the complete culture medium to terminate digestion.After blowed using Pap straw,cells were seeded on polylysine-coated dishes.Four hours later,neuron culture medium was added and refreshed culture medium half every 3 days.Results Adipose-derived stem cells cultured in vitro were adherent and spindle shaped.ADSCs highly expressed CD44(86.69%)and CD29(98.49%),but did not express hematopoietic stem cell markers CD45 and extremely low expression of endothelial cell marker CD34(8.61%).The primary neurons isolated and cultured in vitro could be matural for 11 days.They had large bodies,rich protrusions and connected with each other.Conclusions Adipose-derived stem cells and functional matural neurons can be cultured in vitro.Par ? Neural directional differentiation of adipose-derived stem cells and preliminary functional identificationBackgrounds Adipose-derived stem cells can be directionally differentiated into neuron-like cells induced by small molecules and cytokines.The potentiality of ADSCs differentiating into neuron-like cells may play a critical role in the treatment of neurological diseases.Whether neuron-like cells has neural morphology and function and whether can be integrated into a new neural network has become focus.Aims To induce adipose-derived stem cells directional differentiate into neuron-like cells in vitro and explore its function.Methods Neural directional differentiation of adipose-derived stem cells was divided into two steps.First,ADSCs was induced into the neural precursor cells in a serum-free medium containing 20ng/ml EGF,20ng/ml b FGF-2 and 2% B27.Immunofluorescence staining(Nestin,DCX,MAP2)was used to identify the cells before and after induction.Secondly,neural precursor cells were induced to adherent neuron-like cells in a serum-free medium containing 2% B27,50ng/ml BDNF,0.5umol/ml RA and 10umol/ml Forskolin.Immunofluorescence staining(Nestin,DCX,MAP2)was used to identify the cells at different stages of differentiation.The preliminary functional study of neuron-like cells was carried out by using fluorescent dye Dio and FM4-64 to label living cells.The Sholl analysis software was used to quantitatively analyze the synapse-like complexity of neuron-like cells at different stages of differentiation.Results Adipose-derived stem cells can grow into spheres under the condition of b FGF and EGF cytokines which were similar to the morphology of neural stem cells.Neural spheres early express Nestin but don not express MAP2 and DCX.Neural precursor cells can further differentiate into neuron-like cells under the condition of RA,Forskolin and BDNF.Neuron-like cells presented neurons morphology and connect with each other at 14 days in vitro.They express the newborn neurons marker DCX and the mature neurons marker MAP2 in the later period.With the prolongation of the time,the synaptic complexity of neuron-like cells increased gradually.At about 14 days,neuron-like cells have preliminary neural function presented by releasing synapse-like vesicles under the stimulation of high potassium Kreb's solution.Conclusions Adipose-derived stem cells can differentiate into neuron-like cells under the action of small molecules and cytokines and have preliminary neural function.Part ? Neuroprotective effects of ADSCs on iron-overload primary cortical neurons and molecular mechanismsBackgrounds ADSCs not only has self-renewal and multipotential differentiation potentials,they also could secret multiple bioactive cytokines and participate in regulating inflammatory reaction.Iron-induced apoptosis is an important way of programmed cell death in a variety of diseases,such as stroke,neurodegenerative diseases.It is a new target to explore the specific mechanism of ADSCs' protective effects on iron-overload neurons.Aims To elucidate neuroprotective effects of ADSCs on iron-overload neurons and its specific mechanisms.Methods Indirectional co-culture system of ADSCs and primary cortical neurons was established by using 0.4?m Transwell culture dishes.Ferrous sulfate(Fe SO4)was added to simulate an in vitro model of secondary iron-overload after intracerebral hemorrhage,and the optimal concentration of Fe SO4 was determined by MTT test.According to the purposes of experiment,cortical neurons were divided into four groups: normal control group(Vehicle),ADSCs co-culture control group(ADSCs),ferrous sulfate treatment group(Fe SO4)and ADSCs treatment group(ADSCs+Fe SO4 group).Cell apoptosis rate of different groups was detected by flow cytometry with Annexin V/PI double labeling.The concentration of lipid peroxidation product MDA was determined by MDA kits.ELISA was used to detect the content of insulin-like growth factor-1 in different groups.The activities of t NOS and i NOS were measured by the nitric oxide synthase kits.Protein expression levels of PI3K/Akt,Nrf2/HO-1 and Bax/Bcl-2 related signaling pathways in cortical neurons were presented by Western-Blots.The expression of HO-1 in ADSCs was measured by immunofluorescence staining.Results Toxic effects of FeSO4 on cortical neurons were concentration dependent,and 8?M was the best concentration.Compared with Vehicle and ADSCs groups,the MDA concentrations,t NOS and i NOS activities of neurons in Fe SO4 group increased significantly.While the MDA concentrations,t NOS and i NOS activities of neurons in ADSCs+ Fe SO4 group were significantly lower than those in Fe SO4 group.The level of IGF-1 in ADSCs and ADSCs+ Fe SO4 groups was significantly higher than that in Vehicle and Fe SO4 groups.Flow cytometry result showed that cells in Vehicle and ADSCs groups had a little apoptosis,and the apoptotic rate was about 16.9%.The apoptotic rate of neurons in Fe SO4 group markedly increased(27.6%),while decreased to 22.4% in ADSCs+ Fe SO4 group.WB results showed that under normal circumstances,Vehicle and ADSCs groups expressed very low p-PI3 K,Nrf2 and HO-1,and expressed some degrees of Akt,p-Akt,Bax and Bcl2 to play a normal physiological role.The expressions of p-PI3 k,p-Akt and Bcl2 in the neurons in Fe SO4 group decreased significantly and Nrf2,HO-1 increased significantly.Compared with the Fe SO4 group,the expressions of p-PI3 k,p-Akt and Bcl2 increased significantly,while the expression of Nrf2 and HO-1 further increased in the ADSCs+ Fe SO4 group.In ADSCs,the expression of HO-1 was significantly increased treated with Fe SO4(*p<0.05 or **p<0.01).Conclusions Co-culturing with ADSCs can reduce the level of Fe SO4 induced lipid peroxidation products,reduce neuronal apoptosis,NOS activities,and down-regulate the levels of inflammation in primary cultured cortical neurons.The specific mechanisms were that ADSCs activate IGF-1/PI3K/Akt signaling pathway to promote neuronal survival by paracrine,activate oxidative stress pathways Nrf2/HO-1 to exert anti-inflammatory effects,and activate mitochondrial apoptosis pathway Bax/Bcl-2 to reduce neuronal apoptosis.