Isolation Of Rat Adipose-derived Stromal Cells And Differentiation Into Neural Cells By Two-step Way In Vitro

Adipose-derived stromal cells(ADSCs) are the one type of stem cells with multiple potencies,existing in adipose tissue,which show the same potency in differentiation with embryonic stem cells(ESCs),and can differentiate into some other of tissue cells such as sarcoblast and adipocyte,all of which are originaly derived from mesoderm and ectoderm.Compared with the neural stem cells(NSCs) from both embryonic tissue and adult central nerve system(CNS),the ADSCs possess the more advantages including abundance sources,easy collection,low immunological rejection and ethics limits.Therefore,ADSCs have the high potential of clinic application in the field of regeneration medicine of CNS diseases.This experiment aimed to explore the methodology of the isolation of ADSCs and their differentiation into the neural cells,to establish one convenient high-efficientive method of culturing NSCs from ADSCs in vitro,and to optimize the experimental process of the induced differentiation of adult ADSCs into neural cells to enhance the ratio of differentiationin vitro,then to offer a research base for cellular transplant therapy of CNS diseases in clinic application.Partâ… :to observe the cultivation and amplifieatory of ADSCs from adult rats in vitro and their biological characteristics AIM:To isolate,purificate and amplify the ADSCs from adult rats in vitro and to observe their biological properties.METHODS:To collect the retroperitoneal adipose tissue of SD rats under aseptic anesthetic state.To shear it into pieces and centrifuge following the enzymatic digestion with collagenase I,then cultivate the cells in DMEM/F12 medium with 10%of fetal bovine serum(FBS) in culture flask 37℃,5%CO₂.To trace and investigate the growth condition of living cells under the CK2 contrast phase microscope.To detect the cellular proliferation condition by methyl thiazolyl tetrazolium(MTT) and draw the cellular growth curve.To detect the cellular surface markers such as CD34,CD106,CD44 and HLA-1 with both flow cytometer and immunofluorescence cytochemical staining.RESULTS:The majority of inoculated cells were globular floating in nutritive medium and a little lipid droplet suspending on the top level layer under the inverted microscope.The pro-ADSCs had diverse morphous,e.x.globular,long fusiform and fiber forming-like.The contaminated cells were eliminated through the adhering transfer of culture,and the cellular morphous were maintain as the uniformity.The long fusiform,whirlpool-like cells lined up in order at the time of 5^th generation.The passage cells proliferated twice in size per 24h and showed stronger chemical positive staining of CD44 by immunocytochemistry.The rates of cell-surface markers under the flow cytometer were CD34 0.8%,CD106 13.6%,CD44 99.7%,HLA-1 96.7% respectly,which all conformed to the phenotype characteristics of ADSCs.CONCLUSIONS:It was convenient and feasible to adopt the method of the isolation,civilization of cells,in this experiment,the acquired ADSCs could be highly purified with thriving cellular vigor and powerful self-renewal capability.Partâ…¡:Differentiation of adult rats ADSCs into neural cells with two-step methodAIM:To investigate the experimental methods of inducment from ADSCs into neurospheres that consisted of NSCs,and the neural differentiation from neurospheres into the neural cells. METHODS:We induced and differentiated passage 5 of ADSCs,combined with basic fibroblast growth factor(bFGF) 20ng/ml,epithelial growth factor(EGF) 20ng/ml and N2(1:100) additives,in the serum-free Neurobasal nutrient medium. The ADSCs were at first induced into the neurospheres,and then blew slightly in order to scatter them by the mechanical method by the time of formative neurospheres,then passaged at the ratio of 1:3 after being re-alligated in the Neurobasal nutrient medium.1%FBS,5%HoS,0.5μmol/L retinoic acid(RA)and 50ng/mL brain-derived neurotrophic factor(BDNF) were added into the 2^nd generation neurospheres to stimulate them differentiating into the neurons and the glials.The expressions of the marker proteins such as Nestin,MAP2ab,β-tubulinâ…¢, glia fibrillary acidic protein(GFAP) and Galactocerebroside(GalC) were assayed with immunocytochemistry(ICC).The cellular morphous were observated and the markers positive cells were shot and counted under the fluorescent microscope.Then the surface topography of the differentiated cells was viewed with the help of scanning electron microscope(SEM).RESULTS:The fusiform ADSCs grew,adhering the plate wall after passaged and purified,with thriving proliferation capability and almost without the expression of positive Nestin.At the 3^rd -4^th day,the cells began to concentrate at the center, forming lots of colonic groups in the serum-free Neurobasal nutrient medium.At the 5^th -7^th day,many of small floating neurospheres emerged,which proliferated so fast that their diameter could amount to 100um at the 14^th day following the inducement. After adding both the serum and the neurotrophic substances such as bFGF,EGF and N2 into the Neurobasal nutrient medium,the NSCs in neurospheres gradually spreaded and grew adhering the plate wall,with different prodiferation and thriving accretion ability.At the beginning of the 4^th-5^th day after induced,part of the cells from the neurospheres presented the classic neuron-like change.Under SEM, compared with undifferetiation condition,some of the applanate ADSCs appeared fiber-like projects from the cellular bodies.The induced neuron-like ADSCs seemed more stereo with more secretion at the cellular surface,and became the long spindle-form with prominences.IMF staining showed that the neurospheres expressed the positive Nestin highly.Majority of the neurospheres differentiated cells to display MAP2 andβ-tubulinâ…¢(+),but with little expression of GFAP and GalC.CONCLUSIONS:By the method in this experiment,high percentage of NSCs clones could be gotten,with neurobasal serum-free medium added N2,bFGF and EGF because of the NSCs proliferation.They could stably show the characters of NSCs and retain the ability of the clone in continuous passage culture.The differentiated cells presented typical neuron-like morphology under light microscope and SEM.The immunocytochemistry test showed that differentiated cells belonged to the high percentage of nerve tissue cells,especially the neurons.All these results implied that the experimental program for differentiaring ADSCs into neural cells in this study is efficient and feasible.