Isolation And Culture Of OPCs Lineage Cells And Induced Differentiation

Objective: Oligodendrocytes, the myelinating glia of the central nervoussystem (CNS), play a crucial role in facilitating the rapid conduction ofneuronal action potentials and supporting axonal survival.Oligodendrocytesare generated from OPCs, which proliferate and migrate throughout the CNSduring late embryonic development,and later differentiate into maturemyelinating oligodendrocytes.Several distinct stages during oligodendrocytematuration have been identified for oligodendroglial lineage cells in vitro3,5.These include proliferating OPCs, characterized by expression of progenitorcell markers such as the platelet-derived growth factor alpha receptor(PDGFaR) with a bipolar or tripoIar morphology,intermediate immatureoligodendrocytes, expressing markers recognized by the O4antibody with amultipolar morphology, and finally mature myelinating oligodendrocytes,expressing myelinspecific proteins such as myelin basic protein.Simplemethods for the isolation and purification of workable quantities of OPCs notonly aid in efforts to better understand oligodendrocyte development, functionand axon-oligodendroglial interactions but also provide an indispensable toolfor myelin repair research.More importently, when transplanted into the brainof hypomyelinated hosts, oligodendrocyte precursors can migrate a significantdistance, and give rise to a large number of mature oligodendrocytes,so thetreatment of planting O2A/OPCs to cure demyelinating desease is in trial. alarge, highly enriched population of OPCs lineage cells are in need.Meanwhile, These methods of Isolation and culture of oligodendrocyteprecursor lineage cells will facilitate the in vitro use of OPCs to address suchissues as effects of drugs on demyelinating desease.Methods:1.mixed glial cultures of new born Sprague DawleyRat:Decapitate a P1-2rat,Anesthetize the rats first by burying them in ice for 1-5minutes. Divide the cerebrum and cut off olfactory bulbs, basal gangliaand the hippocampus. Dice and the Triturate cortical tissues, Pipette thecontents of the Petri dish into a sterile centrifuge tube,Add HBSS with DNaseI stock solution and0.6ml trypsin stock solution, about15minutes later stoptrypsinization, centrifuge,then spread cell suspension over the entire flask,Feed every2-3days with complete medium change of10ml DMEM20S for8-9days.2. Primary OPCs were isolated from mixed glial cultures by using aselective detachment procedure,further purified by differential adhesion,andmaintained in a chemical conditioned medium,with appended platelet-derived growth factor (PDGF),basic fibroblast growth factor (bFGF). MTTassay was used to identify the vitality of OPCs in vitro,immunocytochemicalanalysis evealed that more than95%of the isolated cells expressed the OPCsspecific marker A2B5, and measure the purity of the the OPCs3. change the medium to chemical conditioned medium without serumand with serum seperately, Then the changes among its developmental stageswere observed and the mature cells were immunostained by relevant antibodyResults:1. At7-8days after plating, mixed glial cultures will be confluent, and OPCsappear on top of phase-gray bed layers of astrocytes；2. OPCs were small and have one or two fine processes,Immunocytochemicalanalysis revealed that more than95%of the isolated cells expressed the OPCsspecific marker A2B5,the purity of the the OPCs is95%；3. OPCs had bipotential differentiation features,they could differentiate intooligodendrocytes or type-2-astrocytes according to the ingredient of theculture mediumConclusions: The method for OPC preparation through oligospheregeneration from rat neural progenitor cells is simple and straightforward.It canbe easily followed by a researcher without prior OPC isolation experience.High ratios of purified oligodendrocytes can be obtained by twice orbitalshaking and proper culture medium.Cell output will increase notabily and restin immature phase by appending both PDGF and bFGF.OPCs had bipotential differentiation features,they could differentiate into oligodendrocytes ortype-2-astrocytes,which could be used in other trials such as how drugscontribute to neuroglial cells.