Stage-related capacity for limb chondrogenesis in cell culture☆

[摘要] Cellsfromwingbudsofvarying-stagechickembryosweredissociatedandgrowninculturetotesttheircapacityforcartilagedifferentiation.Micro-masscultureswereinitiatedwithacelllayergreaterthanconfluency,whichoccupiedarestrictedareaoftheculturedishsurface(10–13mm2).Cellsfromstage24chickembryowingbuds(priortotheappearanceofcartilageinvivo)undergocartilagedifferentiationinsuchcultures.Typically,duringthefirst1–2daysofculture,cellsformaggregates(clustersofcellswithadensity1.5timesgreaterthanthatofthesurroundingnonaggregatearea).ByDay3,virtuallyallaggregatesdifferentiateintocartilagenoduleswhichareeasilyrecognizedbytheirAlcianbluestaining(pH1.0)extracellularmatrix.Subsequently,nodulesincreaseinsize,andadjacentnodulesbegintocoalesce.Micro-masscultureswereusedtotestthechondrogeniccapacityofwingbudcellsfromchickembryosrepresentingthedifferentstagesoflimbdevelopmentuptotheappearanceofcartilageinvivo(stages17–25).Cellsfromembryostages21–24formaggregateswhichdifferentiateintocartilagenodulesinvitrowithequalcapacity(scoredasnumberofnodulesperculture).Incontrast,cellsfromembryostages17–19formaggregatesinsimilarnumbers,buttheseaggregatesneverdifferentiateintonodulesunderroutineconditions.However,aggregateswhichforminculturesofstage19wingbudcellsdodifferentiateintocartilagenodulesifexposedtodibutyrylcyclicAMPandtheophylline.Cellsfromstage20embryosmanifestavaryingcapacitytoformcartilagenodules;apparently,thisisatransitionstage.Cellsfromstage25embryosproducecartilageinvitrowithoutformingeitheraggregatesornodules.Basedontheresultspresentedinthispaper,theauthorsproposeamodelforcartilagedifferentiationfromembryonicmesodermcellsinvolving:(1)aggregation,(2)acquisitionoftheabilitytorespondtotheenvironmentintheaggregate,(3)elevatedintracellularcyclicAMPlevels,and(4)stabilizationandexpressionofcartilagephenotype.

[学科分类] 生物科学（综合）