Differentiation Of Mesenchymal Stem Cells Towards A Nucleus Pulposus-like Cell In Vitro Based On The New Definition Of Nucleus Pulposus Cell Phenotype

Background:Intervertebral disc degeneration is the main cause of low back painThe pathological mechanism is not clear.Mesenchymal stem cells as seeds for transplantation can retard,or regenerate degenerated discs in vivo and in vitro.Previously,SOX9,type II collagen and aggrecan were used as specific phenotypes of nucleus pulposus cells.The spine research interest group defined the new NPC phenotype at the 2014 Annual ORS meeting.This research is based on the new specific phenotype of NPC to study the possibility of differentiation of BMSC into NPC-like.Objective:To isolate,culture and observe BMSC and nucleus pulposus cells from rat in vitro.Inducing BMSC with different concentration of culture medium from nucleus pulposus cell simulate indirect co-culture,then compare induced BMSC and nucleus pulposus cells phenotypic markers.Methods:Femur,tibia and caudal intervertebral disc are separated from Sprague Dawley rats aged 6-weeks-old under the sterile condition.BMSC from marrow cavity and NPC from caudal intervertebral disc were cultured by adherent cultivation approach.Two kinds of cells’ morphological changes were obsevered by inverted microscope;BMSC were identified by flow cytometry;The pure BMSC and NPC were used to be control.BMSC were induced with different concentrations of NPC culture medium for 7 days.Messenger RNA expression levels of the new nucleus pulposus phenotypic markers of pure NPC and BMSC which were induced were determined by RT-PCR.The markers of NPC are HIF-1α、GLUT-1、Acan/Co12>20、SHH、Brachyury、Krt8、18、19、CA12、CD24.Results:The expression of CD29 and CD44 in BMSC was postive and CD45 was negative.The expression of HIF-1α,GLUT-1,Col2,Acan,Brachyury(T),Krt8,Krt18 Krt19,CA12,CD24and SHH in the nucleus pulposus cells was postive.RT-PCR showed that compared with the pure nucleus pulposus cells,the expression of HIF-1α,GLUT-1,Col2,Acan,Brachyury(T),Krt8,Krtl8 Krtl9,CA12,CD24and SHH in the pure BMSC were negative.The expression of HIF-la,GLUT-1,Col2,Acan,Brachyury(T),Krt8,Krt18 Krt19,CA12,CD24and SHH in BMSC induced by 50%of concentrations of nucleus pulposus cell culture medium was increased.The expression of Co12,Brachyury(T),Krt8,Krt18 Krt19,CA12and SHH in BMSC induced by 100%of concentrations of nucleus pulposus cell culture medium was increased.Conclusion:1.The mesenchymal stem cells can be induced to differentiate into nucleus pulposus cells through the nucleus pulposus cell culture medium.2.The degree of differentiation which mesenchymal stem cells induced by 50%concentration of nucleus pulposus cell culture medium into nucleus pulposus-like cell is more higher.3.Mesenchymal stem cells can potentially retard,or regenerate degenerated discs structure and function.