Lineage Tracing Study On Postnatal Growth,degeneration And Regeneration Of Nucleus Pulposus Subpopulation

As the global aged tendency of population,the incidence of degenerative diseases of bone such as osteoporosis,osteoarthritis and degeneration diseases of intervertebral disc(DDD)gradually increases.Among them,DDD happens at early age and causes severe symptoms,which not only have influence on the livelihood of people but also bring heavy burden to the society.The pathological changes of degenerative intervertebral disc(IVD)include cell senescence,cell loss and abnormal metabolism of extracellular matrix.Molecular and cellular abnormalities lead to the damages of the structure and function of IVD,which eventually induce clinical outcomes.Conservative therapies and surgical treatment are major treatment means of DDD,bringing symptomatic relief of patients instead of prevention of the degenerative process.Cell therapy is a promising cure for DDD.It is aimed to restore the structure and function of IVD by supplying stem cells or progenitor cells to degenerated disc,especially nucleus pulposus(NP).However,the current curative effect of cell therapy is unsatisfied due to the inefficiency and safety problem of the cell transplant~1.The underlying obstruct is the lack of understanding of in vivo regenerative ability of the disc cells,especially NP cells.The process of tissue regeneration is partly a recreation of developmental stage.Similar cell behaviors are experienced during regeneration and development,including cell proliferation,differentiation and fate determination of tissue-specific cells,usually stem cells or progenitor cells.Finding the origin of adult stem/progenitor cells can provide the source of seed cells for repair.Besides,cell proliferation is also very important.It is the cellular basis of the morphogenesis during development and the key to initiate self-repair.Nowadays,researches indicated a big difference between the in vitro and in vivo proliferating ability of NP cells.Meanwhile,the cellular contribution of the isolated NP cell with stem cell characteristics to development and regeneration of IVD is still unclear.To solve these problems,we investigated in vivo cell behaviors especially cell proliferation of NP cells during postnatal growth and repair.By utilizing lineage tracing strategy,we revealed the cellular mechanism of NP development and degeneration by discovering two subpopulations of NP which play important roles on both processes.Part ⅠThe in vivo proliferation characteristics of NP cells during postnatal growth,degeneration and regenerationMethods:1.Proliferation of NP cells in lumbar and caudal vertebrae by Ki67 immunostaining and Ed U incorporation assay:Three-day-old(P3),two-week-old(2w)and four-week-old(4w)mice were intraperitoneally injected with 1(Ed U×1)or 6 doses(Ed U×6)of Ed U(1 mg/10 g body weight)and then sacrificed.The sections of lumbar and caudal vertebrae were stained with Ki67 and Ed U respectively.The number of proliferative NP cells were counted.2.Ed U long-term labeling assay of NP:Intraperitoneal 6 injections of Ed U(1 mg/10 g body weight)were performed at 1w.The spines were harvested after injection,at 4w,6 w and 7-month-old(7m).After Ed U staining,Ed U~+NP cells were calculated.3.The proliferating characteristics of immature and mature NP cells by clonal lineage tracing of Aggrecan Cre ERT2;Rosa26-Confetti mice:Tamoxifen was injected continuously for 3 days(1 mg/10 g body weight)at 2w(immature)and 2m(mature)respectively.Samples were collected right after injection and after 1 month of tracing.4.Tail-loop mice model:Tails of mice was bent and fixed for 2 weeks(Loop2w group)and 4 weeks(Loop4w group)to induce DDD.The fixator was removed after 2 weeks of looping.Mice were sacrificed after another 2 weeks(Unloop group).The control group was performed a sham operation.5.Tail-loop mice model analysis:6.The morphologic analyses were performed by X-ray.The histological analysis is performed by Safranin O/Fast green staining,Hematoxylin/Eosin staining,and scoring.The cell proliferation was detected by incorporation of Ed U before sacrifice and q PCR analysis of the m RNA expression of cell cycle related genes.Results:1.Ki67 immunostaining and Ed U incorporation assay:Multiple injections of Ed U(Ed U×6)labelled more proliferative NP cells.The number of proliferative NP cells decreased rapidly after birth,but the proliferation of NP cells didn’t cease at 4w.The percentage of proliferating NP cells in caudal vertebrae was higher than that in lumbar vertebrae.Most of the proliferative NP cells were located in the outer region of NP.2.Ed U long-term labeling experiment:The number of long-term labeled Ed U~+NP cells increased indicating the slow self-renewal of NP tissue.The distribution of long-term labeled Ed U~+NP cells gradually moved from the outer region to the inner region in nucleus pulposus suggesting that the proliferated NP cells remained in the inner region3.Clonal lineage tracing of Aggrecan Cre ERT2;Rosa26-Confetti mice:Single-colored fluorescent cell colonies were found in both immature and mature NP.Cell division still exists in mature NP.4.Tail-looping model:Gross phenotype analysis showed that the height of NP was affected by looping,but the height could be completely restored by unlooping.Histopathological analysis showed that looping caused degeneration,scores raises significantly;but the structure of the mice in unloop group was restored,and no significant difference were found both control and unloop groups.The results of cell cycle and cell proliferation analysis showed that the number of Ed U~+cells and the expression of cell cycle related genes in NP of unloop mice were significantly increased.Conclusion:1.Cell proliferation does not cease after the early stage of postnatal growth.It existes in mature NP during homeostasis and regeneration。2.The outer region of NP contains more proliferating cells.Caudal NP cells are be more proliferative than lumbar NP cells.Part Ⅱ Study of the role of FGFR3~+NP cells in postnatal growth,degeneration and regeneration of NPMethods:1.The different expression of FGFR3 in outer and inner region of NP:1.1 FGFR3 immunostaining was performed in mouse disc.1.2 Generation of FGFR3-3*Flag-IRES-GFP mice to detect FGFR3-expressing cells in-situ.1.3 FGFR3Cre ERT2;Rosa26-m T/m G mice and FGFR3Cre ERT2;Rosa26-td Tomato mice were taken intraperitoneal injection of tamoxifen for 1,3 or 5 days(1 mg/10 g body weight)at 2w and sacrificed after tamoxifen administration to label the FGFR3-expressing cells.2.The role of FGFR3~+NP cells in postnatal growth:2.1 FGFR3Cre ERT2;Rosa26-m T/m G mice at age of 2w were injected with tamoxifen for3 days intraperitoneally(1 mg/10 g body weight)and sacrificed at 1m,2m,3m and 7m.The percentage of FGFR3~+NP cells,the distribution of FGFR3~+NP cells,height,area,cell number and cell apoptosis in NP were analyzed and calculated.2.2 FGFR3Cre ERT2;Rosa26-DTA mice at age of 1w were injected with single dose of tamoxifen(1 mg/10 g body weight)and sacrificed after 1w to analyze the height,area,cell number,cell apoptosis and cell proliferation in NP.3.The proliferation of FGFR3~+NP cells revealed the characteristics of postnatal growth:3.1 FGFR3Cre ERT2;Rosa26-Confetti mice were injected with single dose of tamoxifen(1 mg/10 g body weight)at 1w and sacrificed at 4m o follow the clonal cell fate of FGFR3~+NP cells.3.2 FGFR3Cre ERT2;Rosa26-m T/m G mice were injected with Ed U(Ed U×6)at 1w followed by tamoxifen administration at 2w.The co-expression of long-term labeling NP cells and FGFR3~+NP cells were counted at 1m and 7m4.The role of FGFR3~+NP cells in degeneration and regeneration:FGFR3Cre ERT2;Rosa26-m T/m G mice at age of 2m were injected with tamoxifen for 3days(1 mg/10 g body weight)and then performed tail-looping surgeries.5.The different proliferating potential between inner and outer NPTamoxifen was injected into FGFR3Cre ERT2;Rosa26-m T/m G mice at age of 2w.NP cells of caudal vertebrae were isolated at 1m.and extracted.FGFR3~+and FGFR3~-NP cells were sorted by flow cytometry.Then the RNA of FGFR3~+and FGFR3~-NP cells were extracted and sent to RNA-sequencing.Results:1.FGFR3 labelled proliferating NP cells in outer region of NP:Immunostaining revealed higher expression of FGFR3 in outer region of NP.FGFR3Cre ERT2 mice labelled outer NP cells at 2w,4w and 4m.FGFR3~+NP cells expressed higher Ki67 than FGFR3~-NP cells.2.FGFR3~+NP cells participate in postnatal growth of NP:The number of FGFR3~+NP cells increased significantly after chasing.The proportion of FGFR3~+NP cells in the outer region significantly increased to nearly 100%.The proportion of FGFR3~+NP cells in the inner region increased significantly from～0%to～40%with the rise of height,volume and cell number of NP.The kill of FGFR3~+cells resulted in disrupt of IVD structures especially caused severe loss of area,height and cell number of NP.3.The proliferation of FGFR3~+NP cells suggested the characteristics of postnatal growth:Single-colored fluorescent cell colonies were found in the outer region of NP in FGFR3Cre ERT2;Rosa26-Confetti mice after chasing.These cell colonies were arranged in columns and distributed radially along the radius of nucleus pulposus.In FGFR3Cre ERT2;Rosa26-m T/m G with Ed U pulse and chase experiment,After tracing,FGFR3~+NP cells formed clones in the outer region of NP,while the Ed U labeled long-term remaining cells remained in the inner area of NP,and Ed U~+FGFR3~+NP cells were found at the junction zone.4.The possible role of FGFR3~+NP cells in degeneration and regeneration:In tail-looping models,compared with the control group,the number of FGFR3~+NP cells decreased after looping.In unloop group,the percentage of FGFR3~+NP significantly higher than the control group.5.Higher cell cycling activity in outer region of NPRNA-seq showed FGFR3~+NP cells after chasing that higher expression of cell cycle related genes in FGFR3~+NP cells than that in FGFR3~-NP cells The cell cycle pathway was also found in the top20 pathway enriched by KEGG database and in the top10 up-regulated pathway enriched in Panther database by GSEA analysis.Conclusion:1.FGFR3~+NP cells located in the outer region of nucleus pulposus represented a group of proliferative NP cells,which participated in the growth and morphogenesis of NP after birth.2.The growth of postnatal NP cells is mainly along the direction of nucleus pulposus radius.3.FGFR3~+NP cells may participate in postnatal degeneration and regeneration.Part Ⅲ Study of the role of Foxa2~+NP cells in postnatal growth and homeostasis maintenance of NPMethods:1.The localization and fate of Foxa2~+cells in postnatal IVD:1.1 Immunofluorescence assay was used to detect the expression of Foxa2 in FGFR3~+NP cells in FGFR3Cre ERT2;Rosa26-m T/m G mice.1.2 Foxa2Cre ERT2;Rosa26-m T/m G mice were taken intraperitoneal injection of tamoxifen for 3 days(1 mg/10 g body weight)at 2w,4w and 8w and sacrificed after tamoxifen administration and after one month of chasing.Foxa2 immunostaining was also performed in Foxa2Cre ERT2;Rosa26-m T/m G mice.2.Relationship between Foxa2~+NP cells and long-term labeled Ed U~+NP cells:2.1 Foxa2Cre ERT2;Rosa26-m T/m G mice were injected with Ed U(Ed U×6)at P7,then taken injection of tamoxifen for 3 days(1 mg/10 g body weight)at 2m.After administration of tamoxifen,the mice were sacrificed.2.2 One-month-old and 2-month-old mice were intraperitoneally injected with Ed U(Ed U×6)and sacrificed after 1m and 3m of chasing.Results:1.The localization and fate of Foxa2+cells in postnatal IVD:1.1 Immunofluorescence assay found expression of Foxa2 in FGFR3~+NP cells in FGFR3Cre ERT2;Rosa26-m T/m G mice.1.2 Foxa2~+cells were found in the outer region of NP,hypertrophic chondrocyte region of growth plate,and also slightly in annulus fibrosus and endplate in Foxa2Cre ERT2;Rosa26-m T/m G mice.Foxa2~+growth plate cells entered the bone marrow cavity after tracing.Early natal Foxa2~+NP cells can proliferate and form cell clones.Foxa2~+cells in mature NP rarely proliferate to form cell clones.2.Relationship beween Foxa2~+NP cells and long-term labeled Ed U~+NP cells:2.1 Foxa2~+NP cells were co-located with long-term labeled Ed U~+NP cells labeled at not P7.2.2 Ed U~+NP cells labeled at 1m and 2m showed no significant changes in cell number or cell location after 1 month and 3 months of chasing.Conclusion:1.Foxa2 are slightly expressed in postnatal IVD.Foxa2~+growth plate cells have migration capacity and migrated to the bone marrow cavity after tracing.2.Few Foxa2~+NP cells are found in the outer region of postnatal NP.Early natal Foxa2~+NP cells have better proliferating capacity than mature NP and can form cell clones.Foxa2~+cells in mature NP rarely proliferate to form cell clones.3.Foxa2~+NP cells are probably the long-term labeled slow cycling cells in NP and may participate in not only postnatal growth but also homeostasis maintenance.