Differentiation Of Human Ligamentum Flavum Stem Cells Towards Intervertebral Nucleus Pulposus-like Cells Induced By Coculture System And Hypoxia

Objectives: Low back pain(LBP) is one of the most common painful conditions that lead to work absenteeism, medical visits, and hospitalization,which is closely related to intervertebral disc(IVD) degeneration1. IVD degeneration involves a reduction in the function and number of viable cells in the disc, mostly through cellular senescence2-3 and apoptosis4-5. In fact, LBP is usually the first symptom of intervertebral disk degeneration(IDD), which eventually may progress to multiple spinal disorders such as disk herniation, spinal stenosis,spondylolisthesis and even paralysis,with the consequent neurological symptoms have also been reported.Current IVD-degeneration treatments mainly involve medicines application and surgery, which is limited to relieve symptoms. However, in cell-based tissue engineering, the aim is to restore IVD structure and function,which has been attracting a great deal of attention by using minimally invasive approaches to transplant growth factors,ozone therapy,stem cells,and biomaterials.New treatments based on tissue engineering approaches are focused in restoring disk height and biomechanical factors, as these factors can promote IVD regeneration and re-establish its original function, which are mainly related to reduction of hospitalization and side effect.So this new treatments is considered a promising approach for IVD degeneration. Although it is commonly accepted that nucleus pulposus cells would be ideal to use for assessing biological repair therapies,unfortunately they are limited in number and would have to be expanded in culture to get sufficient numbers.So it is difficult to meet clinical requires. One potential solution would be to induce the stem cells cocultured with nucleus pulposus(NP)cells, which can differentiate into NP cells and maintain their NP’s morphology and phenotype for long time. Several types of multipotent stem cells are present in adult tissues(such as in bone marrow,heart,brain,skeletal-muscle, and adipose tissues, synovial membrane, menstrual blood6-15 the most common being bone marrow stromal cells(BM-MSCs) or adipose tissue(adipose derived stem cells).most of them are obtained with difficultly or invasively.these adult stem cells have some defects for research and clinical use, including(1)invasive methods for sample collection,(2) less availability,(3) and lower proliferation capacity compared with human ligamentum flavum stem cells. F or this,it may be a “choke point” to find a new source of stem cell.In this regard, we isolated stem cells from ligamentum flavum(LFSCs) and compared them with BM-MSCs obtained from the same patient in this study.The study is based on identifying and isolating the same hierarchy stem cell. The developing techniques include fluorescence activated cell sorting;isolation on the basis of a side population phenotype;isolation on the basis of cell size and an enzyme; cell attachment to various interface. However,most of them are expensive and few special marker which limite its development and use. LFSCs highly expressed integrinα 5β 1 and α vβ 3. And the cell interaction was mediated via integrin subunit α and β in this case. As we know thrombin that could promote the expresssion ofα 5β 1,α vβ 3 and fibronectin. Also fibronectin coated surfaces can promote the attachment.So we can use fibronectin and thrombin to isolate the very small subpopulation of adult stem cells. Isolation and culture on the basis of this technique,Their morphology, proliferation rate, cell cycle, immunophenotype and stem cell gene expression were tested. In this regards,the presence of a rare population of adult stem cells in LF generally possess a selfrenewal ability and multilineage differentiation potential are able to regenerate the nucleus pulposus-like cells.Here,coculture system and hypoxia was applied to assess the NP differentiation capability of LFSCs. We found that the characteristic NP markers(KRT19, CA12, Fox F1) and special protein were significantly upregulated in differentiated LFSCs. Importantly, we know that Notch signaling plays a critical role in the regulation of cartilage formation and performs that Notch inhibition of chondrogenesis acts via up-regulation of the transcription factor Twist1. To our knowledge, the underlying mechanisms that hypoxic conditions mediate Notch signaling still remain unclear.The study of hypoxia in MSCs enhances nucleus pulposus-like cells should be paid attention to know the further mechanisms.Currently, HIF-1α regulate Notch signaling in human LFSCs induced for differentiation under hypoxia,which has been reported.So, Our findings demonstrate that the special marker and protein of LFSCs changes over time during the process of differentiation to nucleus pulposus-like cells under hypoxia. To address this we performed use of Lentivirus-mediated cell infection and demonstrate that HIF-1α enhance of chondrogenesis acts via down-regulation of the transcription factor Twist-1.This study presents a potential novel mechanism whereby HIF-1α may repress Twist-1 to enhance LFSCs chondrogenic differentiation via direct Notch NICD/RBPjk binding to its promoter region. These findings provide additional support for the potential treatment of IVD by local modification of Notch signaling in LFSCs.Materials and methods1. Technique used to isolate LFSCs:The samples of ligamentum flavum were collected from transforaminal endoscopic surgery or microendoscopic discectomy surgery for lumbar disc degenerative diseases,and primary ligamentum flavum were obtained by mechanical method combined with collagenas I for 6-8 h, then the cells were seeded into 24 well plates(Nunc) pre-coated with 8μg/cm2 Fibronectin and 4 U/ml thrombin, optical density(OD) values were detected at a wavelength of 490 nm to adhesion assay(number of cells in the adherent fraction /number of total cells).The percentage of LFSCs were analyzed on cell surface markers with FACScan.2.The LFSCs generally possess a self-renewal ability and multilineage differentiation potential,which have approved the unique features such as stem cell markers, specific protein, and multidifferentiation potential in vitro. The proliferation capacity, cell cycle and stem cell genes of these LFSCs were compared with BM-MSCs.3. To induce human LFSCs towards intervertebral nucleus pulposus-like cells by coculture system and hypoxia: Using coculture plates with 1μ m pore size polyethylene terephthalate track-etched inserts, LFSCs and NP cells(1:1 ratio) were cocultured with cell-to-cell contact for 14 days in normal(20% O2) or low oxygen tension(2% O2),respectively. Novel characteristic human NP markers cytokeratin-19(KRT19), carbonic anhydrase XII(CA12), Sox-9, collagen-II, aggrecan,HIF-1α and forkhead box F1(Fox F1) were also detected by q-PCR. Immunohistochemical detection of collagen-I, collagen-II and aggrecan at 0,14,21 day.4. To investigate whether HIF-1α regulates Notch signaling, and whether HIF-1αplays a role in the expression of Twist1:c DNAs encoding human HIF-1α was cloned into the EF.v-CMV lentiviral vector. We performed transient transfection experiments using HIF-1α overexpression plasmid. Immunofluorescence using Twist1 anti-body in LFSCs showed that Twist1 labeling in both nuclear areas was observed as early as 48 h after over-expression of HIF-1α.Extracellular matrix accumulation was quantified by alcian blue staining and q-PCR at the time points 3 or 7 days. Western blots showed the protein levels of Twist1 in control cells were highly expressed from 6 h,24 h,48h in micromass culture.Results:1. α 5 β 1 and α v β 3 are expressed on the plasma membrane of LFSCs.The discrepancy of cell morphology among cells of passage 1 was observed,but the cells became comparatively uniform after 8μg/cm2 Fibronectin and 4 U/ml thrombin-mediated adhesion. The adhesive capacity of human LFSCs to fibronectin and thrombin-mediated adhesion was analyzed by means of a static adhesion assay in which fibronectin and bovine serum albumin(BSA) coatings were used as positive and negative controls, respectively. Fibronectin and thrombin-mediated adhesion supports LFSCs adhesion compared with others, which is, at least in part,mediated byα 5β 1andα vβ 3-integrins.2.The results of flow cytometry showed these cells were positive for CD73,CD105,CD44,CD29 and CD90,but not positive for CD34,CD45,CD133 and stro-1.Cells cultured in osteogenic induced produced an alizarin red positive matrix,adipogenic medium were stained with Oil Red O,chondrogenic induction micromass medium formed an aggregate and m RNA analysis of RT-PCR results revealed that the osteogenesis-related genes(ALP, OC, RUNX-2), the adipogenesis-related genes(PPAR-2,APP,LPL)and chondrogenesis-related genes(AGG,COL II,SOX-9) were all markedly increased in the respective induced group as compared to the control(non-induction, growth) group. All LFSCs expressed collagen-I, whereas a subset of BM-MSCs expressed collagen-I, and α-SMA was more abundant in LFSCs than in BMSCs. All LFSCs and BM-MSCs expressed fibronectin, but not collagen-II. The cell cycle shows that more than 80% of the BM-MSCs and LFSCs were in the G0/G1 phase, The OD value increased from day 1 to day 10 and reached a plateau from day 10 to day 14. LFSCs and BM-MSCs did not express significantly different levels of NANOG and Sox2(2-tailed,t=0.37, P=0.68,and t =1.83, P=0.22, respectively)(P>0.05);however, the OCT4 m RNA level was higher in LFSCs than in BM-MSCs(2-tailed, t =12.8, P=0.004)(P<0.05).3. After 14 days’ coculture under hypoxia,real-time PCR and western-blotting results revealed that compared with LFSCs grown under normoxia, hypoxia-treated LFSCs expressed higher levels of Sox-9, collagen-II, aggrecan, KRT19, CA12, FOXF1, and HIF-1α genes and HIF-1α, collagen-II, Sox-9, and aggrecan proteins(P<0.05).4. LFSCs differentiation pathway was identified by cell surface markers,and our data suggest that the surface marker expression would changed during differentiation.At the first day, CD105 and CD271 highly express. GD2,Tie2 and CD24 have low level.At 14 days,Almost all of these markers down-regulate except for CD24,and CD24 express up-regulation.After 21 days,only CD24 and CD271 were positive, but others were negative. Immunocytochemistry staining of special proteins shows that collagen-II and aggrecan express at 14 and 21 days.The expression of collagen-I decreases following the cells differentiation.5. Overexpression of HIF-1α resulted in increase a strong alcian blue staining of cartilage matrix and mature chondrocytes was observed inside the chondrogenic nodules at 3 and 7 days,compared to GFP-lentirvirus infected controls.Real time-PCR shows that HIF-1 α overexpression enhance of chondrogenesis acts via down-regulation of the transcription factor Twist-1,and also enhance the expression of collagen-II and aggrecan(P<0.05). A quick response of Twist-1 to HIF-1α, Immunofluorescence using Twist-1 antibody in LFSCs showed that Twist-1 was decreased 48 h after over-expression of HIF-1α. Quantification of Twist-1 protein level in Western blots was performed using Image J software shows that decrease of Twist1 with over-expression of HIF-1α at 6,24,48 h.Conclusions:1. This study presents the presence of stem cells in the human LF,which obtained by transforaminal endoscopic surgery or microendoscopic discectomy surgery. We found that 8μg/cm2 Fibronectin and 4 U/ml thrombin-mediated adhesion has superiority.We also identify surface marker Stro-1 was negative.Interestingly, our data show that stem cell markers Oct4 amplifications were significantly detected in LFSCs. Oct4 is required for efficient self-renewal and the pluripotency of stem cells,and our results suggest that LFSCs was significantly expressed compared to that of BMSCs to maintain pluripotent properties. All in all,LFSCs has universal stem cell characteristics such as multipotency and self-renewal capacity.2. It is based on coculture system and hypoxia to induce human ligamentum flavum stem cells towards intervertebral nucleus pulposus-like cells.To investigate whether LFSCs potentially provides new cell candidates for cell-based regenerative medicine and tissue engineering.We can conclude these following options:1) Under coculture system and hypoxia, In our study, we tested their morphology, the change of immunophenotype and the level of gene and protein expression. LFSCs were successfully differentiated into NP-like cells and hypoxia promotes chondrogenesis,which may become a new source of seed cells for the treatment of intervertebral disc degeneration in the future.2) To investigate whether HIF-1α regulates Notch signaling, and whether HIF-1αplays a role in the expression of Twist1, which was identified as an important target of canonical Notch signaling that contributes to the repression of chondrogenesis. HIF-1αenhance of chondrogenesis acts via up-regulation of the transcription factor Twist1, further studies are required to know the underlying mechanisms refer to gene expression invivo for long time.3) Here,we identify the change of cell surface markers by FACS analysis,and our data suggest that the surface marker expression would changed during LFSCs’ differentiation pathway, so we can choose the right cell type is used in the right clinical setting,such as nucleus pulposus-like adult stem/progenitor cells.Not only induce LFSCs differentiation into nucleus pulposus cells,but also advoided tumourigenic.LFSCs can be an excellent candidate in clinical application for treatment of a wide range of medical condition.