title: Differentiation of Human Ligamentum Flavum Stem Cells Toward Nucleus Pulposus-Like Cells Induced by Coculture System and Hypoxia.
abstract: METHODS
  Human ligamentum flavum (LF)-derived stem cells (LFSCs) and nucleus pulposus cells (NPCs) were cocultured under normoxia or hypoxia.
  
  
  OBJECTIVE
  To isolate and identify human LFSCs and determine whether they can differentiate into NPCs when cocultured with NPCs under hypoxia.
  
  
  BACKGROUND
  Mesenchymal stem cell (MSC)-based therapies have been proposed as a biological treatment for intervertebral disc degeneration. MSCs derived from various tissues are leading candidates for cell-based therapies, but such cells have not been reported in LF.
  
  
  METHODS
  LF cells were isolated from patient samples and cultured using culture flasks coated with fibronectin, and their identity was confirmed using flow cytometry. The cells were induced to differentiate into osteoblasts, chondrocytes, and adipocytes, and their morphology, immunophenotype, cell proliferation capacity, cell cycle, and expression of stem cell-specific genes were compared with those of bone marrow-MSCs (BM-MSCs) derived from the same patients. NPCs and LFSCs were cocultured in 1-μm-pore-size insert transwell-culture systems under hypoxia (2% O2) or normoxia. CD24 expression was measured by flow cytometry and confocal microscopy assay. On day 14, reverse transcription-polymerase chain reaction was used for comparing the expression of chondrogenic genes (Sox-9, collagen-II, aggrecan) and novel marker genes (KRT19, CA12, FOXF1, HIF-1α) between the 2 groups.
  
  
  RESULTS
  LFSCs were obtained using the fibronectin differential-adhesion assay. The morphology of LFSCs was altered, and their immunophenotype, multilineage induction, cell proliferation capacity, cell cycle, and stem cell-specific gene expression were closely related-but not identical-to BM-MSCs, CD24 expression was highly significant in the differentiated LFSCs. RT/Real-time polymerase chain reaction revealed that compared with LFSCs grown under normoxia, hypoxia-treated LFSCs expressed higher levels of Sox-9, collagen-II, aggrecan, KRT19, CA12, and HIF-1α genes except FOXF1.
  
  
  CONCLUSIONS
  Stem cells were identified in human LF, and LFSCs cocultured with NPCs were successfully differentiated into NP-like cells under hypoxia. This potentially provides new cell candidates for cell-based regenerative medicine and tissue engineering.
  
  
  METHODS
  N/A.
authors.affiliation: *Department of Orthopedics, Xinqiao Hospital, Third Military Medical University, Chongqing, People's Republic of China; and †The 12th Hospital of PLA, Kashgar, People's Republic of China.
authors.forename: Xiao-Bo
authors.initials: XB
authors.lastname: Han
authors.forename: Yan-Ling
authors.initials: YL
authors.lastname: Zhang
authors.forename: Hai-Yin
authors.initials: HY
authors.lastname: Li
authors.forename: Bin
authors.initials: B
authors.lastname: Chen
authors.forename: Xian
authors.initials: X
authors.lastname: Chang
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.forename: Kuang
authors.initials: K
authors.lastname: Yang
authors.forename: Yue
authors.initials: Y
authors.lastname: Zhou
authors.forename: Chang-Qing
authors.initials: CQ
authors.lastname: Li
mesh_terms: D050156:Adipogenesis; D000328:Adult; D000368:Aged; D002450:Cell Communication; D002453:Cell Cycle; D002454:Cell Differentiation / Q000235:genetics; D015687:Cell Hypoxia; D049109:Cell Proliferation; D002469:Cell Separation; D048430:Cell Shape; D002478:Cells, Cultured; D020219:Chondrogenesis / Q000235:genetics; D018920:Coculture Techniques; D005260:Female; D018507:Gene Expression Regulation, Developmental; D005819:Genetic Markers; D006801:Humans; D007403:Intervertebral Disc / Q000378:metabolism / Q000473:pathology*; D017843:Ligamentum Flavum / Q000378:metabolism / Q000473:pathology*; D008297:Male; D059630:Mesenchymal Stem Cells / Q000378:metabolism / Q000473:pathology*; D008875:Middle Aged; D010641:Phenotype; D023822:Tissue Engineering
chemical_list: D005819:Genetic Markers