Selection and identification of tissue engineering human ligamentum flavum-derived stem cells

English Keywords:【Key words】 Human  Ligamentum flavum-derived stem cells  Bone mesenchymal stem cells  Nucleus pulposus  Fibronectin differential adhesion assay  Tissue engineering.

English Abstract:

  【Abstract】 Objectives: To select ligamentum flavum-derived stem cells by fibronectin differential adhesion assay, and to determine whether MSCs exist in the human ligamentum flavum which provide cell candidates for cell-based regenerative medicine and tissue engineering. Methods: The samples of ligamentum flavum were collected from transforaminal endoscopic surgery or microendoscopic discectomy surgery for lumbar degenerative disease, and primary ligamentum flavum was obtained by mechanical method combined with collagenas Ⅰ． Primitive cells proliferating to passage 2 were transferred to culture flasks coated with fibronectin． The cells′ special markers were detected by flow cytometry. Cell clusters were analyzed by Immunohistochemical Staining for expression of stem cell-related proteins and were induced to differentiate into osteoblasts, chondrocytes and adipocytes under differentiation medium(experimental group)． Negative control wells were maintained in basal Medium. The two groups were stained with alizarin red staining, oil red O and alcian blue respectively. RT-PCR was used to analyze the mRNA expressions of osteogenic, adipogenic- and chondrogenic-related genes. Human BM-MSCs were isolated from bone marrow aspirates taken from the iliac crest of the same patient. Statistical analysis was used to test the proliferation capacity, cell cycle and stem cell gene expression, and the results were compared with bone marrow MSCs(BM-MSCs). Results: The primary cell morphology was triangle and multiangualr, but the cells became comparatively uniformly fibroblastic-like． The percentages of CD73 and CD90 positive cells were more than 96.8%, while the percentage of CD1051-positive cells was more than 95.9%, but negative for stro-1． Osteogenic differentiation group was strongly positive by alizarin red staining. In the adipogenic assay, it exhibited characteristic intracellular lipid droplets that could be stained with Oil Red O. In the chondrogenic micromass culture medium, chondrogenic induction micromass formed a sulfated proteoglycan-rich extracellular matrix which exhibited a positive alcian blue staining, while the non-induction group was negative. The mRNA 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) all markedly increased in induced group compared with the control(non-induction, growth) group(P>0.05), interestingly, the low-level expression of RUNX-2 was detected in the non-induction group. Immunocytochemistry staining further confirmed that all LFSCs and BMMSCs expressed type Ⅰ a-SMA, fibronectin, collagen-Ⅰ, but no collagen-Ⅱ. The cell cycle showed remarkably capable of self-renewal of more than 80% of the BMMSCs 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(P>0.05), both stem cell types exhibited similar growth tendencies. The stem cell gene(NONAG, OCT-4, SOX2) expression in the LFSCs was similar to that in BM-MSCs and the differences were not significant(P>0.05). Conclusions: The ligamentum flavum-derived stem cells obtained by fibronectin differential adhesion assay have capacity for osteogenic, adipogenic and chondrogenic differentiation, which can be used as a new candidate for cell-based regenerative medicine and tissue engineering.