Browsing by Author "Tang, Xinyan"

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    A new non-enzymatic method for isolating human intervertebral disc cells preserves the phenotype of nucleus pulposus cells.
    (Cytotechnology, 2014-12) Tang, Xinyan; Richardson, William J; Fitch, Robert D; Brown, Christopher R; Isaacs, Robert E; Chen, Jun
    Cells isolated from intervertebral disc (IVD) tissues of human surgical samples are one of potential sources for the IVD cellular therapy. The purpose of this study was to develop a new non-enzymatic method, "tissue incubation", for isolating human IVD cells. The IVD tissues of annulus fibrosus (AF) and nucleus pulposus (NP) were incubated separately in tissue culture flasks with culture medium. After 7-10 days incubation, cells were able to migrate out of IVD tissues and proliferate in vitro. After 3-4 weeks culture, expanded cells were harvested by trypsinization, and the remaining tissues were transferred to a new flask for another round of incubation. The molecular phenotype of IVD cells from juvenile and adult human samples was evaluated by both flow cytometry analysis and immunocytochemical staining for the expression of protein markers of NP cells (CD24, CD54, CD239, integrin α6 and laminin α5). Flow cytometry confirmed that both AF and NP cells of all ages positively expressed CD54 and integrin α6, with higher expression levels in NP cells than in AF cells for the juvenile group sample. However, CD24 expression was only found in juvenile NP cells, and not in AF or older disc cells. Similar expression patterns for NP markers were also confirmed by immunocytochemistry. In summary, this new non-enzymatic tissue incubation method for cell isolation preserves molecular phenotypic markers of NP cells and may provide a valuable cell source for the study of NP regeneration strategies.
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    Identifying molecular phenotype of nucleus pulposus cells in human intervertebral disc with aging and degeneration.
    (Journal of orthopaedic research : official publication of the Orthopaedic Research Society, 2016-08) Tang, Xinyan; Jing, Liufang; Richardson, William J; Isaacs, Robert E; Fitch, Robert D; Brown, Christopher R; Erickson, Melissa M; Setton, Lori A; Chen, Jun
    Previous study claimed that disc degeneration may be preceded by structure and matrix changes in the intervertebral disc (IVD) which coincide with the loss of distinct notochordally derived nucleus pulposus (NP) cells. However, the fate of notochordal cells and their molecular phenotype change during aging and degeneration in human are still unknown. In this study, a set of novel molecular phenotype markers of notochordal NP cells during aging and degeneration in human IVD tissue were revealed with immunostaining and flow cytometry. Furthermore, the potential of phenotype juvenilization and matrix regeneration of IVD cells in a laminin-rich pseudo-3D culture system were evaluated at day 28 by immunostaining, Safranin O, and type II collagen staining. Immunostaining and flow cytometry demonstrated that transcriptional factor Brachyury T, neuronal-related proteins (brain abundant membrane attached signal protein 1, Basp1; Neurochondrin, Ncdn; Neuropilin, Nrp-1), CD24, and CD221 were expressed only in juvenile human NP tissue, which suggested that these proteins may be served as the notochordal NP cell markers. However, the increased expression of CD54 and CD166 with aging indicated that they might be referenced as the potential biomarker for disc degeneration. In addition, 3D culture maintained most of markers in juvenile NP, and rescued the expression of Basp1, Ncdn, and Nrp 1 that disappeared in adult NP native tissue. These findings provided new insight into molecular profile that may be used to characterize the existence of a unique notochordal NP cells during aging and degeneration in human IVD cells, which will facilitate cell-based therapy for IVD regeneration. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1316-1326, 2016.