Identifying molecular phenotype of nucleus pulposus cells in human intervertebral disc with aging and degeneration

Tang, Xinyan; Jing, Liufang; Richardson, William J.; Isaacs, Robert E.; Fitch, Robert D.; Brown, Christopher R.; Erickson, Melissa; Setton, Lori A.; Chen, Jun

doi:10.1002/jor.23244

Cited by 60 publications

(65 citation statements)

References 55 publications

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“…qPCR analysis demonstrated significantly higher expression of a panel of previously‐identified NC markers (KRT8, KRT18, KRT19, CD24, and T) in porcine NC cells compared to AF cells (Figure B). This paralleled the expression patterns of these genes in the human system as previously described …”

Section: Resultssupporting

confidence: 79%

An optimized culture system for notochordal cell expansion with retention of phenotype

et al. 2018

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Background Notochordal (NC) cells display therapeutic potential in treating degeneration of the intervertebral disc. However, research on their phenotype and function is limited by low‐cell yields and a lack of appropriate methodology for cell expansion. Utilizing porcine cells, this study aimed to develop an optimized culture system which allows expansion of NC cell populations with retention of phenotype. Methods Post‐natal porcine and foetal human nucleus pulposus tissue was compared histologically and expression of known NC cell marker genes by porcine NC cells was analyzed. Porcine NC cells were isolated from six‐week post‐natal discs and cultured in vitro under varied conditions: (1) DMEM vs αMEM; (2) laminin‐521, fibronectin, gelatin and uncoated tissue culture‐treated polystyrene (TCP); (3) 2% O 2 vs normoxia; (4) αMEM (300 mOsm/L) vs αMEM (400 mOsm/L); (5) surface stiffness of 0.5 and 4 kPa and standard TCP. Adherence, proliferation, morphology and expression of NC cell markers were assessed over a 14‐day culture period. Results Native porcine nucleus pulposus tissue demonstrated similar morphology to human foetal tissue and porcine NC cells expressed known notochordal markers (CD24, KRT8, KRT18, KRT19, and T). Use of αMEM media and laminin‐521‐coated surfaces showed the greatest cell adherence, proliferation and retention of NC cell morphology and phenotype. Proliferation of NC cell populations was further enhanced in hypoxia (2%) and phenotypic retention was improved on 0.5 kPa culture surfaces. Discussion Our model has demonstrated an optimized system in which NC cell populations may be expanded while retaining a notochordal phenotype. Application of this optimized culture system will enable NC cell expansion for detailed phenotypic and functional study, a major advantage over current culture methods described in the literature. Furthermore, the similarities identified between porcine and human NC cells suggest this system will be applicable in human NC cell culture for investigation of their therapeutic potential.

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Section: Resultssupporting

confidence: 79%

An optimized culture system for notochordal cell expansion with retention of phenotype

et al. 2018

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“…As we all know, the inflammation environment is one of the key factors for DDD, CD54 can be used as a biomarker to evaluate the inflammation-associated disc degeneration, because the expression of CD54 was insignificant in younger NP tissue, and showed stronger expression in aged NP tissue. 33 There is no significant increase of CD54 expression after differentiation in the hydrogel, which indicated that there is no inflammation reaction occurring in our constructed hydrogel, and will cause no harm to native disc after implantation.…”

Section: Discussionmentioning

confidence: 88%

Development of kartogenin-conjugated chitosan–hyaluronic acid hydrogel for nucleus pulposus regeneration

et al. 2017

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Injectable constructs for in vivo gelation have many advantages in the regeneration of degenerated nucleus pulposus. In this study, an injectable hydrogel consisting of chitosan (CS) and hyaluronic acid (HA) crosslinked with glycerol phosphate (GP) at different proportions (CS : GP : HA, 6 : 3 : 1, 5 : 3 : 2, 4 : 3 : 3, 3 : 3 : 4, 2 : 3 : 5, 1 : 3 : 6, V : V : V) was developed and employed as a delivery system for kartogenin (KGN), a biocompound that can activate chondrocytes. In vitro gelation time, morphologies, swelling, weight loss, compressive modulus and cumulative release of KGN in hydrogels were studied.For biocompatibility assessments, human adipose-derived stem cells (ADSCs) were encapsulated in these hydrogels. The effects of KGN on stem cell proliferation and differentiation into nucleus pulposus-like cells were examined. The hydrogels with higher concentrations of HA showed a slightly shorter gelation time, higher water uptake, faster weight loss and faster KGN release compared to the hydrogels with lower concentrations of HA. As the KGN-conjugated hydrogel prepared with the proportions 5 : 3 : 2 displayed good mechanical properties, it was chosen as the optimal gel to promote cell proliferation and differentiation. No significant difference was seen in the expression levels of nucleus pulposus markers induced by KGN or TGF-β. Additionally, inclusion of KGN and TGF-β together did not produce a synergistic effect in inducing nucleus pulposus properties. In conclusion, we have developed a KGN-conjugated CS/HA hydrogel (5 : 3 : 2) with sustained release of KGN in hydrogel that can promote ADSC proliferation and nucleus pulposus differentiation. This kind of hydrogel may be a simple and effective candidate for the repair of degenerative NP tissue after minimally invasive surgery.

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“…In terms of IVD biology, CD24 has previously been identified in rat notochordal NP cells and in human chordomas (tumors arising from remnants of the notochord), which corroborates the findings in this study confirming this protein as a notochordal marker. Additionally CD24 expression has been reported in discs from children and adolescents undergoing surgery for scoliosis . Furthermore, a subpopulation of CD24 positive mouse embryoid body cells showed spontaneous differentiation to cells with notochordal characteristics .…”

Section: Discussionmentioning

confidence: 93%

Spatiotemporal analysis of putative notochordal cell markers reveals CD24 and keratins 8, 18, and 19 as notochord‐specific markers during early human intervertebral disc development

Rodrigues-Pinto

Berry

Hanley

et al. 2016

Journal Orthopaedic Research

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In humans, the nucleus pulposus (NP) is composed of large vacuolated notochordal cells in the fetus but, soon after birth, becomes populated by smaller, chondrocyte‐like cells. Although animal studies indicate that notochord‐derived cells persist in the adult NP, the ontogeny of the adult human NP cell population is still unclear. As such, identification of unique notochordal markers is required. This study was conducted to determine the spatiotemporal expression of putative human notochordal markers to aid in the elucidation of the ontogeny of adult human NP cells. Human embryos and fetuses (3.5–18 weeks post‐conception (WPC)) were microdissected to isolate the spine anlagens (notochord and somites/sclerotome). Morphology of the developing IVD was assessed using hematoxylin and eosin. Expression of keratin (KRT) 8, KRT18, KRT19, CD24, GAL3, CD55, BASP1, CTGF, T, CD90, Tie2, and E‐cadherin was assessed using immunohistochemistry. KRT8, KRT18, KRT19 were uniquely expressed by notochordal cells at all spine levels at all stages studied; CD24 was expressed at all stages except 3.5 WPC. While GAL3, CD55, BASP1, CTGF, and T were expressed by notochordal cells at specific stages, they were also co‐expressed by sclerotomal cells. CD90, Tie2, and E‐cadherin expression was not detectable in developing human spine cells at any stage. This study has identified, for the first time, the consistent expression of KRT8, KRT18, KRT19, and CD24 as human notochord‐specific markers during early IVD development. Thus, we propose that these markers can be used to help ascertain the ontogeny of adult human NP cells. © 2016 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. J Orthop Res 34:1327–1340, 2016.

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Identifying molecular phenotype of nucleus pulposus cells in human intervertebral disc with aging and degeneration

Cited by 60 publications

References 55 publications

An optimized culture system for notochordal cell expansion with retention of phenotype

An optimized culture system for notochordal cell expansion with retention of phenotype

Development of kartogenin-conjugated chitosan–hyaluronic acid hydrogel for nucleus pulposus regeneration

Spatiotemporal analysis of putative notochordal cell markers reveals CD24 and keratins 8, 18, and 19 as notochord‐specific markers during early human intervertebral disc development

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