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, Ricardo; Berry, Andrew; Hanley, Karen Piper; Hanley, Neil A.; Richardson, Stephen M.; Hoyland, Judith A.

doi:10.1002/jor.23205

Cited by 53 publications

(81 citation statements)

References 49 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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“…Tie2 + preservation could, however, be augmented under hypoxic conditions, by supplementation of fibroblast growth factor 2 (FGF2) or synergistic FGF2 and hypoxic conditions (Figure C). Rodrigues‐Pinto et al demonstrated a lack of Tie2 expression during the development of the fetal notochord up to 18 weeks post‐conception, indicating that Tie2 might not play a role during development. Nonetheless, after fetal IVD explantation, isolation, and culture we were able to detect Tie2 + NPCs from both human and canine fetal tissue (Figure S1).…”

Section: Introductionmentioning

confidence: 99%

Successful fishing for nucleus pulposus progenitor cells of the intervertebral disc across species

et al. 2018

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Background Recently, Tie2/TEK receptor tyrosine kinase (Tie2 or syn. angiopoietin‐1 receptor) positive nucleus pulposus progenitor cells were detected in human, cattle, and mouse. These cells show remarkable multilineage differentiation capacity and direct correlation with intervertebral disc (IVD) degeneration and are therefore an interesting target for regenerative strategies. Nevertheless, there remains controversy over the presence and function of these Tie2 + nucleus pulposus cells (NPCs), in part due to the difficulty of identification and isolation. Purpose Here, we present a comprehensive protocol for sorting of Tie2 + NPCs from human, canine, bovine, and murine IVD tissue. We describe enhanced conditions for expansion and an optimized fluorescence‐activated cell sorting‐based methodology to sort and analyze Tie2 + NPCs. Methods We present flow cytometry protocols to isolate the Tie2 + cell population for the aforementioned species. Moreover, we describe crucial pitfalls to prevent loss of Tie2 + NPCs from the IVD cell population during the isolation process. A cross‐species phylogenetic analysis of Tie2 across species is presented. Results Our protocols are efficient towards labeling and isolation of Tie2 + NPCs. The total flow cytometry procedure requires approximately 9 hours, cell isolation 4 to 16 hours, cell expansion can take up to multiple weeks, dependent on the application, age, disease state, and species. Phylogenetic analysis of the TEK gene revealed a strong homology among species. Conclusions Current identification of Tie2 + cells could be confirmed in bovine, canine, mouse, and human specimens. The presented flow cytometry protocol can successfully sort these multipotent cells. The biological function of isolated cells based on Tie2 + expression needs to be confirmed by functional assays such as in vitro differentiation. in vitro culture conditions to maintain and their possible proliferation of the Tie2 + fraction is the subject of future research.

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“…In this regard, functional analysis of the T transcription factor using U‐CH1‐N must be undertaken with caution. Furthermore, largely due to a paucity of data on IVD biology, we do not fully understand what criteria define “notochordal NP cell‐like cell line.” Although further studies are mandatory to address these issues, based on the observations of the past and present studies, we would like to propose the expression of notochordal NP cell‐specific markers, especially T and CD24, and the chondrogenic capacity as potentially the necessary conditions for “notochordal NP cell‐like cell line.” Nevertheless, bearing the limitations in mind, U‐CH1‐N is probably one of the best cell lines available to date to study the biology of notochordal NP cells.…”

Section: Discussionmentioning

confidence: 91%

Chordoma‐derived cell line U‐CH1‐N recapitulates the biological properties of notochordal nucleus pulposus cells

Fujita¹,

Suzuki²,

Watanabe³

et al. 2016

Journal Orthopaedic Research

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Intervertebral disc degeneration proceeds with age and is one of the major causes of lumbar pain and degenerative lumbar spine diseases. However, studies in the field of intervertebral disc biology have been hampered by the lack of reliable cell lines that can be used for in vitro assays. In this study, we show that a chordoma‐derived cell line U‐CH1‐N cells highly express the nucleus pulposus (NP) marker genes, including T (encodes T brachyury transcription factor), KRT19, and CD24. These observations were further confirmed by immunocytochemistry and flow cytometry. Reporter analyses showed that transcriptional activity of T was enhanced in U‐CH1‐N cells. Chondrogenic capacity of U‐CH1‐N cells was verified by evaluating the expression of extracellular matrix (ECM) genes and Alcian blue staining. Of note, we found that proliferation and synthesis of chondrogenic ECM proteins were largely dependent on T in U‐CH1‐N cells. In accordance, knockdown of the T transcripts suppressed the expression of PCNA, a gene essential for DNA replication, and SOX5 and SOX6, the master regulators of chondrogenesis. On the other hand, the CD24‐silenced cells showed no reduction in the mRNA expression level of the chondrogenic ECM genes. These results suggest that U‐CH1‐N shares important biological properties with notochordal NP cells and that T plays crucial roles in maintaining the notochordal NP cell‐like phenotype in this cell line. Taken together, our data indicate that U‐CH1‐N may serve as a useful tool in studying the biology of intervertebral disc. © 2016 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 34:1341–1350, 2016.

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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

Cited by 53 publications

References 49 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

Successful fishing for nucleus pulposus progenitor cells of the intervertebral disc across species

Chordoma‐derived cell line U‐CH1‐N recapitulates the biological properties of notochordal nucleus pulposus cells

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