Novel immortal human cell lines reveal subpopulations in the nucleus pulposus

Introduction: Relatively little is known about cellular subpopulations in the mature nucleus pulposus (NP). Detailed understanding of the ontogenetic, cellular and molecular characteristics of functional intervertebral disc (IVD) cell populations is pivotal to the successful development of cell replacement therapies and IVD regeneration. In this study, we aimed to investigate whether phenotypically distinct clonal cell lines representing different subpopulations in the human NP could be generated using immortalization strategies. Methods: Nondegenerate healthy disc material (age range, 8 to 15 years) was obtained as surplus surgical material. Early passage NP monolayer cell cultures were initially characterized using a recently established NP marker set. NP cells were immortalized by simian virus 40 large T antigen (SV40LTag) and human telomerase reverse transcriptase expression. Immortalized cells were clonally expanded and characterized based on collagen type I, collagen type II, alpha 1 (COL2A1), and SRY-box 9 (SOX9) protein expression profiles, as well as on expression of a subset of established in vivo NP cell lineage markers. Results: A total of 54 immortal clones were generated. Profiling of a set of novel NP markers (CD24, CA12, PAX1, PTN, FOXF1 and KRT19 mRNA) in a representative set of subclones substantiated successful immortalization of multiple cellular subpopulations from primary isolates and confirmed their NP origin and/or phenotype. We were able to identify two predominant clonal NP subtypes based on their morphological characteristics and their ability to induce SOX9 and COL2A1 under conventional differentiation conditions. In addition, cluster of differentiation 24 (CD24)-negative NP responder clones formed spheroid structures in various culture systems, suggesting the preservation of a more immature phenotype compared to CD24-positive nonresponder clones. Conclusions: Here we report the generation of clonal NP cell lines from nondegenerate human IVD tissue and present a detailed characterization of NP cellular subpopulations. Differential cell surface marker expression and divergent responses to differentiation conditions suggest that the NP subtypes may correspond to distinct maturation stages and represent distinct NP cell subpopulations. Hence, we provide evidence that the immortalization strategy that we applied is capable of detecting cell heterogeneity in the NP. Our cell lines yield novel insights into NP biology and provide promising new tools for studies of IVD development, cell function and disease

Production of ω mesons in pp collisions at √s=7TeV

The invariant differential cross section of inclusive ω(782) meson production at midrapidity (| y| < 0.5) in pp collisions at s=7TeV was measured with the ALICE detector at the LHC over a transverse momentum range of 2<pT<17GeV/c. The ω meson was reconstructed via its ω→ π+π-π decay channel. The measured ω production cross section is compared to various calculations: PYTHIA 8.2 Monash 2013 describes the data, while PYTHIA 8.2 Tune 4C overestimates the data by about 50%. A recent NLO calculation, which includes a model describing the fragmentation of the whole vector-meson nonet, describes the data within uncertainties below 6GeV/c, while it overestimates the data by up to 50% for higher pT. The ω/ π ratio is in agreement with previous measurements at lower collision energies and the PYTHIA calculations. In addition, the measurement is compatible with transverse mass scaling within the measured pT range and the ratio is constant with Cω/π0=0.67±0.03(stat)±0.04(sys) above a transverse momentum of 2.5GeV/c. © 2020, CERN for the benefit of the ALICE collaboration