Advertisement

Single-Cell Phenotyping of Complex Heterogeneous Tissue

  • Petra Kraus
  • Kangning Li
  • Darren Sipes
  • Lara Varden
  • Rachel Yerden
  • Althea Henderson
  • Shantanu Sur
  • Thomas LufkinEmail author
Living reference work entry

Abstract

The intervertebral disc (IVD) is a focus of regenerative medicine, with many ongoing clinical trials exploring disc regeneration through injection of stem cells. Anatomically a simple organ, the IVD is primarily comprised of two major tissue types: the annulus fibrosus (AF), which encapsulates the nucleus pulposus (NP). However, cellular composition and signaling cascades resulting in the different cell types present in the mature IVD are still a field of investigation and remain to be fully elucidated. The adult bovine IVD is a suitable model system that anatomically and histologically reflects the situation in human. We have repeatedly isolated primary cell lineages from the AF and NP tissues of bovine IVDs. Cells isolated from AF or NP tissue are typically heterogeneous in culture. Some cells exhibit several features of stem or progenitor cells and have been subjected to physiological and transcriptome-based phenotyping methods with single-cell resolution, which will be described here.

Keywords

Intervertebral disc Annulus fibrosus Nucleus pulposus PISH Single-cell gene expression Single-cell velocity Single-cell proliferation Bos taurus 

Notes

Acknowledgments

This work was supported by the Bayard and Virginia Clarkson Endowment Fund granted to Thomas Lufkin.

References

  1. Alini M, Eisenstein SM, Ito K et al (2008) Are animal models useful for studying human disc disorders/degeneration? Eur Spine J 17:2–19.  https://doi.org/10.1007/s00586-007-0414-yCrossRefGoogle Scholar
  2. Antoniou J, Steffen T, Nelson F et al (1996) The human lumbar intervertebral disc: evidence for changes in the biosynthesis and denaturation of the extracellular matrix with growth, maturation, ageing, and degeneration. J Clin Invest 98:996–1003.  https://doi.org/10.1172/JCI118884CrossRefGoogle Scholar
  3. Baksh D, Song L, Tuan RS (2004) Adult mesenchymal stem cells: characterization, differentiation, and application in cell and gene therapy. J Cell Mol Med 8:301–316CrossRefGoogle Scholar
  4. Bayliss MT, Johnstone B, O’Brien JP (1988) 1988 Volvo award in basic science. Proteoglycan synthesis in the human intervertebral disc. Variation with age, region and pathology. Spine (Phila Pa 1976) 13:972–981CrossRefGoogle Scholar
  5. Bedore J, Leask A, Seguin CA (2014) Targeting the extracellular matrix: matricellular proteins regulate cell-extracellular matrix communication within distinct niches of the intervertebral disc. Matrix Biol 37:124–130.  https://doi.org/10.1016/j.matbio.2014.05.005CrossRefGoogle Scholar
  6. Bibby SR, Urban JP (2004) Effect of nutrient deprivation on the viability of intervertebral disc cells. Eur Spine J 13:695–701.  https://doi.org/10.1007/s00586-003-0616-xCrossRefGoogle Scholar
  7. Bushell GR, Ghosh P, Taylor TF et al (1977) Proteoglycan chemistry of the intervertebral disks. Clin Orthop Relat Res 115–123CrossRefGoogle Scholar
  8. Chatterjee S, Sivakamasundari V, Yap SP et al (2014) In vivo genome-wide analysis of multiple tissues identifies gene regulatory networks, novel functions and downstream regulatory genes for Bapx1 and its co-regulation with Sox9 in the mammalian vertebral column. BMC Genomics 15:1072.  https://doi.org/10.1186/1471-2164-15-1072CrossRefGoogle Scholar
  9. Chelberg MK, Banks GM, Geiger DF et al (1995) Identification of heterogeneous cell populations in normal human intervertebral disc. J Anat 186(Pt 1):43–53Google Scholar
  10. Cheung KM, Karppinen J, Chan D et al (2009) Prevalence and pattern of lumbar magnetic resonance imaging changes in a population study of one thousand forty-three individuals. Spine (Phila Pa 1976) 34:934–940.  https://doi.org/10.1097/BRS.0b013e3181a01b3fCrossRefGoogle Scholar
  11. Craig L, Sanschagrin PC, Rozek A et al (1998) The role of structure in antibody cross-reactivity between peptides and folded proteins. J Mol Biol 281:183–201.  https://doi.org/10.1006/jmbi.1998.1907CrossRefGoogle Scholar
  12. Daley WP, Peters SB, Larsen M (2008) Extracellular matrix dynamics in development and regenerative medicine. J Cell Sci 121:255–264.  https://doi.org/10.1242/jcs.006064CrossRefGoogle Scholar
  13. Demers CN, Antoniou J, Mwale F (2004) Value and limitations of using the bovine tail as a model for the human lumbar spine. Spine (Phila Pa 1976) 29:2793–2799CrossRefGoogle Scholar
  14. Deng Q, Ramskold D, Reinius B et al (2014) Single-cell RNA-seq reveals dynamic, random monoallelic gene expression in mammalian cells. Science 343:193–196.  https://doi.org/10.1126/science.1245316CrossRefGoogle Scholar
  15. Emmert-Buck MR, Bonner RF, Smith PD et al (1996) Laser capture microdissection. Science 274:998–1001CrossRefGoogle Scholar
  16. Errington RJ, Puustjarvi K, White IR et al (1998) Characterisation of cytoplasm-filled processes in cells of the intervertebral disc. J Anat 192(Pt 3):369–378CrossRefGoogle Scholar
  17. Erwin WM, Hood KE (2014) The cellular and molecular biology of the intervertebral disc: a clinician’s primer. J Can Chiropr Assoc 58:246–257Google Scholar
  18. Etienne-Manneville S (2004) Actin and microtubules in cell motility: which one is in control? Traffic 5:470–477.  https://doi.org/10.1111/j.1600-0854.2004.00196.xCrossRefGoogle Scholar
  19. Eyre DR (1979) Biochemistry of the intervertebral disc. Int Rev Connect Tissue Res 8:227–291CrossRefGoogle Scholar
  20. Eyre DR, Muir H (1976) Types I and II collagens in intervertebral disc. Interchanging radial distributions in annulus fibrosus. Biochem J 157:267–270CrossRefGoogle Scholar
  21. Gilson A, Dreger M, Urban JP (2010) Differential expression level of cytokeratin 8 in cells of the bovine nucleus pulposus complicates the search for specific intervertebral disc cell markers. Arthritis Res Ther 12:R24.  https://doi.org/10.1186/ar2931CrossRefGoogle Scholar
  22. Grunhagen T, Shirazi-Adl A, Fairbank JC et al (2011) Intervertebral disk nutrition: a review of factors influencing concentrations of nutrients and metabolites. Orthop Clin North Am 42:465–477, vii.  https://doi.org/10.1016/j.ocl.2011.07.010CrossRefGoogle Scholar
  23. Guilak F, Cohen DM, Estes BT et al (2009) Control of stem cell fate by physical interactions with the extracellular matrix. Cell Stem Cell 5:17–26.  https://doi.org/10.1016/j.stem.2009.06.016CrossRefGoogle Scholar
  24. Hedlund E, Deng Q (2018) Single-cell RNA sequencing: technical advancements and biological applications. Mol Aspects Med 59:36–46.  https://doi.org/10.1016/j.mam.2017.07.003CrossRefGoogle Scholar
  25. Humzah MD, Soames RW (1988) Human intervertebral disc: structure and function. Anat Rec 220:337–356.  https://doi.org/10.1002/ar.1092200402CrossRefGoogle Scholar
  26. Kolodziejczyk AA, Kim JK, Svensson V et al (2015) The technology and biology of single-cell RNA sequencing. Mol Cell 58:610–620.  https://doi.org/10.1016/j.molcel.2015.04.005CrossRefGoogle Scholar
  27. Kraus P, Lufkin T (1999) Mammalian Dlx homeobox gene control of craniofacial and inner ear morphogenesis. J Cell Biochem Suppl 32–33:133–140CrossRefGoogle Scholar
  28. Kraus P, Lufkin T (2017) Implications for a stem cell regenerative medicine based approach to human intervertebral disk degeneration. Front Cell Dev Biol 5:17.  https://doi.org/10.3389/fcell.2017.00017CrossRefGoogle Scholar
  29. Kraus P, Fraidenraich D, Loomis CA (2001) Some distal limb structures develop in mice lacking Sonic hedgehog signaling. Mech Dev 100:45–58CrossRefGoogle Scholar
  30. Kraus P, Xing X, Lim SL et al (2012) Mouse strain specific gene expression differences for illumina microarray expression profiling in embryos. BMC Res Notes 5:232.  https://doi.org/10.1186/1756-0500-5-232CrossRefGoogle Scholar
  31. Kraus P, Sivakamasundari V, Lim SL et al (2013) Making sense of Dlx1 antisense RNA. Dev Biol 376:224–235.  https://doi.org/10.1016/j.ydbio.2013.01.035CrossRefGoogle Scholar
  32. Kraus P, Sivakamasundari V, Xing X et al (2014) Generating mouse lines for lineage tracing and knockout studies. Methods Mol Biol 1194:37–62.  https://doi.org/10.1007/978-1-4939-1215-5_3CrossRefGoogle Scholar
  33. Kraus P, Kocsis V, Williams C et al. (2015) Plate in situ hybridization (PISH) as a time and cost effective RNA expression assay to study phenotypic heterogeneity in a population of cultured murine cells at single cell resolution. Biotechnol Lett 37:1573–1577.  https://doi.org/10.1007/s10529-015-1833-1CrossRefGoogle Scholar
  34. Kraus P, Yerden R, Kocsis V et al (2017) RNA in situ hybridization characterization of non-enzymatic derived bovine intervertebral disc cell lineages suggests progenitor cell potential. Acta Histochem 119:150–160.  https://doi.org/10.1016/j.acthis.2016.12.004CrossRefGoogle Scholar
  35. Kraus P, Sivakamasundari V, Olsen V et al (2018a) Klhl14 antisense RNA is a target of key skeletogenic transcription factors in the developing intervertebral disc. Spine (Phila Pa 1976). in pressGoogle Scholar
  36. Kraus P, Yerden R, Sipes D et al (2018b) A quantitative and qualitative RNA expression profiling assay for cell culture with single cell resolution. Cytotechnology 70:185–192.  https://doi.org/10.1007/s10616-017-0132-1CrossRefGoogle Scholar
  37. Lama P, Le Maitre CL, Harding IJ et al (2018) Nerves and blood vessels in degenerated intervertebral discs are confined to physically disrupted tissue. J Anat 233:86–97.  https://doi.org/10.1111/joa.12817CrossRefGoogle Scholar
  38. Lander AD (2009) The ‘stem cell’ concept: is it holding us back? J Biol 8:70.  https://doi.org/10.1186/jbiol177CrossRefGoogle Scholar
  39. Lee WJ, Chatterjee S, Yap SP et al (2017) An integrative developmental genomics and systems biology approach to identify an in vivo Sox trio-mediated gene regulatory network in murine embryos. Biomed Res Int 2017:8932583.  https://doi.org/10.1155/2017/8932583CrossRefGoogle Scholar
  40. Li K, Kapper D, Youngs B et al (2019) Potential biomarkers of the mature intervertebral disc identified at the single cell level. J Anat 234:16–32.  https://doi.org/10.1111/joa.12904CrossRefGoogle Scholar
  41. Liang CZ, Li H, Tao YQ et al (2012) The relationship between low pH in intervertebral discs and low back pain: a systematic review. Arch Med Sci 8:952–956.  https://doi.org/10.5114/aoms.2012.32401CrossRefGoogle Scholar
  42. Liao TW (2005) Clustering of time series data – a survey. Pattern Recogn 38:1857–1874.  https://doi.org/10.1016/j.patcog.2005.01.025CrossRefzbMATHGoogle Scholar
  43. Lv F, Leung VY, Huang S et al (2014) In search of nucleus pulposus-specific molecular markers. Rheumatology (Oxford) 53:600–610.  https://doi.org/10.1093/rheumatology/ket303CrossRefGoogle Scholar
  44. Minogue BM, Richardson SM, Zeef LA et al (2010a) Characterization of the human nucleus pulposus cell phenotype and evaluation of novel marker gene expression to define adult stem cell differentiation. Arthritis Rheum 62:3695–3705.  https://doi.org/10.1002/art.27710CrossRefGoogle Scholar
  45. Minogue BM, Richardson SM, Zeef LA et al (2010b) Transcriptional profiling of bovine intervertebral disc cells: implications for identification of normal and degenerate human intervertebral disc cell phenotypes. Arthritis Res Ther 12:R22.  https://doi.org/10.1186/ar2929CrossRefGoogle Scholar
  46. Molinos M, Almeida CR, Caldeira J et al (2015) Inflammation in intervertebral disc degeneration and regeneration. J R Soc Interface 12:20141191.  https://doi.org/10.1098/rsif.2014.1191CrossRefGoogle Scholar
  47. Moriguchi Y, Alimi M, Khair T et al (2016) Biological treatment approaches for degenerative disk disease: a literature review of in vivo animal and clinical data. Global Spine J 6:497–518.  https://doi.org/10.1055/s-0036-1571955CrossRefGoogle Scholar
  48. Oegema TR Jr (1993) Biochemistry of the intervertebral disc. Clin Sports Med 12:419–439Google Scholar
  49. Oshima H, Ishihara H, Urban JP et al (1993) The use of coccygeal discs to study intervertebral disc metabolism. J Orthop Res 11:332–338.  https://doi.org/10.1002/jor.1100110304CrossRefGoogle Scholar
  50. Pattappa G, Li Z, Peroglio M et al (2012) Diversity of intervertebral disc cells: phenotype and function. J Anat 221:480–496.  https://doi.org/10.1111/j.1469-7580.2012.01521.xCrossRefGoogle Scholar
  51. Pennicooke B, Moriguchi Y, Hussain I et al (2016) Biological treatment approaches for degenerative disc disease: a review of clinical trials and future directions. Cureus 8:e892.  https://doi.org/10.7759/cureus.892CrossRefGoogle Scholar
  52. Petrie RJ, Gavara N, Chadwick RS et al (2012) Nonpolarized signaling reveals two distinct modes of 3D cell migration. J Cell Biol 197:439–455.  https://doi.org/10.1083/jcb.201201124CrossRefGoogle Scholar
  53. Risbud MV, Schoepflin ZR, Mwale F et al (2015) Defining the phenotype of young healthy nucleus pulposus cells: recommendations of the Spine Research Interest Group at the 2014 annual ORS meeting. J Orthop Res 33:283–293.  https://doi.org/10.1002/jor.22789CrossRefGoogle Scholar
  54. Sakai D, Andersson GB (2015) Stem cell therapy for intervertebral disc regeneration: obstacles and solutions. Nat Rev Rheumatol 11:243–256.  https://doi.org/10.1038/nrrheum.2015.13CrossRefGoogle Scholar
  55. Sakai D, Schol J (2017) Cell therapy for intervertebral disc repair: clinical perspective. J Orthop Translat 9:8–18.  https://doi.org/10.1016/j.jot.2017.02.002CrossRefGoogle Scholar
  56. Schindelin J, Arganda-Carreras I, Frise E et al (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676–682.  https://doi.org/10.1038/nmeth.2019CrossRefGoogle Scholar
  57. Sivakamasundari V, Lufkin T (2013) Stemming the degeneration: IVD stem cells and stem cell regenerative therapy for degenerative disc disease. Adv Stem Cells 2013.  https://doi.org/10.5171/2013.724547
  58. Sivakamasundari V, Kraus P, Sun W et al (2017) A developmental transcriptomic analysis of Pax1 and Pax9 in embryonic intervertebral disc development. Biol Open 6:187–199.  https://doi.org/10.1242/bio.023218CrossRefGoogle Scholar
  59. Thorpe AA, Binch AL, Creemers LB et al (2016) Nucleus pulposus phenotypic markers to determine stem cell differentiation: fact or fiction. Oncotarget 7:2189–2200.  https://doi.org/10.18632/oncotarget.6782CrossRefGoogle Scholar
  60. Tinevez JY, Perry N, Schindelin J et al (2017) TrackMate: an open and extensible platform for single-particle tracking. Methods 115:80–90.  https://doi.org/10.1016/j.ymeth.2016.09.016CrossRefGoogle Scholar
  61. Turner SA, Wright KT, Jones PN et al (2016) Temporal analyses of the response of intervertebral disc cells and mesenchymal stem cells to nutrient deprivation. Stem Cells Int 2016:5415901.  https://doi.org/10.1155/2016/5415901CrossRefGoogle Scholar
  62. Urban JP, Holm S, Maroudas A et al (1977) Nutrition of the intervertebral disk. An in vivo study of solute transport. Clin Orthop Relat Res 101–114CrossRefGoogle Scholar
  63. van den Akker GGH, Koenders MI, van de Loo FAJ et al (2017) Transcriptional profiling distinguishes inner and outer annulus fibrosus from nucleus pulposus in the bovine intervertebral disc. Eur Spine J 26:2053–2062.  https://doi.org/10.1007/s00586-017-5150-3CrossRefGoogle Scholar
  64. Wang W, Lo P, Frasch M et al (2000) Hmx: an evolutionary conserved homeobox gene family expressed in the developing nervous system in mice and Drosophila. Mech Dev 99:123–137CrossRefGoogle Scholar
  65. Waterman BR, Belmont PJ Jr, Schoenfeld AJ (2012) Low back pain in the United States: incidence and risk factors for presentation in the emergency setting. Spine J 12:63–70.  https://doi.org/10.1016/j.spinee.2011.09.002CrossRefGoogle Scholar
  66. Wills QF, Mellado-Gomez E, Nolan R et al (2017) The nature and nurture of cell heterogeneity: accounting for macrophage gene-environment interactions with single-cell RNA-Seq. BMC Genomics 18:53.  https://doi.org/10.1186/s12864-016-3445-0CrossRefGoogle Scholar
  67. Wuertz K, Godburn K, Neidlinger-Wilke C et al (2008) Behavior of mesenchymal stem cells in the chemical microenvironment of the intervertebral disc. Spine (Phila Pa 1976) 33:1843–1849.  https://doi.org/10.1097/BRS.0b013e31817b8f53CrossRefGoogle Scholar

Copyright information

© Springer Nature Singapore Pte Ltd 2019

Authors and Affiliations

  • Petra Kraus
    • 1
  • Kangning Li
    • 1
  • Darren Sipes
    • 1
  • Lara Varden
    • 1
  • Rachel Yerden
    • 1
  • Althea Henderson
    • 1
  • Shantanu Sur
    • 1
  • Thomas Lufkin
    • 1
    Email author
  1. 1.Department of BiologyClarkson UniversityPotsdamUSA

Personalised recommendations