title: Regenerative and immunogenic characteristics of cultured nucleus pulposus cells from human cervical intervertebral discs.
abstract: Cell-based regenerative approaches have been suggested as primary or adjuvant procedures for the treatment of degenerated intervertebral disc (IVD) diseases. Our aim was to evaluate the regenerative and immunogenic properties of mildly and severely degenerated cervical nucleus pulposus (NP) cells with regard to cell isolation, proliferation and differentiation, as well as to cell surface markers and co-cultures with autologous or allogeneic peripheral blood mononuclear cells (PBMC) including changes in their immunogenic properties after 3-dimensional (3D)-culture. Tissue from the NP compartment of 10 patients with mild or severe grades of IVD degeneration was collected. Cells were isolated, expanded with and without basic fibroblast growth factor and cultured in 3D fibrin/poly (lactic-co-glycolic) acid transplants for 21 days. Real-time reverse-transcription polymerase chain reaction (RT-PCR) showed the expression of characteristic NP markers ACAN, COL1A1 and COL2A1 in 2D- and 3D-culture with degeneration- and culture-dependent differences. In a 5,6-carboxyfluorescein diacetate N-succinimidyl ester-based proliferation assay, NP cells in monolayer, regardless of their grade of degeneration, did not provoke a significant proliferation response in T cells, natural killer (NK) cells or B cells, not only with donor PBMC, but also with allogeneic PBMC. In conjunction with low inflammatory cytokine expression, analyzed by Cytometric Bead Array and fluorescence-activated cell sorting (FACS), a low immunogenicity can be assumed, facilitating possible therapeutic approaches. In 3D-culture, however, we found elevated immune cell proliferation levels, and there was a general trend to higher responses for NP cells from severely degenerated IVD tissue. This emphasizes the importance of considering the specific immunological alterations when including biomaterials in a therapeutic concept. The overall expression of Fas receptor, found on cultured NP cells, could have disadvantageous implications on their potential therapeutic applications because they could be the targets of cytotoxic T-cell activity acting by Fas ligand-induced apoptosis.
authors.affiliation: Tissue Engineering Laboratory and Berlin-Brandenburg Center for Regenerative Therapies, Department of Rheumatology and Clinical Immunology, Charité - Universitätsmedizin Berlin, Berlin, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Stich
authors.affiliation: Institute of Medical Immunology and Berlin-Brandenburg Center for Regenerative Therapies, Charité - Universitätsmedizin Berlin, Berlin, Germany.
authors.forename: Meaghan
authors.initials: M
authors.lastname: Stolk
authors.affiliation: Department of Neurosurgery, Innsbruck Medical University, Innsbruck, Austria.
authors.forename: Pierre Pascal
authors.initials: PP
authors.lastname: Girod
authors.affiliation: Department of Neurosurgery, Innsbruck Medical University, Innsbruck, Austria.
authors.forename: Claudius
authors.initials: C
authors.lastname: Thomé
authors.affiliation: Tissue Engineering Laboratory and Berlin-Brandenburg Center for Regenerative Therapies, Department of Rheumatology and Clinical Immunology, Charité - Universitätsmedizin Berlin, Berlin, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Sittinger
authors.affiliation: Tissue Engineering Laboratory and Berlin-Brandenburg Center for Regenerative Therapies, Department of Rheumatology and Clinical Immunology, Charité - Universitätsmedizin Berlin, Berlin, Germany.
authors.forename: Jochen
authors.initials: J
authors.lastname: Ringe
authors.affiliation: Institute of Medical Immunology and Berlin-Brandenburg Center for Regenerative Therapies, Charité - Universitätsmedizin Berlin, Berlin, Germany.
authors.forename: Martina
authors.initials: M
authors.lastname: Seifert
authors.affiliation: Department of Neurosurgery, University Medical Center Mannheim, Heidelberg University, Mannheim, Germany.
authors.forename: Aldemar Andres
authors.initials: AA
authors.lastname: Hegewald
mesh_terms: D000328:Adult; D000368:Aged; D002478:Cells, Cultured; D002574:Cervical Vertebrae; D018920:Coculture Techniques; D020869:Gene Expression Profiling; D006801:Humans; D007403:Intervertebral Disc / Q000166:cytology / Q000276:immunology / Q000502:physiology*; D008875:Middle Aged