Stich Stefan, Stolk Meaghan, Girod Pierre Pascal, Thomé Claudius, Sittinger Michael, Ringe Jochen, Seifert Martina, Hegewald Aldemar Andres
Tissue Engineering Laboratory and Berlin-Brandenburg Center for Regenerative Therapies, Department of Rheumatology and Clinical Immunology, Charité - Universitätsmedizin Berlin, Berlin, Germany.
Institute of Medical Immunology and Berlin-Brandenburg Center for Regenerative Therapies, Charité - Universitätsmedizin Berlin, Berlin, Germany.
PLoS One. 2015 May 19;10(5):e0126954. doi: 10.1371/journal.pone.0126954. eCollection 2015.
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.
基于细胞的再生方法已被建议作为治疗退变椎间盘（IVD）疾病的主要或辅助手段。我们的目的是评估轻度和重度退变的颈椎髓核（NP）细胞在细胞分离、增殖和分化方面的再生及免疫原性特性，以及细胞表面标志物和与自体或异体外周血单个核细胞（PBMC）共培养的情况，包括三维（3D）培养后其免疫原性特性的变化。收集了10例轻度或重度IVD退变患者NP区的组织。分离细胞，在有和无碱性成纤维细胞生长因子的情况下进行扩增，并在3D纤维蛋白/聚（乳酸 - 乙醇酸）酸移植物中培养21天。实时逆转录聚合酶链反应（RT-PCR）显示，在二维和三维培养中，特征性NP标志物ACAN、COL1A1和COL2A1的表达存在退变和培养依赖性差异。在基于5,6 - 羧基荧光素二乙酸琥珀酰亚胺酯的增殖试验中，单层NP细胞，无论其退变程度如何，不仅对供体PBMC，而且对异体PBMC，在T细胞、自然杀伤（NK）细胞或B细胞中均未引发显著的增殖反应。通过细胞计数珠阵列和荧光激活细胞分选（FACS）分析，结合低炎症细胞因子表达，可以推测其免疫原性较低，这有利于可能的治疗方法。然而，在3D培养中，我们发现免疫细胞增殖水平升高，并且来自严重退变IVD组织的NP细胞总体上有更高反应的趋势。这强调了在治疗概念中纳入生物材料时考虑特定免疫改变的重要性。在培养的NP细胞上发现的Fas受体的总体表达可能对其潜在的治疗应用有不利影响，因为它们可能成为通过Fas配体诱导的凋亡起作用的细胞毒性T细胞活性的靶标。
