Effect of indirect co-culture system on differentiation of bone marrow mesenchymal stem cells into nucleus pulposus-like cells

doi:10.3969/j.issn.2095-4344.2016.45.003

Abstract

Abstract:

BACKGROUND: Mesenchymal stem cells (MSCs) for the treatment of degenerative disc diseases present a new therapeutic strategy for the structural and functional recovery of the degenerative intervertebral disc.
OBJECTIVE: To study the differentiation potential of rabbit bone marrow MSCs (BMSCs) into nucleus pulposus-like cells.
METHODS: Passage 3 BMSCs and nucleus pulosus cells (NPCs) extracted from rabbits were assigned into simple BMSCs culture, simple NPCs culture or co-culture group. In the former two groups, BMSCs or NPCs were cultured in 6-well culture plates. In the co-culture group, passage 3 BMSCs and NPCs were cultured on the upper or lower layer of the 6-well Transwell chamber, respectively. Cell morphology was observed by inverted contrast phase microscope. At 7 days of culture, immunocytochemistry, RT-PCR and western blot were used to examine the expression levels of type II collagen and aggrecan.
RESULTS AND CONCLUSION: At 7 days of co-culture, BMSCs were polygonal or short spindle-shaped, with no fiber-like changes, and cell morphology was close to that of NPCs. Additionally, the expression levels of type II collagen and aggrecan in the BMSCs co-cultured with NPCs were similar to those in the NPCs cultured alone, but significantly higher than those in the BMSCs culture only. Overall, these results show that coculture of BMSCs with NPCs can induce BMSCs differentiating into an NPCs phenotype, indicating that BMSCs may provide sufficient sources of cells for the biological treatment of degenerative disc diseases.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Bone Marrow, Mesenchymal Stem Cells, Coculture Techniques, Intervertebral Disk, Tissue Engineering

CLC Number:

R394.2

Wu Hai-jun, Yin He-ping, Hu Ji-ping, Liu Cong, Li Shu-wen, Bai Ming, Du Zhi-cai . Effect of indirect co-culture system on differentiation of bone marrow mesenchymal stem cells into nucleus pulposus-like cells[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(45): 6706-6713.

Figures/Tables 2

2.1 兔骨髓间充质干细胞的形态及鉴定原代骨髓间充质干细胞贴壁大约需24 h，7 d后细胞长满融合可进行传代继续培养，经传代得到的骨髓间充质干细胞生长迅速。原代骨髓间充质干细胞在倒置相差显微镜下观察表现为不规则生长，大部分细胞呈多角形，传到第3代时表现为集落样生长，大部分细胞呈长梭形(图1A)。第3代细胞经免疫细胞化学染色后CD29、CD44呈阳性表达，胞浆为棕色或棕褐色，CD34呈阴性表达，证明提取、纯化的细胞是骨髓间充质干细胞而不是成纤维细胞或造血干细胞(图1B-D)。 2.2 兔髓核细胞的形态及鉴定倒置相差显微镜观察发现原代髓核细胞贴壁时间平均为7 d，贴壁后长出类圆形及多角形的伪足，表现为集落样生长(图2A)，髓核细胞生长缓慢，生长至95%融合时呈长梭形，平均需15 d；第3代髓核细胞的贴壁时间平均为12 h，生长迅速，生长至95%融合平均需7 d，大部分髓核细胞呈梭形和多角形(以两三个角为主)(图2B)。经甲苯胺蓝染色的髓核细胞呈天蓝色，在胞质内的聚集蛋白聚糖被染成天蓝色，越接近细胞核，天蓝色越深(图2C)。对髓核细胞进行免疫细胞化学染色后位于髓核细胞胞质内的Ⅱ型胶原呈黄褐色，离核越近，染色越深(图2D)。 2.3 各组细胞的形态单纯培养骨髓间充质干细胞组第2天可见细胞贴壁生长，形态为长梭形，7 d后形成集落，细胞融合达到90%，形态一致，呈漩涡状生长(图3A)；单纯培养髓核细胞组第3，4天即可贴壁生长，细胞形态呈圆形或多角形，7-9 d即可传代(图3B)；共培养组第2天可见骨髓间充质干细胞被髓核细胞诱导向类髓核细胞分化，骨髓间充质干细胞由长梭形分化为类圆形，细胞边缘残存有伪足，被诱导分化的骨髓间充质干细胞逐渐融合聚集分化成类髓核细胞的细胞集落(图3C)，共同培养第7天，骨髓间充质干细胞呈多角形、短梭形改变，无纤维样变化，形态接近髓核细胞(图3D)。 2.4 免疫细胞化学检测细胞Ⅱ型胶原蛋白的表达单纯培养骨髓间充质干细胞第7天，免疫细胞化学检测Ⅱ型胶原蛋白表达为阴性(图4A)；单纯培养髓核细胞第7天，免疫细胞化学检测Ⅱ型胶原蛋白表达为阳性，胞浆中越靠近细胞核，棕褐色越深(图4B)；共培养第7天，免疫细胞化学检测Ⅱ型胶原蛋白表达为阳性，而且与单纯培养髓核细胞组Ⅱ型胶原蛋白表达无明显区别(图4C)。 2.5 甲苯胺蓝染色检测各组细胞聚集蛋白聚糖的表达单纯培养骨髓间充质干细胞第7天，甲苯胺蓝染色检测聚集蛋白聚糖的表达为阴性(图5A)；单纯培养髓核细胞第7天，甲苯胺蓝染色检测聚集蛋白聚糖的表达为阳性，胞浆中越靠近细胞核，蓝染程度越深(图5B)；共培养第7天，甲苯胺蓝染色检测骨髓间充质干细胞聚集蛋白聚糖的表达为阳性，而且与单纯培养髓核细胞组聚集蛋白聚糖的表达无明显差别(图5C)。 2.6 RT-PCR法检测各组细胞Ⅱ型胶原与聚集蛋白聚糖mRNA的表达共培养组中骨髓间充质干细胞Ⅱ型胶原蛋白和聚集蛋白聚糖的表达量明显高于单纯培养骨髓间充质干细胞组，吸光度值比较差异有显著性意义(P < 0.01)，而共培养组中骨髓间充质干细胞Ⅱ型胶原蛋白和聚集蛋白聚糖的表达量与单纯培养髓核细胞组无明显差别，吸光度值比较差异无显著性意义(P > 0.05)，见图6，表2。 2.7 Western blot测定各组细胞Ⅱ型胶原与聚集蛋白聚糖的表达共培养组中骨髓间充质干细胞Ⅱ型胶原蛋白和聚集蛋白聚糖的表达量明显高于单纯培养骨髓间充质干细胞组，吸光度值比较差异有显著性意(P < 0.01)，而共培养组中骨髓间充质干细胞Ⅱ型胶原蛋白和聚集蛋白聚糖的表达量与单纯培养髓核细胞组无明显差别，吸光度值比较差异无显著性意义(P > 0.05)，见图7，表3。"

References

[1] Vadalà G, Studer RK, Sowa G, et al. Coculture of bone marrow mesenchymal stem cells and nucleus pulposus cells modulate gene expression profile without cell fusion. Spine (Phila Pa 1976). 2008;33(8):870-876.
[2] Goffin J, Geusens E, Vantomme N, et al. Long-term follow-up after interbody fusion of the cervical spine. J Spinal Disord Tech. 2004;17(2):79-85.
[3] Colombini A, Lombardi G, Corsi MM, et al. Pathophysiology of the human intervertebral disc. Int J Biochem Cell Biol. 2008;40(5):837-842.
[4] Diwan AD, Parvataneni HK, Khan SN, et al. Current concepts in intervertebral disc restoration. Orthop Clin North Am. 2000;31(3):453-464.
[5] Okuma M, Mochida J, Nishimura K, et al. Reinsertion of stimulated nucleus pulposus cells retards intervertebral disc degeneration: an in vitro and in vivo experimental study. J Orthop Res. 2000;18(6): 988-997.
[6] Bianco P, Robey PG, Simmons PJ. Mesenchymal stem cells: revisiting history, concepts, and assays. Cell Stem Cell. 2008;2(4):313-319.
[7] Chamberlain G, Fox J, Ashton B, et al. Concise review: mesenchymal stem cells: their phenotype, differentiation capacity, immunological features, and potential for homing. Stem Cells. 2007;25(11): 2739-2749.
[8] Zanini C, Bruno S, Mandili G, et al. Differentiation of mesenchymal stem cells derived from pancreatic islets and bone marrow into islet-like cell phenotype. PLoS One. 2011;6(12):e28175.
[9] Greco SJ, Zhou C, Ye JH, et al. An interdisciplinary approach and characterization of neuronal cells transdifferentiated from human mesenchymal stem cells. Stem Cells Dev. 2007;16(5):811-826.
[10] 武海军,银和平,李树文,等. 密度梯度离心和贴壁筛选法分离培养兔骨髓间充质干细胞的形态学观察[J]. 中华临床医师杂志:电子版, 2012, 6(22): 7261-7265.
[11] Luoma K, Riihimäki H, Luukkonen R, et al. Low back pain in relation to lumbar disc degeneration. Spine (Phila Pa 1976). 2000;25(4):487-492.
[12] Cheung KM, Karppinen J, Chan D, et al. Prevalence and pattern of lumbar magnetic resonance imaging changes in a population study of one thousand forty-three individuals. Spine (Phila Pa 1976). 2009; 34(9):934-940.
[13] Urban MR, Fairbank JC, Bibby SR, et al. Intervertebral disc composition in neuromuscular scoliosis: changes in cell density and glycosaminoglycan concentration at the curve apex. Spine (Phila Pa 1976). 2001;26(6): 610-617.
[14] Le Maitre CL, Pockert A, Buttle DJ, et al. Matrix synthesis and degradation in human intervertebral disc degeneration. Biochem Soc Trans. 2007;35(Pt 4): 652-655.
[15] Pockert AJ, Richardson SM, Le Maitre CL, et al. Modified expression of the ADAMTS enzymes and tissue inhibitor of metalloproteinases 3 during human intervertebral disc degeneration.Arthritis Rheum. 2009; 60(2):482-491.
[16] Richardson SM, Hoyland JA, Mobasheri R, et al. Mesenchymal stem cells in regenerative medicine: opportunities and challenges for articular cartilage and intervertebral disc tissue engineering. J Cell Physiol. 2010;222(1):23-32.
[17] Hohaus C1, Ganey TM, Minkus Y, et al. Cell transplantation in lumbar spine disc degeneration disease. Eur Spine J. 2008;17 Suppl 4:492-503.
[18] Fassett DR, Kurd MF, Vaccaro AR. Biologic solutions for degenerative disk disease. J Spinal Disord Tech. 2009;22(4):297-308.
[19] Bruder SP, Jaiswal N, Ricalton NS, et al. Mesenchymal stem cells in osteobiology and applied bone regeneration. Clin Orthop Relat Res. 1998;(355 Suppl): S247-256.
[20] Sobajima S, Vadala G, Shimer A, et al. Feasibility of a stem cell therapy for intervertebral disc degeneration. Spine J. 2008;8(6):888-896.
[21] Richardson SM, Walker RV, Parker S, et al. Intervertebral disc cell-mediated mesenchymal stem cell differentiation. Stem Cells. 2006;24(3):707-716.
[22] Yamamoto Y, Mochida J, Sakai D, et al. Upregulation of the viability of nucleus pulposus cells by bone marrow-derived stromal cells: significance of direct cell-to-cell contact in coculture system. Spine (Phila Pa 1976). 2004;29(14):1508-1514.
[23] Sakai D, Mochida J, Yamamoto Y, et al. Transplantation of mesenchymal stem cells embedded in Atelocollagen gel to the intervertebral disc: a potential therapeutic model for disc degeneration. Biomaterials. 2003;24(20):3531-3541.
[24] Ni WF, Yin LH, Lu J, et al. In vitro neural differentiation of bone marrow stromal cells induced by cocultured olfactory ensheathing cells. Neurosci Lett. 2010;475(2): 99-103.
[25] Xu J, Liu X, Chen J, et al. Cell-cell interaction promotes rat marrow stromal cell differentiation into endothelial cell via activation of TACE/TNF-alpha signaling. Cell Transplant. 2010;19(1):43-53.
[26] Kumai Y, Kobler JB, Park H, et al. Modulation of vocal fold scar fibroblasts by adipose-derived stem/stromal cells. Laryngoscope. 2010;120(2):330-337.