Cartilage repair using induced pluripotent stem cell derived chondrocytes in osteoarthriti

doi:10.3969/j.issn.2095-4344.2017.33.015

Abstract

Abstract:

BACKGROUND: Stem cell-based therapy has been proposed for the treatment of osteoarthritis (OA) and induced pluripotent stem cells (iPSCs) are becoming a promising stem cell source as they have strong proliferation and differentiation potentials and no ethics problem.
OBJECTIVE: To explore an effective method of iPSCs differentiating into chondrocytes and to study the therapeutic effect of iPSCs derived chondrocytes on osteoarthritis.
METHODS: In this study, three steps were developed to induce human iPSCs to differentiate into chondrocytes which were then transplanted into rat OA models induced by monosodium iodoacetate (MIA). There were four groups in the experiment: control group with normal saline injection, model group with MIA injection, iPSCs group with iPSCs transplantation following MIA injection, and differentiated iPSCs group with transplantation of iPSCs derived chondrocytes following MIA injection. At 15 weeks after transplantation, micro-CT was used and histological analysis of the knee joint was performed.
RESULTS AND CONCLUSION: Compared with the iPS group, the expression of chondrocyte specific genes and proteins (Col2A1, GAG and Sox9) were significantly increased in the differentiated iPSCs group after 6 days of embryoid formation and after 2 weeks of cell differentiation. At 15 weeks after cell transplantation, no immune responses were observed, micro-CT showed an improvement in subchondral bone integrity, and histological examinations demonstrated the production of articular cartilage matrix. iPSCs derived chondrocytes showed better effects on articular cartilage repair than the iPSCs. To conclude, iPSCs derived chondrocytes can be effective via transplantation approach for cartilage tissue regeneration in OA rats.

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: Osteoarthritis, Induced Pluripotent Stem Cells, Cell Differentiation, Chondrocytes, Tissue Engineering

CLC Number:

R394.2

Wu Zhang-song, Zhu Hong-xia, Luo Zhi-qiang, Wu Xiao-min, Lin Guang-yao, Zhang Ming-yu, Gu Hong-sheng,Zhu Yan-xia. Cartilage repair using induced pluripotent stem cell derived chondrocytes in osteoarthriti[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(33): 5339-5347.

Figures/Tables 3

2.1 iPS细胞形态学改变在分化之前，iPS细胞呈单层，细胞较小，呈纤维状，当细胞在甲基纤维素上培养后，细胞呈克隆样生长，选择大小相似的拟胚体进行诱导实验。在将拟胚体细胞贴壁培养之前，在拟胚体细胞悬液中加入软骨诱导剂进行预诱导。在预诱导过程中，拟胚体表现出良好的细胞形态，很少有内部细胞坏死。将拟胚体转移到明胶包被的培养皿中，用软骨诱导剂培养一两周后，会有软骨样的较大球形细胞从拟胚体上萌芽出来，见图1。 2.2 免疫化学分析诱导分化2周后，整个带有萌芽细胞的拟胚体都被甲苯胺蓝染上较深蓝色(图2A)，表明有软骨外基质形成。而未分化组并没有明显的蓝色出现。为了确定iPS细胞的软骨分化能力，检测到分化的拟胚体有明显的Ⅱ型胶原的表达(红色荧光，图2A)。为了进一步确认软骨细胞分化程度，在分化的第7天和第14天，分别用荧光定量PCR对软骨特异性基因和干性相关基因进行检测，结果发现，诱导2周后Col2a1，Aggrecan和Sox9的表达量显著升高。与未分化的iPS细胞相比，分化后细胞Oct4和Nanog的表达量显著性下降。而未分化的拟胚体和分化了7 d的iPS细胞这两种基因表达水平差异无显著性意义(图2B)。 2.3 骨和软骨下骨定量变化细胞移植后2，5，7，10，15周，采用micro-CT观察骨密度、骨小梁厚度(Tb.Th)、骨小梁数目(Tb.N)和骨小梁间距(Tb.Sp)变化，随着时间的推移，谷氨酸钠碘乙酸注射组和对照组大鼠胫骨软骨下软骨的骨密度、骨小梁厚度和骨小梁数目明显上升，15周后，两组胫骨骨小梁间距与第2周相比，明显减小(图3)。细胞移植后2周，与对照组相比，谷氨酸钠碘乙酸注射组骨密度和骨小梁数目均明显下降。细胞移植后5周，骨密度值略有上升，而第7周可能已经出现骨质疏松症状，骨密度明显较低。但是第10周骨密度值又显著上升，并且与对照组值接近，这可能是软骨内骨化造成的。细胞移植后第5周，骨小梁数目值突然上升，软骨下骨的骨小梁断裂可能是导致骨小梁数目上升的原因。移植未分化iPS细胞组和移植分化后iPS细胞组骨密度和骨小梁数目没有显著性差异。到第15周，两个细胞移植组和对照组胫骨骨小梁数目没有明显差异。细胞移植后第7周，骨小梁厚度值下降至与对照组接近。细胞移植后第5周，尽管骨小梁间距值略有升高，但在接下来几周时间里迅速下降。15周之后，移植软骨分化后iPS细胞组与移植未分化的iPS细胞组相比，其骨密度、骨小梁厚度、骨小梁数目和骨小梁间距值均接近于对照组。 2.4 软骨下骨形态学改变谷氨酸钠碘乙酸注射组所有大鼠都表现出软骨下骨的病理学改变，然而在整个研究过程中(15周)对照组胫骨的软骨下骨没有任何骨关节炎样变化。随着时间的推移，对谷氨酸钠碘乙酸注射组关节形态进行追踪，发现注射谷氨酸钠碘乙酸后的第15周胫骨软骨下骨出现硬化，特别是胫骨内髁部位(图4)。注射谷氨酸钠碘乙酸的胫骨软骨终板在核心区域出现断裂，组织学分析也可见这些断裂的出现。从micro-CT图像可以观察到，谷氨酸钠碘乙酸注射15周后，其膝盖中心胫骨软骨下骨出现空隙，这些空隙通过组织切片观察被确认为是囊肿。通过对所有膝盖的3个空间区域进行分析发现，谷氨酸钠碘乙酸注射组与对照组相比，其胫骨软骨下骨终板的空隙率显著上升(P < 0.01)。谷氨酸钠碘乙酸注射组膝关节三维重构图显示胫骨软骨下骨板出现侵蚀和点蚀，其内侧胫骨平台更严重，而对照组膝关节软骨下骨板表面完整。细胞移植后第15周观察到移植2种细胞的膝关节没有表现出明显形态学差异。但与谷氨酸钠碘乙酸注射组相比，细胞移植组胫骨软骨下骨没有观察到断裂或者空隙，只是胫骨软骨下骨终板的孔隙率略有下降(图4)。 2.5 大体观察注射谷氨酸钠碘乙酸的关节都表现出软骨损伤，软骨终板很容易从关节部位剥落，但谷氨酸钠碘乙酸注射后再进行细胞移植，软骨终板会紧紧地贴合在关节部位，很难剥落。谷氨酸钠碘乙酸注射组和细胞移植组膝关节胫骨软骨表现出褪色的局灶性病变，表明软骨出现退行性变(图5，黑色箭头)，该现象在内侧胫骨平台的中央部位表现得尤为明显，而边缘部位的软骨退变较轻。对照组胫骨中间和边缘区域均未表现出软骨损伤(图5，红色箭头)。移植iPS细胞和iPS来源软骨细胞后，软骨损伤得到部分修复。移植iPS来源的软骨细胞与移植iPS细胞相比，表现出更强的修复能力(图5，白色箭头)。 2.6 组织学分析结果对照组的关节软骨表现出典型的四层完整细胞层，包括表层、过渡层、径向深层和钙化区域，细胞外基质的主要成分是均匀分布的蛋白多糖(图6)。然而，所有注射谷氨酸钠碘乙酸的关节软骨都表现退化。在第15周时，软骨下骨终板出现软骨损伤，在中心区域出现裂痕。组织切片出现骨关节炎样特征，例如表面不连续性，蛋白多糖含量下降，软骨细胞减少以及软骨下骨硬化。移植iPS细胞来源软骨细胞后，与谷氨酸钠碘乙酸注射组相比，软骨表面变得连续，蛋白多糖含量上升，出现了软骨细胞增殖，表明正在修复软骨。但是iPS细胞移植组的效果并没有这样明显(图6)。对新的软骨组织进行Ⅱ型胶原免疫染色分析，结果表明iPS细胞来源软骨细胞可修复软骨组织(图6)。与对照组软骨相比，谷氨酸钠碘乙酸注射组的软骨表达很少的胶原蛋白，当注射iPS细胞和iPS细胞来源软骨细胞后，Ⅱ型胶原蛋白明显上升，移植iPS细胞来源软骨细胞组比移植iPS细胞组可表达更多的Ⅱ型胶原蛋白，表明iPS细胞来源软骨细胞比iPS细胞具有更好的软骨修复效果，但仍然无法达到对照组水平。番红-固绿复染的胫骨软骨切片图像如图6所示。谷氨酸钠碘乙酸注射组软骨出现大量的蛋白多糖流失，对照组并没有观察到此现象。移植iPS细胞之后，中间的胫骨平台变厚出现局部退化，骨头表面仍然有斜裂现象，软骨下骨出现骨吸收现象，此外可观察到软骨下骨重建及变厚现象。但总体来说iPS细胞来源软骨细胞组的修复效率要明显高于iPS细胞组。"

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