Neri Simona, Guidotti Serena, Lilli Nicoletta Libera, Cattini Luca, Mariani Erminia
Immunorheumatology and Tissue Regeneration Laboratory, Rizzoli Orthopedic Institute, Via di Barbiano 1/10, Bologna, 40136, Italy.
Department of Medical and Surgical Sciences, University of Bologna, Bologna, Italy.
J Orthop Res. 2017 May;35(5):1029-1037. doi: 10.1002/jor.23349. Epub 2016 Jul 3.
Different sources of mesenchymal stromal cells can be considered for regenerative medicine applications. Here we analyzed human adipose-derived stromal cells from infrapatellar fat pad (IFPSC) of osteoarthritis patients, representing a very interesting candidate for cartilage regeneration. No data are available concerning IFPSC stability after in vitro expansion. Indeed, replicative potential and multipotency progressively decrease during culture passages while DNA damage and cell senescence increase, thus possibly affecting clinical applications. To investigate whether in vitro expansion influences the genetic stability and replicative senescence of IFPSC, we performed long-term cultures and comparatively analyzed cells at different culture passages. Stromal vascular fraction was harvested from infrapatellar fat pad of 11 osteoarthritis patients undergoing knee replacement surgery. Cell recovery, growth kinetics, surface marker profile, and differentiation ability in inductive culture conditions were recorded. Genetic integrity maintenance was estimated by microsatellite instability analysis and mismatch repair gene expression, whereas telomere length and telomerase activity were assessed to evaluate replicative senescence. Anchorage-dependent growth was tested by soft agar culture. IFPSC displayed a phenotype similar to mesenchymal stromal cells from subcutaneous fat and showed differentiation ability. No microsatellite instability was documented even at advanced culture times in accordance to a sustained expression of mismatch repair genes, thus highlighting stability of short repeated sequences in the genome. No significant telomere attrition nor telomerase activity were documented during culture and cells did not lose anchorage-dependent growth ability. The presented data support the suitability and safety of in vitro expanded IFPSC from osteoarthritis patients for applications in regenerative medicine approaches. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1029-1037, 2017.
间充质基质细胞的不同来源可用于再生医学应用。在此，我们分析了骨关节炎患者髌下脂肪垫（IFPSC）来源的人脂肪间充质基质细胞，其是软骨再生的一个非常有吸引力的候选细胞。目前尚无关于IFPSC体外扩增后稳定性的数据。实际上，在培养传代过程中，细胞的复制潜力和多能性逐渐降低，而DNA损伤和细胞衰老增加，从而可能影响临床应用。为了研究体外扩增是否会影响IFPSC的遗传稳定性和复制性衰老，我们进行了长期培养，并对不同传代的细胞进行了比较分析。从11例接受膝关节置换手术的骨关节炎患者的髌下脂肪垫中获取基质血管成分。记录细胞回收率、生长动力学、表面标志物谱以及诱导培养条件下的分化能力。通过微卫星不稳定性分析和错配修复基因表达评估遗传完整性维持情况，同时评估端粒长度和端粒酶活性以评价复制性衰老。通过软琼脂培养检测贴壁依赖性生长。IFPSC表现出与皮下脂肪来源的间充质基质细胞相似的表型，并具有分化能力。即使在培养后期，根据错配修复基因的持续表达，也未发现微卫星不稳定性，从而突出了基因组中短重复序列的稳定性。培养过程中未发现明显的端粒损耗和端粒酶活性，且细胞未丧失贴壁依赖性生长能力。所呈现的数据支持从骨关节炎患者体外扩增的IFPSC在再生医学方法中的适用性和安全性。© 2016骨科研究协会。由Wiley Periodicals, Inc.出版。《矫形外科研究杂志》35:1029 - 1037, 2017年。
