Department of Otolaryngology, School of Medicine, Fukushima Medical University, Fukushima City, Japan.
Cell Transplant. 2013;22(2):341-53. doi: 10.3727/096368912X653147. Epub 2012 Aug 2.
The treatment of laryngotracheal stenosis remains a challenge as treatment often requires multistaged procedures, and successful decannulation sometimes fails after a series of operations. Induced pluripotent stem cells (iPSCs) were generated in 2006. These cells are capable of unlimited symmetrical self-renewal, thus providing an unlimited cell source for tissue-engineering applications. We have previously reported tracheal wall regeneration using a three-dimensional (3D) scaffold containing iPSCs. However, the efficiency of differentiation into cartilage was low. In addition, it could not be proven that the cartilage tissues were in fact derived from the implanted iPSCs. The purpose of this study was to evaluate and improve the use of iPSCs for the regeneration of tracheal cartilage. iPSCs were cultured in vitro in a 3D scaffold in chondrocyte differentiation medium. After cultivation, differentiation into chondrocytes was examined. The ratio of undifferentiated cells was analyzed by flow cytometry. The 3D scaffolds were implanted into tracheal defects, as an injury site, in 24 nude rats. Differentiation into chondrocytes in vitro was confirmed histologically, phenotypically, and genetically. Flow cytometric analysis demonstrated that the population of undifferentiated cells was decreased. Cartilage tissue was observed in the regenerated tracheal wall in 6 of 11 rats implanted with induced iPSCs, but in none of 13 rats implanted with the control and noninduced iPSCs. The expression of cartilage-specific protein was also demonstrated in vivo in 3D scaffolds containing iPSCs. The presence of the GFP gene derived from iPSCs was confirmed in samples of cartilage tissue by the combination of laser microdissection (LMD) and polymerase chain reaction (PCR) techniques. Our study demonstrated that iPSCs have the potential to differentiate into chondrogenic cells in vitro. Cartilage tissue was regenerated in vivo. Our results suggest that iPSCs could be a new cell source for the regeneration of tracheal cartilage.
喉气管狭窄的治疗仍然是一个挑战，因为治疗通常需要多阶段的手术，而且在一系列手术后，成功的拔管有时也会失败。诱导多能干细胞（iPSCs）于 2006 年被成功生成。这些细胞具有无限对称自我更新的能力，因此为组织工程应用提供了无限的细胞来源。我们之前曾报道过使用含有 iPSCs 的三维（3D）支架进行气管壁再生。然而，向软骨分化的效率很低。此外，不能证明这些软骨组织实际上是来自植入的 iPSCs。本研究旨在评估和改进 iPSCs 用于气管软骨再生的用途。iPSCs 在 3D 支架中的软骨细胞分化培养基中进行体外培养。培养后，检查向软骨细胞的分化情况。通过流式细胞术分析未分化细胞的比例。将 3D 支架植入 24 只裸鼠的气管缺损处，作为损伤部位。体外分化为软骨细胞在组织学、表型和遗传学上得到证实。流式细胞术分析表明未分化细胞群体减少。在 11 只植入诱导 iPSCs 的大鼠中有 6 只的再生气管壁中观察到软骨组织，但在 13 只植入对照和非诱导 iPSCs 的大鼠中均未观察到。在含有 iPSCs 的 3D 支架中也在体内证实了软骨特异性蛋白的表达。通过激光微切割（LMD）和聚合酶链反应（PCR）技术的结合，在软骨组织样本中证实了源自 iPSCs 的 GFP 基因的存在。我们的研究表明 iPSCs 具有在体外向软骨细胞分化的潜力。在体内再生了软骨组织。我们的结果表明，iPSCs 可能成为气管软骨再生的新细胞来源。
