Chen JunLong, Kataoka Oki, Tsuchiya Kazeto, Oishi Yoshie, Takao Ayumi, Huang Yen-Chih, Komura Hiroko, Akiyama Saeko, Itou Ren, Inui Masafumi, Enosawa Shin, Akutsu Hidenori, Komura Makoto, Fuchimoto Yasushi, Umezawa Akihiro
Center for Regenerative Medicine, National Center for Child Health and Development Research Institute, Tokyo, Japan.
Department of Advanced Pediatric Medicine, Tohoku University School of Medicine, Sendai, Japan.
Regen Ther. 2024 Sep 10;26:889-900. doi: 10.1016/j.reth.2024.09.007. eCollection 2024 Jun.
Repairing damaged cartilage poses significant challenges, particularly in cases of congenital cartilage defects such as microtia or congenital tracheal stenosis, or as a consequence of traumatic injury, as the regenerative potential of cartilage is inherently limited. Stem cell therapy and tissue engineering offer promising approaches to overcome these limitations in cartilage healing. However, the challenge lies in the size of cartilage-containing organs, which necessitates a large quantity of cells to fill the damaged areas. Therefore, pluripotent stem cells that can proliferate indefinitely are highly desirable as a cell source. This study aims to delineate the differentiation conditions for cartilage derived from human embryonic stem cells (ESCs) and to develop an automated cell culture system to facilitate mass production for therapeutic applications.
Cartilage cell sheets were derived from human ESCs (SEES2, clinical trial-compatible line) by forming embryoid bodies (EBs) with either conventional manual culture or a benchtop multi-pipetter and an automated medium exchange integrated cell incubator, using xeno-free media. Cell sheets were implanted into the subcutaneous tissue of immunodeficient NOG mice to obtain cartilage tissue. The properties of cartilage tissues were examined by histological staining and quantitative PCR analysis.
We have optimized an efficient xeno-free system for cartilage production with the conventional culture method and successfully transitioned to an automated system. Differentiated cartilage was histologically uniform with cartilage-specific elasticity and strength. The cartilage tissues were stained by Alcian blue, safranin O, and toluidine blue, and quantitative PCR showed an increase in differentiation markers such as ACAN, COL2A1, and Vimentin. Automation significantly enhanced the efficiency of human ESC-derived chondrocyte differentiation. The number of constituent cells within EBs and the seeding density of EBs were identified as key factors influencing chondrogenic differentiation efficiency. By automating the process of chondrogenic differentiation, we achieved scalable production of chondrocytes.
By integrating the differentiation protocol with an automated cell culture system, there is potential to produce cartilage of sufficient size for clinical applications in humans. The resulting cartilage tissue holds promise for clinical use in repairing organs such as the trachea, joints, ears, and nose.
修复受损软骨面临重大挑战，尤其是在先天性软骨缺陷的情况下，如小耳畸形或先天性气管狭窄，或因创伤性损伤所致，因为软骨的再生潜力本质上是有限的。干细胞疗法和组织工程为克服软骨愈合中的这些限制提供了有前景的方法。然而，挑战在于含软骨器官的大小，这需要大量细胞来填充受损区域。因此，能够无限增殖的多能干细胞作为细胞来源非常理想。本研究旨在描绘源自人类胚胎干细胞（ESC）的软骨的分化条件，并开发一种自动化细胞培养系统以促进用于治疗应用的大规模生产。
使用无血清培养基，通过传统手动培养或台式多通道移液器和自动换液一体式细胞培养箱形成胚状体（EB），从人类ESC（SEES2，临床试验兼容系）中获得软骨细胞片。将细胞片植入免疫缺陷的NOG小鼠的皮下组织以获得软骨组织。通过组织学染色和定量PCR分析检查软骨组织的特性。
我们用传统培养方法优化了一种高效的无血清软骨生产系统，并成功过渡到自动化系统。分化的软骨在组织学上是均匀的，具有软骨特异性的弹性和强度。软骨组织用阿尔辛蓝、番红O和甲苯胺蓝染色，定量PCR显示分化标志物如ACAN、COL2A1和波形蛋白增加。自动化显著提高了人类ESC来源的软骨细胞分化效率。EB内的组成细胞数量和EB的接种密度被确定为影响软骨生成分化效率的关键因素。通过使软骨生成分化过程自动化，我们实现了软骨细胞的可扩展生产。
通过将分化方案与自动化细胞培养系统相结合，有可能生产出足够大小的软骨用于人类临床应用。所得软骨组织有望用于修复气管、关节、耳朵和鼻子等器官的临床治疗。
