Department of Regenerative Science, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, 700-8558, Japan.
Department of Regenerative Science, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, 700-8558, Japan.
Biochem Biophys Res Commun. 2023 Dec 20;687:149146. doi: 10.1016/j.bbrc.2023.149146. Epub 2023 Oct 24.
Production of cartilaginous particles for regenerative medicine requires a large supply of chondrocytes and development of suitable production techniques. Previously, we successfully produced human induced pluripotent stem cell (hiPSC)-derived limb bud mesenchymal cells (ExpLBM cells) with a high chondrogenic differentiation potential that stably proliferate. It may be possible to use these cells in combination with a stirred bioreactor to develop a tissue-engineered cell culture technology with potential for scale-up to facilitate production of large amounts of cartilaginous particles. ExpLBM cells derived from 414C2 and Ff-I 14s04 (human leukocyte antigen homozygous) hiPSCs were seeded into a stirred bioreactor containing cartilage induction medium. To characterize the cartilaginous particles produced, we performed real-time quantitative reverse transcription-polymerase chain reaction and histological analyses. Additionally, we transplanted the cartilage tissue into osteochondral defects of immunocompromised rats to assess its functionality, and evaluated engraftment of the grafted tissue. We successfully produced large amounts of cartilaginous particles via cartilage induction culture in a stirred bioreactor. This tissue exhibited significantly increased expression levels of type II collagen (COL2), aggrecan (ACAN), and SRY-box transcription factor 9 (SOX9), as well as positive Safranin O and Toluidine blue staining, indicating that it possesses characteristics of hyaline cartilage. Furthermore, engrafted tissues in osteochondral knee defects of immunodeficient rats were positively stained for human vimentin, COL2, and ACAN as well as with Safranin O. In this study, we successfully generated large amounts of hiPSC-derived cartilaginous particles using a combination of tissue engineering techniques. This method is promising as a cartilage regeneration technology with potential for scale-up.
为了再生医学的需要，大量供应软骨细胞并开发合适的生产技术。在此之前，我们成功地生产出具有高软骨分化潜能且稳定增殖的人诱导多能干细胞（hiPSC）衍生的肢芽间充质细胞（ExpLBM 细胞）。这些细胞可能与搅拌式生物反应器结合使用，开发出具有潜在可扩展性的组织工程细胞培养技术，以方便大量软骨颗粒的生产。将 414C2 和 Ff-I 14s04（人类白细胞抗原纯合子）hiPSC 衍生的 ExpLBM 细胞接种到含有软骨诱导培养基的搅拌式生物反应器中。为了表征所产生的软骨颗粒，我们进行了实时定量逆转录聚合酶链反应和组织学分析。此外，我们将软骨组织移植到免疫缺陷大鼠的骨软骨缺损中，以评估其功能，并评估移植组织的植入情况。我们通过在搅拌式生物反应器中进行软骨诱导培养成功地生产出大量的软骨颗粒。这种组织表现出显著增加的 II 型胶原（COL2）、聚集蛋白聚糖（ACAN）和性别决定区 Y 框转录因子 9（SOX9）的表达水平，以及阳性的番红 O 和甲苯胺蓝染色，表明其具有透明软骨的特征。此外，免疫缺陷大鼠膝关节骨软骨缺损中植入的组织对人波形蛋白、COL2 和 ACAN 以及番红 O 呈阳性染色。在这项研究中，我们成功地使用组织工程技术组合生成了大量的 hiPSC 衍生的软骨颗粒。这种方法有望成为一种具有潜在可扩展性的软骨再生技术。
