Department of Regenerative Science, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine, 2-5-1 Shikata-Cho, Kita-Ku, Okayama, 700-8558, Japan.
Department of Orthopaedic Surgery, Surgical Science, Tokai University School of Medicine, Isehara, Japan.
Stem Cell Res Ther. 2023 Feb 24;14(1):34. doi: 10.1186/s13287-023-03252-4.
Cell sheet fabrication for articular cartilage regenerative medicine necessitates a large number of chondrocytes of consistent quality as a cell source. Previously, we have developed human-induced pluripotent stem cell (iPSC)-derived expandable PRRX1 limb-bud mesenchymal cells (ExpLBM) with stable expansion and high chondrogenic capacity, while in this study; our ExpLBM technology was combined with cell sheet engineering to assess its potential as a stable cell source for articular cartilage regeneration.
ExpLBM cells derived from human-induced pluripotent stem cells (hiPSCs), including 414C2 and Ff-KVs09 (HLA homozygous), were seeded onto a culture plate and two-dimensional chondrogenic induction (2-DCI) was initiated. After 2-DCI, ExpLBM-derived chondrocytes were stripped and transferred to temperature-responsive culture inserts and the chondrocyte sheets were histologically examined or transplanted into osteochondral knee defects of immunodeficient rats.
Immunohistochemistry revealed that ExpLBM-derived cell sheets were positive for Safranin O, COL2, and ACAN but that they were negative for COL1 and RUNX2. Furthermore, the engrafted tissues in osteochondral knee defects in immunodeficient rats were stained with SafO, human VIMENTIN, ACAN, and COL2.
The present study is the first to report the chondrocyte sheet fabrication with hiPSC-derived cell source. hiPSC-derived ExpLBM would be a promising cell source for cell sheet technology in articular cartilage regenerative medicine.
关节软骨再生医学需要大量质量一致的软骨细胞作为细胞来源。此前，我们已经开发了具有稳定扩增和高软骨形成能力的人诱导多能干细胞（iPSC）衍生的可扩增 PRRX1 肢芽间充质细胞（ExpLBM），而在这项研究中；我们的 ExpLBM 技术与细胞片工程相结合，评估其作为关节软骨再生稳定细胞来源的潜力。
从人诱导多能干细胞（hiPSCs）中分离出 ExpLBM 细胞，包括 414C2 和 Ff-KVs09（HLA 纯合子），接种到培养板上并开始二维软骨诱导（2-DCI）。2-DCI 后，ExpLBM 衍生的软骨细胞被剥离并转移到温度响应培养插入物中，对软骨细胞片进行组织学检查或移植到免疫缺陷型大鼠的骨软骨膝关节缺损中。
免疫组织化学显示 ExpLBM 衍生的细胞片对番红 O、COL2 和 ACAN 呈阳性，但对 COL1 和 RUNX2 呈阴性。此外，免疫缺陷型大鼠骨软骨膝关节缺损中的植入组织被 SafO、人 VIMENTIN、ACAN 和 COL2 染色。
本研究首次报道了用人诱导多能干细胞衍生细胞源制备的软骨细胞片。hiPSC 衍生的 ExpLBM 将成为关节软骨再生医学中细胞片技术的有前途的细胞来源。
