Kikuchi Takeshi, Nishimura Masuhiro, Komori Natsuki, Iizuka Naho, Otoi Takeshige, Matsumoto Shinichi
Research and Development Center, Otsuka Pharmaceutical Factory, Inc., Naruto, Tokushima, Japan.
Bio-Innovation Research Center, Tokushima University, Myozai-gun, Tokushima, Japan.
Xenotransplantation. 2024 Jan-Feb;31(1):e12831. doi: 10.1111/xen.12831. Epub 2023 Oct 17.
Porcine tissues display a great potential as donor tissues in xenotransplantation, including cell therapy. Cryopreserving clinical grade porcine tissue and using it as a source for establishing therapeutic cells should be advantageous for transportation and scheduled manufacturing of MSCs. Of note, we previously performed encapsulated porcine islet transplantation for the treatment of unstable type 1 diabetes mellitus in the clinical setting. It has been reported that co-transplantation of islets and Mesenchymal stem cells (MSCs) enhanced efficacy. We assume that co-transplantation of porcine islets and porcine islet-derived MSCs could improve the efficacy of clinical islet xenotransplantation.
MSCs were established from fresh and cryopreserved non-clinical grade neonatal porcine islets and bone marrow (termed non-clinical grade npISLET-MSCs and npBM-MSCs, respectively), as well as from cryopreserved clinical grade neonatal porcine islets (termed clinical grade npISLET-MSCs). Subsequently, the cell proliferation rate and diameter, surface marker expression, adipogenesis, osteogenesis, and colony-forming efficiency of the MSCs were assessed.
Cell proliferation rate and diameter did not differ between clinical grade and non-clinical grade npISLET-MSCs. However, non-clinical grade npBM-MSCs were significantly shorter and smaller than both npISLET-MSCs (p < 0.05). MSC markers (CD29, CD44, and CD90) were strongly expressed in clinical grade npISLET-MSCs and non-clinical grade npISLET-MSCs and npBM-MSCs. The expression of MSC-negative markers CD31, CD34, and SLA-DR was low in all MSCs. Clinical grade npISLET-MSCs derived from adipose and osteoid tissues were positive for Oil Red and alkaline phosphatase staining. The results of colony-forming assay were not significantly different between clinical grade npISLET-MSCs and non-clinical grade npBM-MSCs.
The method described herein was successful in of developing clinical grade npISLET-MSCs from cryopreserved islets. Cryopreserved clinical grade porcine islets could be an excellent stable source of MSCs for cell therapy.
猪组织在异种移植（包括细胞治疗）中作为供体组织具有巨大潜力。冷冻保存临床级猪组织并将其用作建立治疗性细胞的来源，对于间充质干细胞（MSC）的运输和规模化生产应该是有利的。值得注意的是，我们之前在临床环境中进行了封装猪胰岛移植以治疗不稳定型1型糖尿病。据报道，胰岛与间充质干细胞（MSC）的共移植可提高疗效。我们推测猪胰岛与猪胰岛来源的MSC的共移植可提高临床胰岛异种移植的疗效。
从新鲜和冷冻保存的非临床级新生猪胰岛及骨髓中分离出MSC（分别称为非临床级npISLET-MSC和npBM-MSC），以及从冷冻保存的临床级新生猪胰岛中分离出MSC（称为临床级npISLET-MSC）。随后，评估了MSC的细胞增殖率、直径、表面标志物表达、脂肪生成、成骨能力和集落形成效率。
临床级和非临床级npISLET-MSC的细胞增殖率和直径没有差异。然而，非临床级npBM-MSC明显比两种npISLET-MSC更短更小（p < 0.05）。MSC标志物（CD29、CD44和CD90）在临床级npISLET-MSC、非临床级npISLET-MSC和npBM-MSC中均强烈表达。所有MSC中MSC阴性标志物CD31、CD34和SLA-DR的表达均较低。临床级npISLET-MSC来源的脂肪和类骨组织经油红和碱性磷酸酶染色呈阳性。临床级npISLET-MSC和非临床级npBM-MSC的集落形成试验结果无显著差异。
本文所述方法成功地从冷冻保存的胰岛中培养出临床级npISLET-MSC。冷冻保存的临床级猪胰岛可能是用于细胞治疗的MSC的优质稳定来源。
