Choi Jungju, Kim Nam Gyo, Kong Dasom, Kim Min-Ji, Kang Byeong-Cheol, Kwon Daekee, Kim Da-Hyun, Kang Kyung-Sun
Adult Stem Cell Research Center and Research Institute for Veterinary Medicine, College of Veterinary Medicine, Seoul National University, Seoul, 08826, Republic of Korea.
Department of Experimental Animal Research, Biomedical Research Institute, Seoul National University Hospital, Seoul, Republic of Korea.
Stem Cell Res Ther. 2025 Aug 6;16(1):430. doi: 10.1186/s13287-025-04554-5.
Peripheral arterial disease (PAD) is a leading cause of limb disability due to ischemia caused by atherosclerotic plaques. Cell-based therapies using endothelial cells (ECs) have shown promise in promoting angiogenesis for PAD, but challenges remain in obtaining sufficient ECs from human tissues. Induced pluripotent stem cells (iPSCs) provide a potential solution, though immune rejection issues arise due to human leukocyte antigen (HLA) mismatches. The depletion of HLA class I and II through gene editing aims to broadly avoid lymphocyte recognition and can be achieved by inactivating β2-microglobulin (B2M) and class II transactivator (CIITA). However, B2M inactivation can lead to a 'missing self' killing response by NK cells and macrophages. To overcome this, we proposed universal iPSCs by knocking out B2M and CIITA and over-expressing CD24 to reduce immune rejection.
Universal iPSCs were tested for their pluripotency and ability to differentiate into ECs. The stimulation of universal iPSC-derived endothelial cells (U-ECs) to T and NK cells was evaluated by activation marker using flow cytometry. We generated a humanized mouse model by intravenously injecting CD34 hematopoietic stem cells isolated from umbilical cord blood into NSG mice. Finally, we induced a humanized PAD mouse model by removing the femoral artery of the left hindlimb. Then we injected U-ECs, demonstrating the therapeutic efficacy of U-ECs.
We generated hypoimmunogenic universal iPSC by knocking out B2M and CIITA, along with over-expressing CD24, and confirmed that their pluripotency was maintained. We demonstrated that U-ECs exhibit functional endothelial properties and reduced immunogenicity, effectively mitigating immune recognition from both adaptive and innate immune responses. U-ECs survived in significantly greater numbers after transplantation and elicited a weaker immune response in humanized mice. Then we induced hindlimb ischemia in humanized mice to establish a humanized PAD model. U-ECs induced effective angiogenic capabilities, leading to significant blood flow restoration in ischemic limbs.
This study demonstrates the feasibility of creating hypoimmunogenic iPSCs and their derivatives that can reduce immune response and function effectively in vivo.
外周动脉疾病（PAD）是由动脉粥样硬化斑块导致的缺血引起肢体残疾的主要原因。使用内皮细胞（ECs）的细胞疗法在促进PAD血管生成方面显示出前景，但从人体组织获取足够的ECs仍存在挑战。诱导多能干细胞（iPSCs）提供了一种潜在的解决方案，不过由于人类白细胞抗原（HLA）不匹配会出现免疫排斥问题。通过基因编辑消除HLA I类和II类分子旨在广泛避免淋巴细胞识别，这可以通过使β2-微球蛋白（B2M）和II类反式激活因子（CIITA）失活来实现。然而，B2M失活会导致自然杀伤细胞（NK细胞）和巨噬细胞的“缺失自我”杀伤反应。为克服这一问题，我们通过敲除B2M和CIITA并过表达CD24来减少免疫排斥，从而构建通用型iPSCs。
对通用型iPSCs的多能性及其分化为ECs的能力进行测试。使用流式细胞术通过激活标志物评估通用型iPSC衍生的内皮细胞（U-ECs）对T细胞和NK细胞的刺激作用。我们通过将从脐带血中分离的CD34造血干细胞静脉注射到NSG小鼠体内构建了人源化小鼠模型。最后，通过切除左后肢的股动脉诱导出人源化PAD小鼠模型。然后我们注射U-ECs，证明了U-ECs的治疗效果。
我们通过敲除B2M和CIITA并过表达CD24构建了低免疫原性的通用型iPSCs，并证实其多能性得以维持。我们证明U-ECs具有功能性内皮特性且免疫原性降低，能有效减轻适应性免疫反应和先天性免疫反应的免疫识别。U-ECs在移植后存活数量显著更多，并且在人源化小鼠中引发的免疫反应较弱。然后我们在人源化小鼠中诱导后肢缺血以建立人源化PAD模型。U-ECs诱导出有效的血管生成能力，使缺血肢体的血流显著恢复。
本研究证明了创建低免疫原性iPSCs及其衍生物的可行性，这些衍生物可减少免疫反应并在体内有效发挥作用。
