Biodistribution and persistence of human umbilical cord-derived mesenchymal stem cells in NCG mice: a comparative study

This study aims to investigate the biodistribution and persistence of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) in NCG mice post-intravenous injection, utilizing ⁸⁹Zr-PET/CT, bioluminescence imaging, multiplex immunohistochemistry (mIHC), and quantitative polymerase chain reaction (qPCR) hUC-MSCs were labeled with ⁸⁹Zr-oxine (⁸⁹Zr-MSCs) or transduced with luciferase gene (Luc-MSCs). Real-time tracking of ⁸⁹Zr-MSCs lasted for 14-days followed by mIHC staining of hCD73. Real-time tracking of Luc-MSCs lasted for 7-days, followed by mIHC staining of hCD73 and human Alu-based qPCR. All methods adhered to ICH and other regulatory guidelines for development of cell-based drugs. A biodistribution and persistence pattern was observed in the order of lung &gt; liver &gt; kidney &gt; &gt;spleen, although discrepancies were noted for the liver and kidney. Each method exhibited strengths and weaknesses: ⁸⁹Zr-PET/CT enabled long-term tracking but encountered issues with ⁸⁹Zr shedding and dead cells; bioluminescence provided specific detection but was hampered by a rapid decline in signal; mIHC identified cells but relied on antigen abundance; qPCR detected minimal cell quantities but was unable to differentiate between live and dead cells. These limitations may obscure the true fate of cells <i>in vivo</i>, highlighting the need for more accurate and reliable assessment techniques. For cell-based therapy, where the exogenous cells will go and how long they will stay are of interested. This study investigated the biodistribution and persistence of hUC-MSCs in NCG mice. hUC-MSCs gradually diminished after intravenous injection, but this process underwent at least two weeks. The data demonstrated that some hUC-MSCs could persist in lung as long as 14 days. The dead hUC-MSCs were transferred to liver and kidney. Whether other place, such as knee joint, would have dead cells is waiting for exploration. In this study, the role of macrophages in cleaning hUC-MSCs was explored, but more work are needed to effectively identify viable MSCs, dead MSCs, and engulfed MSCs. All of the methods indicated that the lung was the primary distribution site for hUC-MSCs after intravenous injection.While ⁸⁹Zr-PET/CT demonstrated that dead hUC-MSCs could be detected in the liver and kidney shortly after injection, it does not exclude the possibility that a small proportion of live MSCs may also migrate to these organs. More precise methodologies are required to distinguish live and dead cells over there.Neither hUC-MSCs nor genetic material from hUC-MSCs could be detected in the spleen over one day after injection using mIHC or Alu-qPCR.The persistent signal of ⁸⁹Zr in the liver and spleen might partially result from enrichment; therefore, careful consideration is necessary to accurately assess the background of radioisotopes in long-term biodistribution studies.The decline of bioluminescence occurred rapidly, and the intensity of the bioluminescent signal was insufficient to detect small quantities of cells <i>in vivo</i>.To identify the presence of cells, staining multiple antigens by mIHC may be a good choice. To quantify the presence of hUC-MSCs or genetic material of hUC-MSCs, qPCR is cost efficient and is able to detect small number of cells. All of the methods indicated that the lung was the primary distribution site for hUC-MSCs after intravenous injection. While ⁸⁹Zr-PET/CT demonstrated that dead hUC-MSCs could be detected in the liver and kidney shortly after injection, it does not exclude the possibility that a small proportion of live MSCs may also migrate to these organs. More precise methodologies are required to distinguish live and dead cells over there. Neither hUC-MSCs nor genetic material from hUC-MSCs could be detected in the spleen over one day after injection using mIHC or Alu-qPCR. The persistent signal of ⁸⁹Zr in the liver and spleen might partially result from enrichment; therefore, careful consideration is necessary to accurately assess the background of radioisotopes in long-term biodistribution studies. The decline of bioluminescence occurred rapidly, and the intensity of the bioluminescent signal was insufficient to detect small quantities of cells <i>in vivo</i>. To identify the presence of cells, staining multiple antigens by mIHC may be a good choice. To quantify the presence of hUC-MSCs or genetic material of hUC-MSCs, qPCR is cost efficient and is able to detect small number of cells.

本研究旨在利用⁸⁹Zr-PET/CT、生物发光成像、多重免疫组化（multiplex immunohistochemistry, mIHC）及定量聚合酶链式反应（quantitative polymerase chain reaction, qPCR），探究人脐带间充质干细胞（hUC-MSCs）在NCG小鼠静脉注射后的生物分布与存续情况。

hUC-MSCs经⁸⁹Zr-奥克辛标记（⁸⁹Zr-MSCs）或转导荧光素酶基因（Luc-MSCs）。⁸⁹Zr-MSCs的实时追踪持续14天，随后进行hCD73的mIHC染色；Luc-MSCs的实时追踪持续7天，随后进行hCD73的mIHC染色及基于人Alu序列的qPCR检测。所有方法均遵循ICH及其他细胞类药物研发相关监管指南。

观察到的生物分布与存续模式为肺＞肝＞肾＞＞脾，尽管肝和肾的结果存在差异。各方法均有优劣：⁸⁹Zr-PET/CT可实现长期追踪，但存在⁸⁹Zr脱落及死细胞干扰问题；生物发光成像可特异性检测，但信号衰减迅速；mIHC可识别细胞，但依赖抗原丰度；qPCR可检测极少量细胞，但无法区分活细胞与死细胞。这些局限性可能掩盖细胞在体内的真实命运，凸显了对更准确可靠的评估技术的需求。

对于细胞疗法而言，外源细胞的归巢位置及存续时间是研究热点。本研究探究了hUC-MSCs在NCG小鼠体内的生物分布与存续情况：静脉注射后hUC-MSCs逐渐减少，但该过程至少持续两周；数据显示部分hUC-MSCs可在肺中存续长达14天；死亡的hUC-MSCs被转移至肝和肾；其他部位（如膝关节）是否存在死亡细胞仍有待探索。

本研究探索了巨噬细胞在清除hUC-MSCs中的作用，但仍需更多研究以有效区分存活、死亡及被吞噬的MSCs。所有方法均表明，肺是静脉注射后hUC-MSCs的主要分布部位。尽管⁸⁹Zr-PET/CT显示注射后短期内肝和肾中可检测到死亡hUC-MSCs，但不排除少量存活MSCs迁移至这些器官的可能性，需更精准的方法区分上述部位的活细胞与死细胞。

注射一天后，通过mIHC或Alu-qPCR无法在脾脏中检测到hUC-MSCs或其遗传物质。肝和脾中持续存在的⁸⁹Zr信号可能部分源于富集效应，因此在长期生物分布研究中需谨慎评估放射性同位素背景。生物发光信号衰减迅速，其强度不足以检测体内极少量细胞。

若需识别细胞存在，通过mIHC进行多抗原染色是较好选择；若需定量hUC-MSCs或其遗传物质，qPCR成本低廉且可检测少量细胞。

所有方法均表明，肺是静脉注射后hUC-MSCs的主要分布部位。尽管⁸⁹Zr-PET/CT显示注射后短期内肝和肾中可检测到死亡hUC-MSCs，但不排除少量存活MSCs迁移至这些器官的可能性，需更精准的方法区分上述部位的活细胞与死细胞。

注射一天后，通过mIHC或Alu-qPCR无法在脾脏中检测到hUC-MSCs或其遗传物质。肝和脾中持续存在的⁸⁹Zr信号可能部分源于富集效应，因此在长期生物分布研究中需谨慎评估放射性同位素背景。生物发光信号衰减迅速，其强度不足以检测体内极少量细胞。

若需识别细胞存在，通过mIHC进行多抗原染色是较好选择；若需定量hUC-MSCs或其遗传物质，qPCR成本低廉且可检测少量细胞。