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.