RT journal article
T1 Assessment of endothelial colony forming cells delivery routes in a murine model of critical limb threatening ischemia using an optimized cell tracking approach
A1 Rojas Torres, Marta
A1 Sánchez Gomar, Ismael
A1 Rosal Vela, Antonio
A1 Beltrán Camacho, Lucía
A1 Eslava Alcón, Sara
A1 Alonso Piñero, José Ángel
A1 Martín-Ramírez, Javier
A1 Moreno Luna, Rafael
A1 Durán Ruiz, María del Carmen
A2 BiomedicinaBiotecnología y Salud Pública
K1 Biodistribution
K1 CLTI
K1 Cell therapy
K1 ECFCs
K1 DiR labeling
AB Background Endothelial colony forming cells (ECFCs), alone or in combination with mesenchymal stem cells, have been selected as potential therapeutic candidates for critical limb-threatening ischemia (CLTI), mainly for those patients considered as "no-option," due to their capability to enhance revascularization and perfusion recovery of ischemic tissues. Nevertheless, prior to translating cell therapy to the clinic, biodistribution assays are required by regulatory guidelines to ensure biosafety as well as to discard undesired systemic translocations. Different approaches, from imaging technologies to qPCR-based methods, are currently applied. Methods In the current study, we have optimized a cell-tracking assay based on DiR fluorescent cell labeling and near-infrared detection for in vivo and ex vivo assays. Briefly, an improved protocol for DiR staining was set up, by incubation of ECFCs with 6.67 mu M DiR and intensive washing steps prior cell administration. The minimal signal detected for the residual DiR, remaining after these washes, was considered as a baseline signal to estimate cell amounts correlated to the DiR intensity values registered in vivo. Besides, several assays were also performed to determine any potential effect of DiR over ECFCs functionality. Furthermore, the optimized protocol was applied in combination with qPCR amplification of specific human Alu sequences to assess the final distribution of ECFCs after intramuscular or intravenous administration to a murine model of CLTI. Results The optimized DiR labeling protocol indicated that ECFCs administered intramuscularly remained mainly within the hind limb muscle while cells injected intravenously were found in the spleen, liver and lungs. Conclusion Overall, the combination of DiR labeling and qPCR analysis in biodistribution assays constitutes a highly sensitive approach to systemically track cells in vivo. Thereby, human ECFCs administered intramuscularly to CLTI mice remained locally within the ischemic tissues, while intravenously injected cells were found in several organs. Our data corroborate the need to perform biodistribution assays in order to define specific parameters such as the optimal delivery route for ECFCs before their application into the clinic.
PB BMC
SN 1757-6512
YR 2021
FD 2021-06
LK http://hdl.handle.net/10498/27466
UL http://hdl.handle.net/10498/27466
LA eng
DS Repositorio Institucional de la Universidad de Cádiz
RD 07-nov-2026