Assessment of endothelial colony forming cells delivery routes in a murine model of critical limb threatening ischemia using an optimized cell tracking approach
Abstract 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 enh...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2022-06-01
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| Series: | Stem Cell Research & Therapy |
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| Online Access: | https://doi.org/10.1186/s13287-022-02943-8 |
| _version_ | 1828316258860269568 |
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| author | Marta Rojas-Torres Ismael Sánchez-Gomar Antonio Rosal-Vela Lucía Beltrán-Camacho Sara Eslava-Alcón José Ángel Alonso-Piñeiro Javier Martín-Ramírez Rafael Moreno-Luna Mª Carmen Durán-Ruiz |
| author_facet | Marta Rojas-Torres Ismael Sánchez-Gomar Antonio Rosal-Vela Lucía Beltrán-Camacho Sara Eslava-Alcón José Ángel Alonso-Piñeiro Javier Martín-Ramírez Rafael Moreno-Luna Mª Carmen Durán-Ruiz |
| author_sort | Marta Rojas-Torres |
| collection | DOAJ |
| description | Abstract 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 µM 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. |
| first_indexed | 2024-04-13T17:11:51Z |
| format | Article |
| id | doaj.art-5fa642859b24408dbf14f7e69bf6bd01 |
| institution | Directory Open Access Journal |
| issn | 1757-6512 |
| language | English |
| last_indexed | 2024-04-13T17:11:51Z |
| publishDate | 2022-06-01 |
| publisher | BMC |
| record_format | Article |
| series | Stem Cell Research & Therapy |
| spelling | doaj.art-5fa642859b24408dbf14f7e69bf6bd012022-12-22T02:38:15ZengBMCStem Cell Research & Therapy1757-65122022-06-0113111410.1186/s13287-022-02943-8Assessment of endothelial colony forming cells delivery routes in a murine model of critical limb threatening ischemia using an optimized cell tracking approachMarta Rojas-Torres0Ismael Sánchez-Gomar1Antonio Rosal-Vela2Lucía Beltrán-Camacho3Sara Eslava-Alcón4José Ángel Alonso-Piñeiro5Javier Martín-Ramírez6Rafael Moreno-Luna7Mª Carmen Durán-Ruiz8Biomedicine, Biotechnology and Public Health Department, Cádiz UniversityBiomedicine, Biotechnology and Public Health Department, Cádiz UniversityBiomedicine, Biotechnology and Public Health Department, Cádiz UniversityBiomedicine, Biotechnology and Public Health Department, Cádiz UniversityBiomedicine, Biotechnology and Public Health Department, Cádiz UniversityBiomedicine, Biotechnology and Public Health Department, Cádiz UniversityPreclinical R&D Department at Ixaka Iberia SLLaboratory of Neuroinflammation, Hospital Nacional de Paraplejicos, SESCAMBiomedicine, Biotechnology and Public Health Department, Cádiz UniversityAbstract 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 µM 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.https://doi.org/10.1186/s13287-022-02943-8BiodistributionCLTICell therapyECFCsDiR labeling |
| spellingShingle | Marta Rojas-Torres Ismael Sánchez-Gomar Antonio Rosal-Vela Lucía Beltrán-Camacho Sara Eslava-Alcón José Ángel Alonso-Piñeiro Javier Martín-Ramírez Rafael Moreno-Luna Mª Carmen Durán-Ruiz Assessment of endothelial colony forming cells delivery routes in a murine model of critical limb threatening ischemia using an optimized cell tracking approach Stem Cell Research & Therapy Biodistribution CLTI Cell therapy ECFCs DiR labeling |
| title | Assessment of endothelial colony forming cells delivery routes in a murine model of critical limb threatening ischemia using an optimized cell tracking approach |
| title_full | Assessment of endothelial colony forming cells delivery routes in a murine model of critical limb threatening ischemia using an optimized cell tracking approach |
| title_fullStr | Assessment of endothelial colony forming cells delivery routes in a murine model of critical limb threatening ischemia using an optimized cell tracking approach |
| title_full_unstemmed | Assessment of endothelial colony forming cells delivery routes in a murine model of critical limb threatening ischemia using an optimized cell tracking approach |
| title_short | Assessment of endothelial colony forming cells delivery routes in a murine model of critical limb threatening ischemia using an optimized cell tracking approach |
| title_sort | assessment of endothelial colony forming cells delivery routes in a murine model of critical limb threatening ischemia using an optimized cell tracking approach |
| topic | Biodistribution CLTI Cell therapy ECFCs DiR labeling |
| url | https://doi.org/10.1186/s13287-022-02943-8 |
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