Basic Study
Copyright ©The Author(s) 2019. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Stem Cells. Feb 26, 2019; 11(2): 100-123
Published online Feb 26, 2019. doi: 10.4252/wjsc.v11.i2.100
Triple-modal imaging of stem-cells labeled with multimodal nanoparticles, applied in a stroke model
Helio Rodrigues da Silva, Javier Bustamante Mamani, Mariana Penteado Nucci, Leopoldo Penteado Nucci, Andrea Tiemi Kondo, Daianne Maciely Carvalho Fantacini, Lucas Eduardo Botelho de Souza, Virginia Picanço-Castro, Dimas Tadeu Covas, José Mauro Kutner, Fernando Anselmo de Oliveira, Nelson Hamerschlak, Lionel Fernel Gamarra
Helio Rodrigues da Silva, Javier Bustamante Mamani, Andrea Tiemi Kondo, José Mauro Kutner, Fernando Anselmo de Oliveira, Nelson Hamerschlak, Lionel Fernel Gamarra, Hospital Israelita Albert Einstein, São Paulo 05651-900, Brazil
Mariana Penteado Nucci, LIM44, Hospital das Clinicas HCFMUSP, Faculdade de Medicina, Universidade de São Paulo, São Paulo 05403-010, Brazil
Leopoldo Penteado Nucci, Centro Universitário do Planalto Central, Brasilia 72445-020, Brazil
Daianne Maciely Carvalho Fantacini, Lucas Eduardo Botelho de Souza, Virginia Picanço-Castro, Dimas Tadeu Covas, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, São Paulo 05403-010, Brazil
Author contributions: da Silva HR and Gamarra LF conceptualized the original idea; da Silva HR, Mamani JB, Nucci MP and Gamarra LF designed and performed the experiments and analyzed the data; Nucci LP and de Oliveira FA performed analysis of the data and animal management; Fantacini DMC, de Souza LEB, Picanço-Castro V and Covas DT performed the cellular culture for transduction of luciferase and the writing of the original draft; Kondo AT, Kutner JM, Nucci LP and Hamerschlak N performed the stem cell isolation and immunophenotype characterization; Mamani JB, Nucci MP, Nucci LP and Gamarra LF did the writing - original draft and writing - review and editing; Gamarra LF performed funding acquisition, investigation, project administration, resources and supervision.
Supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico, No. CNPq-465259/2014-6, and No. CNPq-400856/2016-6; São Paulo State Research Support Foundation, No. 2014/50983-3, and No. 2016/21470-3.
Institutional review board statement: The study was approved by the Ethics Committee for Research at the Instituto Israelita de Ensino e Pesquisa Albert Eisntein, Brazil (CAAE - 27665714.4.0000.0071) for the use of human mesenchymal stem cells after the donors informed consent.
Institutional animal care and use committee statement: The study was approved by the Ethics in Animal Research Committee of the Hospital Israelita Albert Einstein (HIAE) with approval number 1906-13. The vivarium of the Experimental Surgical Training Center (Centro de Experimentação e Treinamento em Cirurgia - CETEC) was accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International (AAALAC International).
Conflict-of-interest statement: The authors have declared that no competing interests exist.
Data sharing statement: The datasets supporting the conclusions of this article are included within in the article.
ARRIVE guidelines statement: The authors have read the ARRIVE guidelines - Animal Research: Reporting In vivo Experiments, and the manuscript was prepared and revised according to the ARRIVE guidelines.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See:
Corresponding author: Lionel Fernel Gamarra, PhD, Senior Scientist, Group of nanobiotechnology of The Albert Einstein Hospital, Hospital Israelita Albert Einstein, Avenida Albert Einstein, 627/701 - Morumbi, São Paulo 05651-900, Brazil.
Telephone: +55-11-21510243
Received: October 22, 2018
Peer-review started: October 23, 2018
First decision: November 14, 2018
Revised: December 5, 2018
Accepted: December 17, 2018
Article in press: December 17, 2018
Published online: February 26, 2019
Research background

Mesenchymal stem cells (MSCs) have been widely tested for therapeutic efficacy in the ischemic brain, providing several benefits. A major obstacle for the clinical translation of these therapies has been the inability to noninvasively monitor the best route, cell doses, and collateral effects, while ensuring survival and effective biological functioning of the transplanted stem cells. Combined image modalities have improved the accuracy of cellular therapy, allowed the in vivo monitoring of the biodistribution and viability of transplanted stem cells due to associated new tracers such as multimodal nanoparticles with new labels and covers, resulting in low toxicity and longtime residence in cells.

Research motivation

Although meta-analyses have examined the benefits of cell transplantation in experimental stroke, low viable cell retention after transplantation in ischemic brain areas still represents the major obstacle to the successful clinical translation of cell-based stroke repair approaches. The multimodal imaging techniques provide morpho-functional information for studying pathological events following ischemia, associated with new tracers. These innovations will contribute to further our comprehension of stem cell transplantation, allowing the assessment of therapeutic effects on a molecular scale.

Research objectives

In this study, we aim to evaluate the sensitivity of triple-modal imaging of stem cells labeled with multimodal nanoparticles with different iron concentrations and their cellular viability in vitro as well as the correlation of the quantification of the iron load between different techniques (ICP-MS, MRI and NIRF. In addition, we seek to verify whether these images of stem cells labeled with multimodal nanoparticles maintain the same properties after application in a stroke model. The results will help us to better understand the biodistribution, sensitivity and viability of stem cells labeled with nanoparticles in sham and stroke models.

Research methods

Isolated and immunophenotypically characterized hBM-MSCs were transduced with a lentivirus for BLI evaluation in vitro and in vivo. In addition, MNP-IR750 that had previously been characterized (hydrodynamic size, zeta potential, and optical properties) were used for labeling these cells and analyzing cell viability via MTT and BLI assays. The internalization process and quantification of the iron load in different concentrations of MNP-IR750 were analyzed via MRI, NIRF, and ICP-MS. In the in vivo study, the same labeled cells were implanted in the sham group and stroke group at different times and MNP-IR750 concentrations (after 4 h and after 6 d of cell implantation) to evaluate the sensitivity of triple-modal images.

Research results

The collection and isolation of hBM-MSCs after immunophenotypic characterization was demonstrated to be adequate for human bone marrow samples. After the transduction of these cells with luciferase, we detected a maximum BLI intensity of 2.0 × 108 photons/s in 106 hBM-MSC samples. Evaluation of the physicochemical characteristics of MNP-IR750 showed an average hydrodynamic diameter of 38.2 ± 0.5 nm, a zeta potential of +29.2 ± 1.9 mV and adequate colloidal stability, without agglomeration over 18 h. The iron load internalization process in hBM-MSCs showed a strong relationship of the signal with the corresponding MNP labeling concentration based on MRI, ICP-MS and NIRF. Cellular viability in the highest MNP-IR750 concentration showed a reduction of less than 10% compared to the control. The correlation analysis of the MNP-IR750 load internalized by hBM-MSCs between the MRI, ICP-MS and NIRF techniques showed the same correlation coefficient of 0.99. The evaluation of BLI, NIRF, and MRI signals in vivo and ex vivo after labeled hBM-MSCs were implanted in the animals showed sensitivity in the detection of MNP-IR750 concentrations of 5, 20 and 50 µg Fe/mL at 4 h and 6 d in the sham groups, with significant results regarding the time and image effect as well as the group effect when the sham and stroke groups were compared at 6 d.

Research conclusions

Our study demonstrates that MNP-IR750 can be used to monitor cell grafts noninvasively, longitudinally, and repetitively, enabling the assessment of cell graft conditions in vivo after stroke for multimodal imaging assessment. The BLI signal of hBM-MSCLuc showed the best imaging technique for functional and longitudinal assessment.

Research perspectives

In summary, we highlight the importance of quantification of multimodal nanoparticles internalized by cells and the efficacy of signal detection under the triple-image modality in a stroke model. However, the applicability of multimodal imaging should always be analyzed in accordance with the limitations and advantages of each technique involved.