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Immunohistochemical toolkit for tracking and quantifying xenotransplanted human stem cells.

ABSTRACT: Biomarker-based tracking of human stem cells xenotransplanted into animal models is crucial for studying their fate in the field of cell therapy or tumor xenografting.Using immunohistochemistry and in situ hybridization, we analyzed the expression of three human-specific biomarkers: Ku80, human mitochondria (hMito) and Alu.We showed that Ku80, hMito and Alu biomarkers are broadly expressed in human tissues with no or low cross-reactivity toward rat, mouse or pig tissues. In vitro, we demonstrated that their expression is stable over time and does not change along the differentiation of human-derived induced pluripotent stem cells or human glial-restricted precursors. We tracked in vivo these cell populations after transplantation in rodent spinal cords using aforementioned biomarkers and human-specific antibodies detecting apoptotic, proliferating or neural-committed cells.This study assesses the human-species specificity of Ku80, hMito and Alu, and proposes useful biomarkers for characterizing human stem cells in xenotransplantation paradigms.

SUBMITTER: Allard J

PROVIDER: S-EPMC4161450 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Immunohistochemical toolkit for tracking and quantifying xenotransplanted human stem cells.

Allard Justine J Li Ké K Lopez Xavier Moles XM Blanchard Stéphane S Barbot Paul P Rorive Sandrine S Decaestecker Christine C Pochet Roland R Bohl Delphine D Lepore Angelo C AC Salmon Isabelle I Nicaise Charles C

Regenerative medicine 20140101 4

<h4>Aim</h4>Biomarker-based tracking of human stem cells xenotransplanted into animal models is crucial for studying their fate in the field of cell therapy or tumor xenografting.<h4>Materials & methods</h4>Using immunohistochemistry and in situ hybridization, we analyzed the expression of three human-specific biomarkers: Ku80, human mitochondria (hMito) and Alu.<h4>Results</h4>We showed that Ku80, hMito and Alu biomarkers are broadly expressed in human tissues with no or low cross-reactivity to ...[more]

PMID: 25159062

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Project description:UnlabelledEvidence suggests that transplanted mesenchymal stem cells (MSCs) can aid recovery of damaged myocardium caused by myocardial infarction. One possible mechanism for MSC-mediated recovery is reprogramming after cell fusion between transplanted MSCs and recipient cardiac cells. We used a Cre/LoxP-based luciferase reporter system coupled to biophotonic imaging to detect fusion of transplanted human pluripotent stem cell-derived MSCs to cells of organs of living mice. Human MSCs, with transient expression of a viral fusogen, were delivered to the murine heart via a collagen patch. At 2 days and 1 week later, living mice were probed for bioluminescence indicative of cell fusion. Cell fusion was detected at the site of delivery (heart) and in distal tissues (i.e., stomach, small intestine, liver). Fusion was confirmed at the cellular scale via fluorescence in situ hybridization for human-specific and mouse-specific centromeres. Human cells in organs distal to the heart were typically located near the vasculature, suggesting MSCs and perhaps MSC fusion products have the ability to migrate via the circulatory system to distal organs and engraft with local cells. The present study reveals previously unknown migratory patterns of delivered human MSCs and associated fusion products in the healthy murine heart. The study also sets the stage for follow-on studies to determine the functional effects of cell fusion in a model of myocardial damage or disease.SignificanceMesenchymal stem cells (MSCs) are transplanted to the heart, cartilage, and other tissues to recover lost function or at least limit overactive immune responses. Analysis of tissues after MSC transplantation shows evidence of fusion between MSCs and the cells of the recipient. To date, the biologic implications of cell fusion remain unclear. A newly developed in vivo tracking system was used to identify MSC fusion products in living mice. The migratory patterns of fusion products were determined both in the target organ (i.e., the heart) and in distal organs. This study shows, for the first time, evidence of fusion products at sites distal from the target organ and data to suggest that migration occurs via the vasculature. These results will inform and improve future, MSC-based therapeutics.

Project description:BackgroundMagnetic resonance imaging (MRI) is a promising tool for monitoring stem cell-based therapy. Conventionally, cells loaded with ironoxide nanoparticles appear hypointense on MR images. However, the contrast generated by ironoxide labeled cells is neither specific due to ambiguous background nor quantitative. A strategy to overcome these drawbacks is (19)F MRI of cells labeled with perfluorocarbons. We show here for the first time that human neural stem cells (NSCs), a promising candidate for clinical translation of stem cell-based therapy of the brain, can be labeled with (19)F as well as detected and quantified in vitro and after brain implantation.Methodology/principal findingsHuman NSCs were labeled with perfluoropolyether (PFPE). Labeling efficacy was assessed with (19)F MR spectroscopy, influence of the label on cell phenotypes studied by immunocytochemistry. For in vitro MRI, NSCs were suspended in gelatin at varying densities. For in vivo experiments, labeled NSCs were implanted into the striatum of mice. A decrease of cell viability was observed directly after incubation with PFPE, which re-normalized after 7 days in culture of the replated cells. No label-related changes in the numbers of Ki67, nestin, GFAP, or ?III-tubulin+ cells were detected, both in vitro and on histological sections. We found that 1,000 NSCs were needed to accumulate in one image voxel to generate significant signal-to-noise ratio in vitro. A detection limit of ?10,000 cells was found in vivo. The location and density of human cells (hunu+) on histological sections correlated well with observations in the (19)F MR images.Conclusion/significanceOur results show that NSCs can be efficiently labeled with (19)F with little effects on viability or proliferation and differentiation capacity. We show for the first time that (19)F MRI can be utilized for tracking human NSCs in brain implantation studies, which ultimately aim for restoring loss of function after acute and neurodegenerative disorders.

Dataset Information

Immunohistochemical toolkit for tracking and quantifying xenotransplanted human stem cells.

Publications

Immunohistochemical toolkit for tracking and quantifying xenotransplanted human stem cells.

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