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Abstract
Xenotransplantation of human cells into immunodeficient mouse models is a very powerful tool and an essential step for the pre-clinical evaluation of therapeutic cell- and gene- based strategies. Here we describe an optimized protocol combining immunofluorescence and real-time quantitative PCR to both quantify and visualize the fate and localization of human myogenic cells after injection in regenerating muscles of immunodeficient mice. Whereas real-time quantitative PCR-based method provides an accurate quantification of human cells, it does not document their specific localization. The addition of an immunofluorescence approach using human-specific antibodies recognizing engrafted human cells gives information on the localization of the human cells within the host muscle fibres, in the stem cell niche or in the interstitial space. These two combined approaches offer an accurate evaluation of human engraftment including cell number and localization and should provide a gold standard to compare results obtained either using different types of human stem cells or comparing healthy and pathological muscle stem cells between different research laboratories worldwide.
Highlights
Investigating the in vivo behaviour of human cells, whether it concerns fundamental aspects, pathological mechanisms or therapeutic strategies, represents a challenging aspect of cell biology
In order to create a regenerative environment in the host and follow the participation of human satellite cells to tissue repair, we induced regeneration in the Tibialis Anterior (TA) muscle of immunodeficient mice using a freeze-injury protocol [7], one of the most commonly used physical procedures for provoking muscle injury and repair in mice [14]
In order to estimate the number of engrafted human myoblasts present in the TA after injection, we quantified the amount of human DNA present in the mouse muscles, using primers recognizing the human Titin (TTN) gene
Summary
Investigating the in vivo behaviour of human cells, whether it concerns fundamental aspects, pathological mechanisms or therapeutic strategies, represents a challenging aspect of cell biology. To better understand muscle maintenance and repair in healthy and dystrophic contexts, as well as to evaluate the efficacy of cell-based therapeutic strategies, it is essential to study the in vivo behaviour of control, dystrophic-derived, and/or modified human satellite cells. Genetic modifications of human stem cells, whether it is through gene therapy or direct genomic correction, must be tested in an in vivo context as a necessary step towards therapy. In this context, accurate methods are needed to evaluate human cell behaviour and participation to muscle regeneration during xenotransplantation. We describe a combined immunofluorescence and real time quantitative PCR-based approach to quantify the grafting efficacy of human myogenic precursors after intramuscular injection in immunodeficient mouse and to analyse their fate in these regenerating muscles
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