Date Published: May 2, 2019
Publisher: Public Library of Science
Author(s): Mona Bensalah, Pierre Klein, Ingo Riederer, Soraya Chaouch, Laura Muraine, Wilson Savino, Gillian Sandra Butler-Browne, Capucine Trollet, Vincent Mouly, Anne Bigot, Elisa Negroni, Atsushi Asakura.
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.
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 recreate in vivo dynamics and provide knowledge about in vivo human cell behaviour, xenotransplantation of human cells in immunodeficient mice has been developed and represents a unique experimental approach. Although xenotransplantation has been widely used in immunology, it also represents a powerful tool to investigate the cell mechanisms involved in both skeletal muscle repair and homeostasis in normal and pathological situations . Post-mitotic and stable during healthy adulthood, skeletal muscle can face rapid and devastating changes following trauma or in dystrophic conditions . At rest, the physiological muscle stem cells, called satellite cells, are mitotically quiescent. After injury or increased load, satellite cells become activated and proliferate thus supplying a large number of new myonuclei ready to fuse to replace damaged myofibers and assure a rapid muscle growth and repair, in addition to generating new satellite cells by self-renewal . Muscle regeneration is an extremely well orchestrated process in which several cell actors (satellite-, immune system- and non-myogenic-cells) [3–5] play distinct roles at different steps of muscle regeneration. Slight modifications in the regeneration kinetics can result in impaired muscle repair. In healthy conditions, muscle regeneration is highly efficient whereas in muscular diseases, regeneration can be delayed or compromised, resulting in progressive muscle wasting and weakness. 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. Indeed, for genetic diseases including muscular dystrophies, cell-based treatment is one of the innovative and promising therapeutic approaches . In addition, 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. Different cell types have been used as possible vectors for cell-based therapy [7–11]. However, the direct comparison of distinct cell types or treatments is often difficult since different techniques are being used in different laboratories to quantify in vivo engraftment. Here 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.
In the present study, we have evaluated two different protocols for the quantification of human cells after transplantation into a muscle of immunodeficient mice. Our results show that both of these methods are equally accurate to quantify human cells within the recipient mouse muscle since results obtained with these two methods are not significantly different (Fig 3E). Each method has its specificity: the qPCR-based method can be used for a quantitative, precise and rapid estimation of human material in human xenografts, but does not give any information about the localization of the engrafted cells and modifications of the architecture of the host tissue. Immunofluorescence, on the other hand, provides spatial information but is more time consuming regarding the quantification of human material. Most importantly, we show that both methods can be combined using the same starting material: a few cryosections can be processed by immunofluorescence to check the localization of human nuclei, and the fate and behaviour of the injected cells while qPCR will provide rapidly quantitative data. We propose the combination of these two methods as a gold standard to compare results obtained with different types of human myogenic stem cells (different precursors, control or pathological, treated or not, etc.) or when different mouse models are used. Such combined methods can be used to assess results obtained in different laboratories, often difficult to compare, in a more objective and rigorous way. Lastly, we believe that the combination of these two approaches can be applied to analyse more generally grafting and cell transplantation experiments, independently of the target tissue and that they may contribute to a variety of transplantation studies aimed at analysing human cells in mouse models.