Techniques to study chimerism at the tissue level in humanized mice

Understanding the origin, distribution, and biology of different cell populations in chimeric mice is critical for interpreting the pathological changes developed in these models. To this aim, the methodological work presented here illustrates the validation and application of a collection of labeling techniques to differentiate between specific mouse and human tissue/cell components in formalin-fixed paraffin-embedded samples from chimeric mice, especially those bearing human tumor and immune cells. First, broad approaches to identify cells of human origin using ubiquitous immunohistochemical targets such as HLA-A, Ku80, and human mitochondrial 60 kDa protein (hMito) were established using specimens from humanized mice and a human tissue microarray including both normal and neoplastic samples. Due to its crisp membranous immunoreactivity, HLA-A was the most useful marker for visual human cell identification; however, Ku80 and hMito may be suitable options when HLA-A is not expressed in the cells of interest. Importantly, using one or more of these markers provides a broad range of coverage for the vast majority of human-derived cells in chimeric mice. Second, tailored immunohistochemical or <i>in situ</i> hybridization methodologies to distinguish specific human or mouse cell subsets are presented, focusing on immune/inflammatory cells and human chimeric antigen receptor (CAR) T-cells. These diverse approaches are accompanied by descriptions of case examples highlighting practical diagnostic and experimental applications in the context of various humanized mouse models. While not comprehensive, this work represents a valuable starting reference for pathologists and investigators working with humanized mouse models and seeking to add spatial resolution to the complex landscape of chimeric tissues.