Methods for analyzing the density, distribution, origin, and phenotypes of restored cell populations in vivo and post-explant

Key challenges in the development of a tissue engineered heart valve (TEHV) include the creation of appropriate scaffold substrates and the selection of phenotypically appropriate cells for seeding or, conversely, the selection of progenitor cells that will appropriately proliferate and differentiate in vivo. Mesenchymal stem cells (MSCs) have come to the forefront as prime contenders for in vivo seeding due to their multilineage potential. However, because MSCs have the innate ability to differentiate into numerous cell types (e.g. osteoblasts, myocytes, adipocytes), methods must be established to accurately characterize the in vitro and in vivo cellular characteristics (cellular origin, phenotype and distribution; gene expression). Such methods can include gene expression analysis, proteomics, traditional quantitative morphology and morphometry, and cellular imaging. Through real-time quantitative PCR, RNA sequencing and microarray technology, the phenotype of differentiated repopulated cells in explanted tissue can be compared to cultured MSCs. Additionally, such methods are useful in tissues or cells harvested from animal models where a lack of specific antibodies for phenotype markers precludes the widespread use of immunoassays. Similarly, while ELISAs and western blots are useful to study the protein products of target genes, alternative methods exist for use in poorly validated animal models (e.g. mass spectrometry, differential gel electrophoresis). Finally, conventional histology is essential to the evaluation of cusp architecture in the TEHV and when coupled with immunohistochemistry or sophisticated cell tracking methods, the ultimate phenotype of repopulated cells can be evaluated.