Optimizing Techniques for Tracking Transplanted Stem Cells In Vivo

Abstract

The potential for bone marrow-derived cells (BMDCs) to contribute to nonhematopoietic tissues has generated considerable debate in recent years. Causes for the controversies include disparities in the techniques used to track engraftment of BMDCs, inappropriate tissue preparation, a lack of appropriate positive and negative controls, and basic misunderstandings about how to properly collect and interpret images from epifluorescent and confocal microscopes. Our laboratory was among the first to use bone marrow transplants from transgenic mice constitutively expressing enhanced green fluorescent protein (GFP) to study the ability of BMDCs to give rise to nonhematopoietic tissue types, a system that is now in widespread use. During our 6 years of experience using GFP, as well as beta-galactosidase and the Y chromosome, to track BMDCs in vivo, we have identified many difficulties and have developed techniques to resolve them. We discuss several of these methods, and, in particular, we describe ratiometric analysis techniques for improving detection of transplanted cells derived from genetically modified bone marrow. Finally, to help resolve reported discrepancies regarding the frequency with which BMDCs contribute to skeletal myofibers, we demonstrate that the pattern of highly autofluorescent myofibers in skeletal muscle is clearly distinct from that of GFP-expressing myofibers and describe how unambiguous conclusions can be drawn from such data. Stem Cells

Sensitive detection of GFP utilizing tyramide signal amplification to overcome gene silencing

“…Since the expression of GFP varies among cells it is likely that using traditional detection methods we miss many GFP+ cells. The methods and problems of GFP detection have been nicely summarized in a recent review [21]. The fact that GFP turns off may account for many publications in the literature that state that no donor cells were detected when using GFP as a reporter gene to label bone marrow [39][40][41].…”

“…Since we also noticed very faintly fluorescence green cells in our experimental samples we decided to try the most sensitive technique to visualize most of the GFP expressing cells. The field has been plagued by controversy mostly due to differences in techniques used by the different groups to follow cell fate as summarized in [21]. In the last decade a new, very sensitive technique became available utilizing tyramide signal amplification [22,23] and its application to immunohistochemistry was reported [1] describing dilutions of primary antibodies for optimal immunohistochemistry [24] as well as its use in dual immunostaining techniques [25].…”

Assessment of cystic fibrosis transmembrane conductance regulator (CFTR) activity in CFTR-null mice after bone marrow transplantation

“…Although several papers strongly support the notion that after BM transplantation epithelial cells can develop from BMDC in mice as well as in humans, there is much controversy regarding the percentage of marrow-derived epithelial cells, the subpopulation of BM cells that is responsible, the mechanism(s) underlying BMDC plasticity, and whether or not BM-derived epithelial cells perform the normal functions of epithelial cells (5).…”

The role of stem cells in the pathogenesis of endometriosis (а review)

“…Стволовые клетки костного мозга способны циркулировать в крови и даже могут быть источником происхожде-ния зрелых, не гемопоэтических клеток различных тканей млекопитающих, в том числе легких, печени, желудочно-кишечного тракта, кожи, ЦНС и эндоте-лия сосудов [36,37].…”