Optimizing techniques for tracking transplanted stem cells in vivo

¹ The University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania
² Stanford University School of Medicine, Stanford, California

^* To whom correspondence should be addressed. E-mail: hblau{at}stanford.edu.

	Abstract

The potential for bone marrow-derived cells (BMDC) to contribute to non-hematopoietic tissues has generated considerable debate in recent years. Causes for the controversies include disparities in the techniques used to track engraftment of BMDC, 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 BMDC to give rise to non-hematopoietic tissue types, a system which is now in widespread use. During our six years of experience using GFP, as well as beta-galactosidase (B-gal) and the Y chromosome, to track BMDC 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 BMDC 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.