Person:

Mortensen, Luke J.

Characterizing how the microenvironment, or niche, regulates stem cell activity is central to understanding stem cell biology and to developing strategies for therapeutic manipulation of stem cells1. Low oxygen tension (hypoxia) is commonly thought to be a shared niche characteristic in maintaining quiescence in multiple stem cell types2–4. However, support for the existence of a hypoxic niche has largely come from indirect evidence such as proteomic analysis5, expression of HIF-1 and related genes6, and staining with surrogate hypoxic markers (e.g. pimonidazole)6–8. Here we perform direct in vivo measurements of local oxygen tension (pO2) in the bone marrow (BM) of live mice. Using two-photon phosphorescence lifetime microscopy (2PLM), we determined the absolute pO2 of the BM to be quite low (<32 mmHg) despite very high vascular density. We further uncovered heterogeneities in local pO2, with the lowest pO2 (~9.9 mmHg, or 1.3%) found in deeper peri-sinusoidal regions. The endosteal region, by contrast, is less hypoxic as it is perfused with small arteries that are often positive for the marker nestin. These pO2 values change dramatically after radiation and chemotherapy, pointing to the role of stress in altering the stem cell metabolic microenvironment.

Abstract. Detection and enumeration of rare circulating cells in mice are important problems in many areas of preclinical biomedical research. Recently, we developed a new method termed “diffuse fluorescence flow cytometry” (DFFC) that uses diffuse photons to increase the blood sampling volume and sensitivity versus existing in vivo flow cytometry methods. In this work, we describe a new DFFC prototype with approximately an order-of-magnitude improvement in sensitivity compared to our previous work. This sensitivity improvement is enabled by a number of technical innovations, which include a method for the removal of motion artifacts (allowing interrogation of mouse hindlegs that was less optically attenuating versus the tail) and improved collection optics and signal preamplification. We validated our system first in limb mimicking optical flow phantoms with fluorescent microspheres and then in nude mice with fluorescently labeled mesenchymal stem cells at injected concentrations of 5×103 cells/mL. In combination, these improvements resulted in an overall cell counting sensitivity of about 1 cell/mL or better in vivo.