Reliability of ROS and RNS detection in hematopoietic stem cells − potential issues with probes and target cell population

Abstract

Many studies have provided evidence for the crucial role of the reactive oxygen species (ROS) and reactive nitrogen species (RNS) in the regulation of differentiation and/or self-renewal, and the balance between quiescence and proliferation of hematopoietic stem cells (HSCs). Several metabolic regulators have been implicated in the maintenance of HSC redox homeostasis; however, the mechanisms that are regulated by ROS and RNS, as well as their downstream signaling are still elusive. This is partially owing to a lack of suitable methods that allow unequivocal and specific detection of ROS and RNS. In this Opinion, we first discuss the limitations of the commonly used techniques for detection of ROS and RNS, and the problem of heterogeneity of the cell population used in redox studies, which, together, can result in inaccurate conclusions regarding the redox biology of HSCs. We then propose approaches that are based on single-cell analysis followed by a functional test to examine ROS and RNS levels specifically in HSCs, as well as methods that might be used in vivo to overcome these drawbacks, and provide a better understanding of ROS and RNS function in stem cells.

α-Tocopherol Acetate Attenuates Mitochondrial Oxygen Consumption and Maintains Primitive Cells within Mesenchymal Stromal Cell Population

“…The possible response may be in a mtROS-driven mechanism, which has been suggested as a critical regulator of stem cell fate. These reports assume the physiological mtROS concentration in the micro-environmental low O 2 as being crucial for the maintenance of stemness while the enhancement of mtROS over this niche-relevant level leads to the commitment and differentiation [52][53][54][55]. In fact, this physiological mtROS level corresponds to the "steady state" in situ mtROS levels as ones found at physiologically relevant O 2 concentrations [22,35,38].…”

ROS-induced ROS release orchestrated by Nox4, Nox2, and mitochondria in VEGF signaling and angiogenesis

“…Separate previous studies indicate that NADPH oxidases Nox4 or Nox2 or that mitochondria is the major source of ROS that serve as signaling molecules to promote switching from a quiescent to an angiogenic phenotype in ECs (1,11,13,18,44,50,56,58,62 this question, we applied several methods to measure intracellular redox status due to limitation of each ROS measurement (15,24,30,59 to promote mtROS production via pSer36-p66Shc, thereby enhancing VEGFR2 activation and angiogenic responses. This may represent a novel positive feed-forward ROS-induced ROS release mechanism orchestrated by the Nox4/Nox2/pS36-p66Shc/mtROS axis, which promotes sustained activation of VEGFR2 angiogenesis signaling program (Fig.…”

Oxidative stress regulates progenitor behavior and cortical neurogenesis

“…To further measure the ROS level in live RGPs during mouse cortical development, we collected the VZ tissues at E11.5, E13.5, E15.5 and E17.5, and prepared neurospheres in culture. We stained the neurospheres with CellROX Green, a cell-permeant dye that measures ROS levels and oxidative stress in live cells (Vlaski-Lafarge and Ivanovic, 2015), and found that ROS levels and oxidative stress progressively increased in the neurospheres prepared at different developmental stages (Fig. S1).…”