High post-transplantation cell mortality is the main limitation of various approaches that are aimed at improving regeneration of injured neural tissue by an injection of neural stem cells (NSCs) and mesenchymal stromal cells (MStroCs) in and/or around the lesion. Therefore, it is of paramount importance to identify efficient ways to increase cell transplant viability. We have previously proposed the "evolutionary stem cell paradigm," which explains the association between stem cell anaerobic/microaerophilic metabolic set-up and stem cell self-renewal and inhibition of differentiation. Applying these principles, we have identified the main critical point in the collection and preparation of these cells for experimental therapy: exposure of the cells to atmospheric O, that is, to oxygen concentrations that are several times higher than the physiologically relevant ones. In this way, the primitive anaerobic cells become either inactivated or adapted, through commitment and differentiation, to highly aerobic conditions (20%-21% O in atmospheric air). This inadvertently compromises the cells' survival once they are transplanted into normal tissue, especially in the hypoxic/anoxic/ischemic environment, which is typical of central nervous system (CNS) lesions. In addition to the findings suggesting that stem cells can shift to glycolysis and can proliferate in anoxia, recent studies also propose that stem cells may be able to proliferate in completely anaerobic or ischemic conditions by relying on anaerobic mitochondrial respiration. In this systematic review, we propose strategies to enhance the survival of NSCs and MStroCs that are implanted in hypoxic/ischemic neural tissue by harnessing their anaerobic nature and maintaining as well as enhancing their anaerobic properties via appropriate ex vivo conditioning.

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.

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The feasibility of ex vivo expansion allows us to consider the steady-state peripheral blood as an alternative source of hematopoietic stem progenitor cells for transplantation when growth factor-induced cell mobilization is contraindicated or inapplicable. Ex vivo expansion dramatically enhances the in vivo reconstituting cell population from steady-state blood. In order to investigate phenotype and the expression of homing molecules, the expression of CD34, CD133, CD90, CD45RA, CD26 and CD9 was determined on sorted CD34+ cells according to CXCR4 (“neg”, “low” “bright”) and CD133 expression before and after ex vivo expansion. Hematopoietic stem cell activity was determined in vivo on the basis of hematopoietic repopulation of primary and secondary recipients - NSG immuno-deficient mice. In vivo reconstituting cells in the steady-state blood CD34+ cell fraction before expansion belong to the CD133+ population and are CXCR4low or, to a lesser extent, CXCR4neg, while after ex vivo expansion they are contained only in the CD133+CXCR4low cells. The failure of the CXCR4bright population to engraft is probably due to the exclusive expression of CD26 by these cells. The limiting-dilution analysis showed that both repopulating cell number and individual proliferative capacity were enhanced by ex vivo expansion. Thus, steady-state peripheral blood cells exhibit a different phenotype compared to mobilized and cord blood cells, as well as to those issued from the bone marrow. These data represent the first phenotypic characterization of steady-state blood cells exhibiting short- and long-term hematopoietic reconstituting potential, which can be expanded ex vivo, a sine qua non for their subsequent use for transplantation.