Hypoxia/Hypercapnia‐Induced Adaptation Maintains Functional Capacity of Cord Blood Stem and Progenitor Cells at 4°C

Abstract

We analyzed the effect of exposure to hypoxic/hypercapnic (HH) gas mixture (5% O2 /9% CO2 ) on the maintenance of functional cord blood CD34(+) hematopoietic stem and progenitor cells in severe hypothermia (4°C) employing the physiological and proteomic approaches. Ten-day exposure to HH maintained the Day 0 (D-0) level of hematopoietic stem cells as detected in vivo on the basis of hematopoietic repopulation of immunodeficient mice-short-term scid repopulating cells (SRC). Conversely, in the atmospheric air (20% O2 /0.05% CO2 ), usual condition used for cell storage at 4°C, stem cell activity was significantly decreased. Also, HH doubled the survival of CD34(+) cells and committed progenitors (CFCs) with respect to the atmospheric air (60% vs. 30%, respectively). Improved cell maintenance in HH was associated with higher proportion of aldehyde dehydrogenase (ALDH) positive cells. Cell-protective effects are associated with an improved maintenance of the plasma and mitochondrial membrane potential and with a conversion to the glycolytic energetic state. We also showed that HH decreased apoptosis, despite a sustained ROS production and a drop of ATP amount per viable cell. The proteomic study revealed that the global protein content was better preserved in HH. This analysis identified: (i) proteins sensitive or insensitive to hypothermia irrespective of the gas phase, and (ii) proteins related to the HH cell-protective effect. Among them are some protein families known to be implicated in the prolonged survival of hibernating animals in hypothermia. These findings suggest a way to optimize short-term cell conservation without freezing.

Chronic myeloid leukemia progenitor cells require autophagy when leaving hypoxia-induced quiescence

“…Indeed, the concentrations of oxygen in the organism are very different from the atmospheric concentration and varies according to the tissues [ 8 ]. The hematopoietic niche is characterized by low oxygen concentrations ranging from 4 to 0.1% [ 9 – 11 ]. TKI used in CML patient treatment are not able to eliminate these CSL and treatment abortion is followed by 60% of relapse [ 12 – 14 ].…”

Normal Hematopoetic Stem and Progenitor Cells Can Exhibit Metabolic Flexibility Similar to Cancer Cells

“…CD34 + or CD34 + CD38 low CD133 + CD90 + CD45RA − cells were plated in Stem-alpha A medium without glucose (Stem Alpha SA, Saint-Genis-l'Argentiere, France), supplemented with penicillin/streptomycin (PS) (100 ng/L), and cytokines: SCF 100 ng/mL, IL-3 0.5 ng/mL, TPO 10 ng/mL. Cells were incubated under physiological conditions (3% O 2 , with glucose 1 g/L), anoxia (0% O 2 , with glucose 1 g/L), or anoxia/aglycemia (AA, 0% O 2 , without glucose) for 5-7 days at 37 • C. The conditions with 3% O 2 were obtained in an O 2 and CO 2 controller-culture chamber (PRO-OX and PRO-CO 2 , Biospherix, NY) (15). Anoxia was achieved using a hermetically sealed modular incubator chamber (Billups-Rothenberg, CA) in which ambient air was replaced with a mixture of 95% nitrogen and 5% CO 2 (Air Liquide, Paris, France).…”

“…As a negative control, an aliquot of ALDF-stained cells was incubated with the ALDH inhibitor diethylaminobenzaldehyde. Cells were washed, resuspended in 200 µL of assay buffer, and analyzed with a flow cytometer (BD Bioscience, FACS Canto II) (15).…”

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

“…The peroxidase substrate Amplex Red is used to detect H 2 O 2 that has been extracellularly generated, as well as any that is produced inside the cell and has diffused out (Hole et al, 2010;Skinner et al, 2008;Thompson et al, 2008;Vlaski et al, 2014). Amplex Red can be oxidized to the fluorescent product resorufin by H 2 O 2 or other electron oxidants (e.g.…”