Reactive Oxygen Species Impair the Function of CD90+ Hematopoietic Progenitors Generated from Human Pluripotent Stem Cells

Abstract

Cell stressors, such as elevated levels of reactive oxygen species (ROS), adversely affect hematopoietic stem cell (HSC) reconstituting ability. However, the effects of ROS have not been evaluated in the context of hematopoietic development from human pluripotent stem cells (hPSCs). Using our previously described in vitro system for efficient derivation of hematopoietic cells from hPSCs, we show that the vast majority of generated hematopoietic cells display supraphysiological levels of ROS compared to fresh cord blood cells. Elevated ROS resulted in DNA damage of the CD34 1 hematopoietic fraction and, following functional assays, reduced colony formation and impaired proliferative capacity. Interestingly, all the proliferative potential of the most primitive hematopoietic cells was limited to a small fraction with low ROS levels. We show that elevation of ROS in hPSC-derived hematopoietic cells is contributed by multiple distinct cellular processes. Furthermore, by targeting these molecular processes with 4 unique factors, we could reduce ROS levels significantly, yielding a 22-fold increase in the most primitive CD90 1 CD34 1 hematopoietic cells with robust growth capacity. We demonstrate that the ROS reducing factors specifically reduced ROS in more primitive hematopoietic fractions, in contrast to endothelial cells that maintained low ROS levels in the cultures. We conclude that high levels of ROS in in vitro differentiation systems of hPSCs is a major determinant in the lack of ability to generate hematopoietic cells with similar proliferation/differentiation potential to in vivo hematopoietic progenitors, and suggest that elevated ROS is a significant barrier to generating hPSC-derived repopulating HSCs. STEM CELLS 2017;35:197-206 SIGNIFICANCE STATEMENTCell stressors, such as elevated levels of reactive oxygen species (ROS), adversely affect hematopoietic stem cell reconstituting ability. However, the effects of ROS have not been evaluated in the context of hematopoietic development from human pluripotent stem cells (hPSCs). Our results show for the first time that high levels of ROS, resulting from mechanisms associated with in vitro hPSC differentiation systems, occurs and functionally impairs the most primitive of the newly generated hematopoietic cells, and that ROS regulation is a requirement for generating functional primitive hematopoietic cells in vitro. We provide strong evidence that efforts to further normalize ROS toward physiological levels will be crucial to generate functionally equivalent human hematopoietic stem and progenitor cells in vitro as compared to their in vivo counterparts.

Hyperbaric oxygen affects endothelial progenitor cells proliferation in vitro

“…In physiological conditions, reactive oxygen species (ROS) are subproducts of oxidative phosphorylation inside the mitochondria (Taniyama and Griendling, ; Liu et al, ; Zhou et al, ; Rönn et al, ). However, in inflammatory conditions, activated macrophages secrete metalloproteinases that increases ROS concentration and the intracellular oxidative stress promoted by this situation can blockade eNOS activity (Vaddi et al, ).…”

Oxidative stress response induced by chemotherapy in leukemia treatment (Review)

“…Hypoxia has emerged as a key regulator of stem cell biology and maintains HSC quiescence with a condition of metabolic dormancy based on anaerobic glycolysis, which causes low production of ROS and high antioxidant defense ( 47 , 48 ). While hematopoietic cell differentiation is accompanied by changes in oxidative metabolism, including a decrease in anaerobic glycolysis and an increase in oxidative phosphorylation, thus producing high levels of ROS ( 49 – 51 ). Furthermore, evidences have indicated that leukemia stem cells (LSC) are more dependent on oxidative respiration and are more sensitive to OS, compared with normal HSCs ( 16 ).…”

The Genomic Health of Human Pluripotent Stem Cells: Genomic Instability and the Consequences on Nuclear Organization

“…As human pluripotent stem cells are metabolically very active and can be maintained in cultures for long periods of time, the increased age and the fast metabolism required in these cells could also be an aspect that factors in the genomic instability often observed. In contrast, it has been reported that both high and low levels of ROS can impair the reprogramming ability of cells into iPSCs (Zhou et al, 2016) and elevated levels can impair their differentiation ability as well (Rönn et al, 2017). These studies suggest that optimal levels of ROS may be required for the cells to grow stably in culture.…”