NAC Improves the Differentiation of IPS Cells Into Hematopoietic Progenitors

Abstract

Abstract 2349In vitro differentiation of human induced pluripotent stem (iPS) cells is a new way to obtain donor material for blood transplantation. However, this is a long process in the course of which cells are exposed to alien environmental factors, capable to change cellular properties and decrease cellular viability. Indeed, cells in vitro are exposed to mechanical stress, artificial growth surface, unnatural gas composition etc. This fact could partially explain why cells obtained during directed iPS cells differentiation have different qualities in comparison with the analogous population from in vivo.It is supposed that oxidative stress is the major underlying mechanism of negative influence of various in vitro environmental factors on cells. N-acetylcysteine (NAC) is a powerful antioxidant. Due to free radical scavenging ability, the principal function of NAC is rendered to the inhibition of cellular damage and cell death in response to reactive oxygen species. Cytoprotective function of NAC is well demonstrated both in vitro and in vivo.We have established a protocol to obtain hematopoietic stem cell-like cells from human iPS cells with a pick of their production at three weeks of differentiation. We analyzed how intracellular accumulation of reactive oxygen species and NO, as well as cell viability, apoptosis, stress resistance, mitochondrial membrane potential were changed during this process by comparing status of cultures at 1 and at 3 weeks of differentiation. We found that during differentiation cells progressively accumulated intracellular reactive oxygen species and increased production of NO. The level of apoptosis in culture was significantly higher at 3 weeks of differentiation than at 1 week. Cell viability, on the contrary, decreased from 1 week till 3 weeks of differentiation. Stress resistance quantified through the amount of cells resistant to the H2O2 treatment was also decreased in 3 weeks old cultures. We also demonstrated that during in vitro culture the mitochondrial membrane potential of the cells under basal conditions and upon stimulation with carbonyl cyanide m-chlorophenylhydrazone was decreased at 3 weeks of differentiation in comparison with that at 1 week. All these phenomena were reversed by NAC supplementation. Remarkably, NAC administration also improved the hematopoietic differentiation of human iPS cells in terms of production of CD34, CD45, CD43 positive cells, that showed normal functionality in colony forming unit assay. CD34+ cells obtained from NAC treated cultures also increased their migration towards SDF1, therefore showing an increased ability of our CD34+ cells to home into bone marrow.Our results suggest that supplementation with NAC is beneficial for the improvement of hematopoietic differentiation of human iPS cells. Disclosures:No relevant conflicts of interest to declare.