Iron Overload Impairs Hematopoiesis by Damaging MSCs Through ROS Signaling Pathway.
Authors: Lu Wenyi , Zhao Mingfeng , Chai Xiao , Meng Juanxia , Xie Fang , Mu Juan , Zhu Haibo , Xu Xinnv , Xiao Xia , Deng Qi , Ma Li 
Published: October 2019

Abstract
Abstract 2097
Iron is a useful component of cytochromes, oxygen-binding molecules and some enzymes due to its capacity to accept and donate electrons readily. However, excessive iron accumulation can damage tissues and cells by catalyzing the conversion of superoxide and hydrogen peroxide to free radical species that can attack cellular membranes, proteins and DNA. Recent multiple data revealed that iron chelation therapy was effective in treating cytopenia in iron overload disease, which supported the idea that iron overload affected hematopoiesis in bone marrow(BM). Based on these findings, We demonstrated that iron overload suppressed hematopoiesis by inhibiting hematopoietic stem/progenitor cells and the effects could be restored by iron chelation or anti-oxidants(Zhao et al., Blood, 2010, 116:4247a). However, it is unclear whether iron overload can impair BM hematopoiesis by injuring the microenvironment. As an important component of the BM microenvironment, Mesenchymal stem cells (MSCs) secrete a large amount of cytokines and extracellular matrix protein which provides a favorable platform for the localization, self-renewal, and differentiation of hematopoietic stem cells. Here we hypothesize that iron overload impairs BM microenvironment by affecting the function and survival of MSCs which is mediated by ROS.
In this study we first established an iron overload model of MSCs by adding ferric ammonium citrae (FAC) to the culture medium. To confirm this model, the labile iron pool (LIP) level of MSCs was detected using the calcein-AM method. We found that the LIP of MSCs was significantly higher than control and reached the highest level when cultured at 400μmol/L FAC for 12h. Next we analyzed whether iron overload can affect proliferation, apoptosis and function of MSCs by the following experiments. Firstly, the proliferation of MSCs was evaluated using population doubling time (DT). Under iron overload, the population doubling time (DT) of MSCs was 24.43± 2.72 hours, which was signifcantly longer than control(16.03± 2.31 hours; P=0.015). However, the difference wasn't significant after two passages (P=0.936). Possible explanation could be that the injury to MSCs is reversible following decreased concentration of iron after passaging. Secondly, the apoptosis of MSCs altered by iron overload was measured by staining Annexin V/PI, and we found the apoptosis rate was higher in the iron overload group(12.75±0.32%) than control (3.63±0.80%)(P<0.05). Finally, mono-nuclear cells were purified from umbilical cord blood and co-cultured with MSCs to assess the hematopoiesis-supportive function of MSCs. Iron overload group showed decreased hematopoietic support capacity than control. Taken together, these findings proved iron overload impaired hematopoietic microenvironment by decreasing proliferation, inducing apoptosis and injuring the hematopoietic support capacity of MSCs.
We then explored the possible mechanism that may take part in this process. It has already been reported that iron overload may result in the generation of reactive oxygen species (ROS). Similarly, we found that ROS level of MSCs could be positively correlated with the concentration of FAC and reached its highest level when cultured at 400μmol/L FAC for 12h. Finally, Western blot analysis of whole cell lysates from umbilical derived MSC using antibodies recognizing known ROS-related signaling pathways revealed robust increases in phospho-p38, p53 in response to FAC compared with control, with inhibition of these signaling pathways noted in response to NAC or GSH at suitable dose, suggesting that antioxidant can inhibit ROS-induced signaling pathway in iron overload.
In conclusion, Our finding indicates that iron overload can injure hematopoiesis by enhancing oxidative stress in MSC. Our data further suggests creatively that antioxidant and cytotherapy may be an effective method in curing deficient hematopoiesis in iron overload.

Disclosures:
No relevant conflicts of interest to declare.