Plasma-activated medium selectively eliminates undifferentiated human induced pluripotent stem cells

Abstract

Human pluripotent stem cells, including human induced pluripotent stem cells (hiPSCs), are promising materials for regenerative medicine and cell transplantation therapy. However, tumorigenic potential of residual undifferentiated stem cells hampers their use in these therapies. Therefore, it is important to develop methods that selectively eliminate undifferentiated stem cells from a population of differentiated cells before their transplantation. In the present study, we investigated whether plasma-activated medium (PAM) selectively eliminated undifferentiated hiPSCs by inducing external oxidative stress. PAM was prepared by irradiating cell culture medium with non-thermal atmospheric pressure plasma. We observed that PAM selectively and efficiently killed undifferentiated hiPSCs cocultured with normal human dermal fibroblasts (NHDFs), which were used as differentiated cells. We also observed that undifferentiated hiPSCs were more sensitive to PAM than hiPSC-derived differentiated cells. Gene expression analysis suggested that lower expression of oxidative stress-related genes, including those encoding enzymes involved in hydrogen peroxide (H 2 O 2 ) degradation, in undifferentiated hiPSCs was one of the mechanisms underlying PAM-induced selective cell death. PAM killed undifferentiated hiPSCs more efficiently than a medium containing the same concentration of H 2 O 2 as that in PAM, suggesting that H 2 O 2 and various reactive oxygen/nitrogen species in PAM selectively eliminated undifferentiated hiPSCs. Thus, our results indicate that PAM has a great potential to eliminate tumorigenic hiPSCs from a population of differentiated cells and that it may be a very useful tool in regenerative medicine and cell transplantation therapy.

State of the art in medical applications using non-thermal atmospheric pressure plasma

“…The scratch treated with 47 mJ nsDBD plasma (right) has greater gap closure and more migrating cells in the gap as compared to the untreated scratch (left) Fig. 64 Effect of PAM on the viability of undifferentiated hiPSCs and RA-induced differentiated hiPSCs (Matsumoto et al 2016). 201B7 hiPSCs were cultured in a medium containing 5 lM RA for 9 days.…”

“…We found that hiPSCs are more sensitive to PAM than are NHDFs [see Fig. 63, (Matsumoto et al 2016)]. We also investigated differences in cell viability between undifferentiated hiPSCs and retinoic acid (RA)-induced differentiated hiPSCs following treatment with PAM and found that undifferentiated cells are more sensitive than are differentiated cells (see Fig.…”

“…For example, Matsumoto et al (2016) investigated oxidative stress-related genes in undifferentiated hiPSCs and differentiated NHDFs using real-time PCR (see Fig. 83).…”

“…The expression levels of GPX1, Fig. 83 Differences in the mRNA expression of oxidative stress-related genes between undifferentiated hiPSCs and differentiated NHDFs (Matsumoto et al 2016). Total RNAs of NHDFs (red bars) and hiPSCs (blue diagonal bars) were extracted from 4-day cultures, and mRNA levels were evaluated by performing real-time PCR.…”

Anticancer Activity of Liquid Treated with Microwave Plasma-Generated Gas through Macrophage Activation

“…Many researchers have tried to understand the working mechanisms of PAM in its tumoricidal activity. Since PAM contains relatively long-lived reactive species compared to direct plasma treatment, hydrogen peroxide and nitric oxide ions were identified as the main players in PAM reactivity [7][8][9][10][11][12][13][14][15][16]. The actions of reactive oxygen species (ROS) and reactive nitrogen species (RNS) are distinguished in biological systems [17].…”