Optimal ROS Signaling Is Critical for Nuclear Reprogramming

Abstract

Efficient nuclear reprogramming of somatic cells to pluripotency requires activation of innate immunity. Because innate immune activation triggers reactive oxygen species (ROS) signaling, we sought to determine whether there was a role of ROS signaling in nuclear reprogramming. We examined ROS production during the reprogramming of doxycycline (dox)-inducible mouse embryonic fibroblasts (MEFs) carrying the Yamanaka factors (Oct4, Sox2, Klf4, and c-Myc [OSKM]) into induced pluripotent stem cells (iPSCs). ROS generation was substantially increased with the onset of reprogramming. Depletion of ROS via antioxidants or Nox inhibitors substantially decreased reprogramming efficiency. Similarly, both knockdown and knockout of p22(phox)-a critical subunit of the Nox (1-4) complex-decreased reprogramming efficiency. However, excessive ROS generation using genetic and pharmacological approaches also impaired reprogramming. Overall, our data indicate that ROS signaling is activated early with nuclear reprogramming, and optimal levels of ROS signaling are essential to induce pluripotency.

Keywords: CRISPR/Cas9; NADPH oxidase; Nrf2; iPSCs; nuclear reprogramming; reactive oxygen species.

Figure 1. iPSCs have low ROS level in comparison to MEFs

(A) Flow cytometry of DHE and CM-H2DCFDA staining for ROS in iPSCs and MEFs — left. Quantitation of flow cytometry data — right. Data are represented as mean ± SD, n=3. See also Figure S1. (B) Ratiometric measurements of redox status by roGFP2. Data are represented as mean ± SE, n=4. *: P<0.05; **: P<0.01; ***: P<0.001. (C) mRNA levels of “oxidant” genes in iPSCs relative to MEFs. Data are represented as mean ± SD, n=3. (D) mRNA levels of “anti-oxidant” genes as indicated in iPSCs relative to MEFs. Data are represented as mean ± SD, n=3.

Figure 2. NF-κB upregulates Nox2 during nuclear reprogramming

(A) mRNA levels of Nox isoforms during nuclear reprogramming process. Data are represented as mean ± SD, n=3. (B) mRNA levels of Nox complex components during nuclear reprogramming. Data are represented as mean ± SD, n=3. (C) Time course of ROS status during nuclear reprogramming. DHE staining for ROS was performed every other day as indicated. Detailed methods were described in Methods section. Data are represented as mean ± SD, n=3. (D) NF-κB inhibitor inhibits Nox2 expression. Reprogramming was initiated by adding ES media containing 2µg/ml of Dox in the absence or presence of BAY117085 (20 µM). Nox2 message was examined at day 1 after reprogramming. Data are represented as mean ± SD, n=3. (E) NF-κB inhibitor impairs reprogramming efficiency. Reprogramming was performed in the absence or presence of BAY117085 (20 µM) for the first 4 days. AP positive colonies were counted at day 21. Data are represented as mean ± SD, n=6. *: P<0.05.

Figure 3. Early ROS repression impairs nuclear reprogramming efficiency

(A) Selective scavengers (25µM EUK134, 50 µM Ebselen and 100 µM Mito-TEMPO) and (B) Nox inhibitors (50 nM DPI and 10 µM Apocynin) decrease AP positive iPSC colonies. Data are represented as mean ± SD, n=6. **: P<0.01; ***: P<0.001; ****: P<0.0001. See also Figure S3. (C) Knockdown of p22^phox. The mRNA level was examined by qRT-PCR at day 3 and 6 of reprogramming. Data are represented as mean ± SD, n=3. p22^phox (D) knockdown and (E) knockout decrease AP positive iPSC colonies. Data are represented as mean ± SD, n=6. See also Figure S3H, I & J.

Figure 4. Accumulation of ROS impairs nuclear reprogramming

(A) Overexpression of Nox2 decreased AP positive colony yield. To overexpress Nox2 in secondary Dox-inducible MEFs, cells were plated in a 6-well plate at 3 × 10⁵ cells/well. After 12 hours serum starvation, Nox2 (Cybb, from Origene) plasmids were introduced via FuGENE 6 according to manufacturer’s instruction. Two days after Nox2 overexpression, reprogramming was initiated and AP positive colony numbers were counted at day 21. Data are represented as mean ± SD, n=6. See also Figure S4. ***: P<0.001. (B) Buthionine sulphoximine (BSO) decreased AP positive colony yield. Nuclear reprogramming was initiated in the presence of 50 µM BSO until day 12. Data are represented as mean ± SD, n=6. *: P<0.05. (C) H₂O₂ decreased AP positive colony yield in a dose-dependent manner. Nuclear reprogramming was initiated in the presence of H₂O₂ until day 12. Data are represented as mean ± SD, n=6. **: P<0.01.