Retinoic Acid Inducible Gene 1 Protein (RIG1)-Like Receptor Pathway Is Required for Efficient Nuclear Reprogramming

Abstract

We have revealed a critical role for innate immune signaling in nuclear reprogramming to pluripotency, and in the nuclear reprogramming required for somatic cell transdifferentiation. Activation of innate immune signaling causes global changes in the expression and activity of epigenetic modifiers to promote epigenetic plasticity. In our previous articles, we focused on the role of toll-like receptor 3 (TLR3) in this signaling pathway. Here, we define the role of another innate immunity pathway known to participate in response to viral RNA, the retinoic acid-inducible gene 1 receptor (RIG-1)-like receptor (RLR) pathway. This pathway is represented by the sensors of viral RNA, RIG-1, LGP2, and melanoma differentiation-associated protein 5 (MDA5). We first found that TLR3 deficiency only causes a partial inhibition of nuclear reprogramming to pluripotency in mouse tail-tip fibroblasts, which motivated us to determine the contribution of RLR. We found that knockdown of interferon beta promoter stimulator 1, the common adaptor protein for the RLR family, substantially reduced nuclear reprogramming induced by retroviral or by modified messenger RNA expression of Oct 4, Sox2, KLF4, and c-MYC (OSKM). Importantly, a double knockdown of both RLR and TLR3 pathway led to a further decrease in induced pluripotent stem cell (iPSC) colonies suggesting an additive effect of both these pathways on nuclear reprogramming. Furthermore, in murine embryonic fibroblasts expressing a doxycycline (dox)-inducible cassette of the genes encoding OSKM, an RLR agonist increased the yield of iPSCs. Similarly, the RLR agonist enhanced nuclear reprogramming by cell permeant peptides of the Yamanaka factors. Finally, in the dox-inducible system, RLR activation promotes activating histone marks in the promoter region of pluripotency genes. To conclude, innate immune signaling mediated by RLR plays a critical role in nuclear reprogramming. Manipulation of innate immune signaling may facilitate nuclear reprogramming to achieve pluripotency. Stem Cells 2017;35:1197-1207.

Keywords: Induced pluripotent stem cells; Pluripotency; Pluripotent stem cells; Reprogramming; Transdifferentiation.

Figure 1. Partial inhibition of pluripotent gene expression and reprogramming in TLR3 KO Tail-tip fibroblasts

Changes in pluripotent gene expression levels after exposure of WT and TLR3KO TTFs to retroviral vector encoding Oct4. (A) Relative fold change in gene expression levels of Nanog following exposure of WT TTFs to vehicle (black line) or pMX-Oct4 (green line) and TLR3KO TTFs to vehicle (blue line) or pMX-Oct4 (red line). This relative fold change was determined after a single pMX-Oct4 infection on Day 0. All data represented as mean ± s.d., n=3, *P <0.005. (B) Summary figure showing the average fold-change in the selected genes (i.e. Tcf4, Gap43, Nanog, Sox2 and Oct4) over time for each condition. All data represented as mean ± s.d., n=3, *P <0.05. Changes in reprogramming levels following retroviral OSKM transduction of WT and TLR3KO TTFs. (C) Protocol for mouse iPSC generation using the reprogramming factors, delivered as retroviral vectors. (D) Representative images of mouse iPSCs on day 19 after initiation of retroviral nuclear reprogramming in WT TTFs (left panel) or TLR3KO TTFs (right panel). (E) Total number of SSEA-1+ miPSCs on day 19 in WT and TLR3KO TTFs. All data represented as mean ± s.d., *P<0.05; WT compared to TRL3KO TTFs. (F) Fold change in Oct4 gene expression in WT and TLR3KO TTFs at day 19.

Figure 2. RLR pathway knockdown inhibits pluripotent gene expression following infection with retroviral vector encoding Oct4

(A) Gene expression levels of Nanog following pMX-Oct4 infection in scramble and IPS1 shRNA-knockdown human fibroblasts. (B) Summary figure showing the average fold-change in the selected genes (i.e. Tcf4, Gap43, Nanog, Sox2 and Oct4) over time for each condition. All data represented as mean ± s.d., n=3, *P <0.05.

Figure 3. RLR pathway knockdown inhibits nuclear reprogramming

(A) Protocol for human iPSC generation using retroviral-reprogramming factors encoding OSKM. (B) Representative images of hiPSCs on day 34 following retroviral nuclear reprogramming for scramble and IPS1 shRNA knockdown fibroblasts. (C) Number of iPSC colonies on day 34 in scramble- and IPS1-shRNA knockdown fibroblasts. All data represented as mean ± s.d., n=3, *P <0.05. (D) Oct4 gene expression in scramble- and IPS1-shRNA knockdown fibroblasts at day 34. All data represented as mean ± s.d., n=3, *P <0.05. (E) Protocol for mmRNA-based nuclear reprogramming. (F) Representative images of hiPSCs on day 11 following mmRNA transduction of the reprogramming factors in scramble- and IPS1-shRNA knockdown fibroblasts. (G) Number of mmRNA-transduced hiPSC colonies on day 11 in scramble- and IPS1-shRNA knockdown fibroblasts. All data represented as mean ± s.d., n=3, *P <0.05. (H) Oct4 gene expression in scramble- and IPS1-shRNA knockdown fibroblasts at day 11 following mmRNA-based nuclear reprogramming. All data represented as mean ± s.d., n=3, *P <0.05.

Figure 4. Elimination of both TLR3 and RLR pathway inhibits nuclear reprogramming

(A) Relative gene expression of IPS1 in WT and TLR3KO TTFs following scramble or shRNA-knockdown of IPS1. (B) Protocol for mouse iPSC generation using the reprogramming factors, delivered as retroviral vectors. (C) Representative images of mouse iPSCs on day 19 after initiation of retroviral nuclear reprogramming in Scramble and IPS1-shRNA knockdown WT TTFs (left panel) and Scramble and IPS1-shRNA knockdown TLR3KO TTFs (right panel). (D) Total number of SSEA-1+ miPSCs on day 19 in Scramble and IPS1-shRNA knockdown WT TTFs or Scramble and IPS1-shRNA knockdown TLR3KO TTFs. All data represented as mean ± s.d., *P<0.05; Scramble WT TTFs compared to IPS1-shRNA WT TTFs. *P<0.05; Scramble TLR3KO TTFs compared to IPS1-shRNA TLR3KO TTFs. #P<0.05; IPS1-shRNA WT TTFs compared to IPS1-shRNA TLR3KO TTFs. (E) Pluripotent genes (Oct4, Sox2, and Nanog) expression in iPSCs derived from Scramble and IPS1-shRNA knockdown WT TTFs and Scramble and IPS1-shRNA knockdown TLR3KO TTFs. *P<0.05; Scramble WT TTFs compared to IPS1-shRNA WT TTFs. *P<0.05; Scramble TLR3KO TTFs compared to IPS1-shRNA TLR3KO TTFs. (F) Immunostaining for Oct4 in iPSCs derived from Scramble and IPS1-shRNA knockdown WT TTFs and Scramble and IPS1-shRNA knockdown TLR3KO TTFs. Scale bars, 100 μm

Figure 5. RLR activation accelerates doxycycline-inducible nuclear reprogramming

MEFs containing the Dox-inducible polycistronic transgene construct encoding OSKM were stimulated by doxycycline (2mg/L), in the absence or presence of 5′ppp-dsRNA. (A) Relative fold change in Oct4 and Sox2 gene expression following treatments with dox alone (black line), dox + control (blue line), dox + 5′ppp-dsRNA (green line) and dox + 5′ppp-dsRNA + PolyIC (red line). All data represented as mean ± s.d., n=3, *P <0.05. (B) Bar graph showing SSEA-1⁺ colonies at day 14 and day 21 following treatments with dox alone, dox + control, dox + 5′ppp-dsRNA and dox + 5′ppp-dsRNA + PolyIC. All data represented as mean ± s.d., n=3, *P <0.05. (C) Representative images of SSEA-1 live staining of iPSC colonies derived from dox-inducible MEFs following treatments with dox alone, dox + control, dox + 5′ppp-dsRNA and dox + 5′ppp-dsRNA + PolyIC. (D) Total number of SSEA-1+ colonies derived from retroviral reprogramming of WT and TLR3KO TTFs with or without treatment of 5′ppp-dsRNA. All data represented as mean ± s.d., n=3, *P <0.05. (E) Pluripotent genes (Oct4, Sox2, and Nanog) expression in iPSC derived from WT and TLR3KO TTFs with or without treatment of 5′ppp-dsRNA. All data represented as mean ± s.d., n=3, *P <0.05. (F) Bar graph showing relative viability at Day 2 of retroviral reprogramming of WT and TLR3KO TTFs with or without treatment of 5′ppp-dsRNA. All data represented as mean ± s.d., n=3, *P <0.05. (G) Relative fold change in IFNb and Casp3 gene expression in WT and TLR3KO TTFs with or without treatment of 5′ppp-dsRNA.

Figure 6. 5′ppp-dsRNA enhance nuclear reprogramming via epigenetic modifications

(A) ChIP assay to assess H3K4me3 of the Oct4 promoters in secondary MEFs on day 2 following treatments with dox alone, dox + control and dox + 5′ppp-dsRNA. Data represented as mean ± S.D, n = 2, *P <0.05 (B) ChIP assay to assess H3K27me3 of the Oct4 promoters in secondary MEFs on day 2 following treatments with dox alone, dox + control and dox + 5′ppp-dsRNA. Data represented as mean ± S.D, n = 2, *P <0.05 (C) ChIP assay to assess H3K4me3 of the Sox2 promoters in secondary MEFs on day 2 following treatments with dox alone, dox + control and dox + 5′ppp-dsRNA. Data represented as mean ± S.D, n = 2, *P <0.05 (D) ChIP assay to assess H3K27me3 of the Sox2 promoters in secondary MEFs on day 2 following treatments with dox alone, dox + control and dox + 5′ppp-dsRNA. Data represented as mean ± S.D, n = 2, *P <0.05.