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. 2016 May;34(5):1198-212.
doi: 10.1002/stem.2327. Epub 2016 Mar 4.

JNK/SAPK Signaling Is Essential for Efficient Reprogramming of Human Fibroblasts to Induced Pluripotent Stem Cells

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Free PMC article

JNK/SAPK Signaling Is Essential for Efficient Reprogramming of Human Fibroblasts to Induced Pluripotent Stem Cells

Irina Neganova et al. Stem Cells. .
Free PMC article

Abstract

Reprogramming of somatic cells to the phenotypic state termed "induced pluripotency" is thought to occur through three consecutive stages: initiation, maturation, and stabilisation. The initiation phase is stochastic but nevertheless very important as it sets the gene expression pattern that permits completion of reprogramming; hence a better understanding of this phase and how this is regulated may provide the molecular cues for improving the reprogramming process. c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPKs) are stress activated MAPK kinases that play an essential role in several processes known to be important for successful completion of the initiation phase such as cellular proliferation, mesenchymal to epithelial transition (MET) and cell cycle regulation. In view of this, we postulated that manipulation of this pathway would have significant impacts on reprogramming of human fibroblasts to induced pluripotent stem cells. Accordingly, we found that key components of the JNK/SAPK signaling pathway increase expression as early as day 3 of the reprogramming process and continue to rise in reprogrammed cells throughout the initiation and maturation stages. Using both chemical inhibitors and RNA interference of MKK4, MKK7 and JNK1, we tested the role of JNK/SAPK signaling during the initiation stage of neonatal and adult fibroblast reprogramming. These resulted in complete abrogation of fully reprogrammed colonies and the emergence of partially reprogrammed colonies which disaggregated and were lost from culture during the maturation stage. Inhibition of JNK/SAPK signaling resulted in reduced cell proliferation, disruption of MET and loss of the pluripotent phenotype, which either singly or in combination prevented establishment of pluripotent colonies. Together these data provide new evidence for an indispensable role for JNK/SAPK signaling to overcome the well-established molecular barriers in human somatic cell induced reprogramming. Stem Cells 2016;34:1198-1212.

Keywords: JNKs; MKK4; MKK7; SAPK; hESC; hiPSC.

Figures

Figure 1
Figure 1
JNK/SAPK signaling is activated during the initiation and maturation stage of reprogramming. (A): Real‐time PCR analysis of MKK4, MKK7, JNK1 and JNK2 expression in H9 (p36), neonatal human fibroblasts (Neo1), adult human fibroblasts (Ad3) and human induced pluripotent stem cell (hiPSC) generated therefrom (Neo1cl1iPSC and Ad3cl1iPSC, respectively). Data represent relative expression to GAPDH and normalized against H9. Data are presented as mean ± SEM. (B): Western blot analysis showing expression of MKK4, MKK7, JNK/SAPKs, pSAPK(Thr183/Tyr185) and pSAPK (Ser63) in hESC (H9), human neonatal fibroblasts (Neo1), human adult fibroblasts (Ad3) at Day 0 and hiPSC derived therefrom (Neo1cl1iPSC and Ad3cl1iPSC, respectively). (C): Western blot analysis of protein expression of MKK4, MKK7, JNK/SAPKs, pSAPK(Thr183/Tyr185) and pSAPK (Ser63) during the reprogramming of Neo1 fibroblasts. Days of transduction are indicated as D3—Day 3 and so on, correspondently. GAPDH served as loading control. Images are representative of at least three independent experiments. (D): Flow cytometric analysis of the distribution of TRA1‐60+/CD44‐, TRA1‐60+/CD44 + and TRA1‐60‐/CD44 + populations during time course of reprogramming of neonatal (Neo1) and adult fibroblasts (Ad3). This is a representative example of at least three independent experiments. (E): Neo1 fibroblasts undergoing reprogramming were sorted in all four different subpopulation by FACS at day 13 of reprogramming and replated. The resulting colonies were stained by alkaline phosphatase at day 28. TRA1‐60 + /CD44– cells formed numerous AP + colonies (upper panel), while TRA1‐60+/CD44 + cells (lower panel) generated partly reprogrammed colonies. (F): Representative examples of flow cytometric analysis showing the distribution of pSAPK + cells among TRA1‐60+/CD44‐, TRA1‐60+/CD44 + and TRA1‐60‐/CD44 + populations at day 10 of reprogramming of Neo1 fibroblasts. (G): Graphic representation of the percentage of p‐SAPK + cells at different cells populations (TRA1‐60+/CD44‐, TRA1‐60+/CD44 + and TRA1‐60‐/CD44+) during the reprogramming of Neo1 fibroblasts assessed by flow cytometric analysis. Data are presented as mean ± SEM. Abbreviations: FACS, Fluorescence‐activated cell sorting; hESC, human embryonic stem cell; iPSC, induced pluripotent stem cell; JNK, c‐Jun N‐terminal kinase; MKK, MAP kinase kinases; SAPK, stress‐activated protein kinase.
Figure 2
Figure 2
Application of JNK/SAPKs inhibitor (SP60015) abrogates human induced pluripotent stem cell generation. (A): Western blot analysis of JNK/SAPKs downregulation by SP60015 (SAPKi) in hESCs (H9). GAPDH used as a loading control. Images are representative of at least three independent experiments. (B): Schematic representation of inhibitor application (SAPKi) at day 8 during the reprogramming process. (C): Graphic representation of flow cytometric analysis data (day 13) indicating a significant impact of SAPKi application (applied at day 8 for 24 hours) on the percentage of TRA1‐60+/CD44‐ cells. Results are presented as mean ± SEM (n = 3). (D): Graphic representation of flow cytometric analysis data (day 13) demonstrating a significant impact of SAPKi application on TRA1‐60+/CD44‐ and TRA1‐60+/CD44 + subpopulations generated during reprogramming of Neo1 fibroblasts. Results are presented as mean ± SEM (n = 3). (E): Phase–contrast observation showing the morphology of partially reprogrammed colonies arising during the reprogramming of Neo1 and Ad3 fibroblasts treated with DMSO or SAPKi for 24 hours at day 8, scale bars = 100 µm. (F): Graphic representation of total colony numbers at day 16 and 28 of reprogramming in SAPKi and DMSO treated Neo1 and Ad3 fibroblasts. Data are presented as mean ± SEM (n = 3). (J): Alkaline phosphatase staining at day 28 confirmed the absence of true AP + colonies from neonatal and adult fibroblasts undergoing reprogramming and treated with SAPKi at day 8 of transduction for 24 hours. (C, D, F): *, p < .05. Abbreviations: AP, alkaline phosphatase; DMSO, dimethyl sulfoxide; FCM, flow cytometric and morphological analysis; JNK, c‐Jun N‐terminal kinase; SAPK, stress‐activated protein kinase.
Figure 3
Figure 3
Assessment of MKK4, MKK7 and JNK1 downregulation in neonatal (A) and adult (B) fibroblasts by real‐time quantitative RT‐PCR analysis. Data represent relative expression to GAPDH and normalized against Control shRNA. Results are presented as average ± SEM (n = 3). t test analysis was carried out to assess differences in gene expression between the control and shRNA groups. Western blot analysis of MKK4, MKK7 and SAPK expression in Control shRNA and MKK4 shRNA, MKK7 shRNA and JNK1 shRNA neonatal cells (C). Analysis of the crosstalk between upstream and downstream MKK4/MKK7‐JNK1 signaling pathways in neonatal (D) and adult fibroblasts (E) revealed by real‐time quantitative RT‐PCR analysis. Results are presented as average ± SEM (n = 3). *, p < .05. Abbreviations: JNK, c‐Jun N‐terminal kinase; MKK, MAP kinase kinases; SAPK, stress‐activated protein kinase; shRNA, short hairpin RNA.
Figure 4
Figure 4
Inhibition of MKK4, MKK7 and JNK1 dependent signaling is detrimental for human induced pluripotent stem cells generation. (A): Graphic representation of flow cytometric analysis on the different cellular subpopulations during reprogramming of MKK4, MKK7 and JNK1 deficient Neo1 fibroblasts. Results are presented as mean ± SEM (n = 3). *, p < .05. (B): Phase‐contrast images of representative colonies arising during reprogramming of the control, MKK4MKK7 and JNK1 deficient Neo1 fibroblasts. Scale bar = 100 µm. (C): Real‐time quantitative RT‐PCR analysis for ectoderm, mesoderm and endoderm markers at day 18 in JNK1 shRNA transduced cells. Data represent relative expression to GAPDH and normalized against Control shRNA. Results are presented as average ± SEM (n = 3). Abbreviations: JNK, c‐Jun N‐terminal kinase; MKK, MAP kinase kinases; SAPK, stress‐activated protein kinase; shRNA, short hairpin RNA.
Figure 5
Figure 5
MKK4, MKK7 and JNK1 downregulation results in changes in the expression of key genes involved in cellular proliferation and cell cycle progression. (A, B): Real‐time quantitative PCR analysis of c‐JUN, E2F2, ATF2 (A) and CDK6, CDK2, CDK1, CCND1 and CCNB1 (B) in Control shRNA (Control) and MKK4, MKK7 and JNK1 deficient Neo1 fibroblasts at Day 1 of reprogramming. Results are presented as mean ± SEM (n = 3). *, p < .05. (C): Flow cytometric cell cycle analysis of MKK4, MKK7 and JNK1 shRNA treated fibroblasts during the reprogramming process. Results are presented as mean ± SEM (n = 3). Abbreviations: JNK, c‐Jun N‐terminal kinase; MKK, MAP kinase kinases; shRNA, short hairpin RNA.
Figure 6
Figure 6
Downregulation of JNK1 expression results in disruption of the E‐cadherin/β‐Catenin network and increased N‐Cadherin junctional staining in emerging partially reprogrammed colonies. Double‐immunofluorescence staining of transduced control and JNK1 deficient Neo1 fibroblast for E‐cadherin (A), β‐Catenin (B) and N‐cadherin (C) together with TRA1‐60 (green) on day 18 post transduction with OCT3/4, SOX2, KLF4, and c‐MYC assessed by confocal microscope. Scale bars represent 50 µm. Images are representative of at least three independent experiments. The percentage of double‐positive colonies is shown on the top right hand corner of the merged images. (D, D1): Real‐time quantitative PCR analysis of SNAIL, ECAD, NCAD and TWIST, VIMENTIN, ZEB1 and SLUG at day 18 of reprogramming. Data represent relative expression to GAPDH and normalized against Control shRNA. Results are presented as mean ± SEM (n = 3). *, p < .05. Abbreviations: JNK, c‐Jun N‐terminal kinase; shRNA, short hairpin RNA.
Figure 7
Figure 7
Schematic summary showing the impacts of MKK4, MKK7 and JNK1 signaling on hiPSC generation. Abbreviations: hiPSC, human induced pluripotent stem cell; JNK, c‐Jun N‐terminal kinase; MET, mesenchymal to epithelial transition; MKK, MAP kinase kinases; SAPK, stress‐activated protein kinase.

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References

    1. Takahashi K, Tanabe K, Ohnuki M et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 2007;131:861–872. - PubMed
    1. Lako M, Armstrong L, Stojkovic M. Induced pluripotent stem cells: It looks simple but can looks deceive? Stem Cells 2010;28:845–850. - PubMed
    1. Beltran AS, Rivenbark AG, Richardson BT et al. Generation of tumor‐initiating cells by exogenous delivery of OCT4 transcription factor. Breast Cancer Res 2011;13:R94. - PMC - PubMed
    1. Liu K, Lin B, Zhao M et al. The multiple roles for Sox2 in stem cell maintenance and tumorigenesis. Cell Signal 2013;25:1264–1271. - PMC - PubMed
    1. Yu F, Li J, Chen H et al. Kruppel‐like factor 4 (KLF4) is required for maintenance of breast cancer stem cells and for cell migration and invasion. Oncogene 2011;30:2161–2172. - PMC - PubMed

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