PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of autophLink to Publisher's site
 
Autophagy. 2016; 12(11): 2000–2008.
Published online 2016 August 11. doi:  10.1080/15548627.2016.1212786
PMCID: PMC5103358

ATG3-dependent autophagy mediates mitochondrial homeostasis in pluripotency acquirement and maintenance

ABSTRACT

Pluripotent stem cells, including induced pluripotent and embryonic stem cells (ESCs), have less developed mitochondria than somatic cells and, therefore, rely more heavily on glycolysis for energy production.1-3 However, how mitochondrial homeostasis matches the demands of nuclear reprogramming and regulates pluripotency in ESCs is largely unknown. Here, we identified ATG3-dependent autophagy as an executor for both mitochondrial remodeling during somatic cell reprogramming and mitochondrial homeostasis regulation in ESCs. Dysfunctional autophagy by Atg3 deletion inhibited mitochondrial removal during pluripotency induction, resulting in decreased reprogramming efficiency and accumulation of abnormal mitochondria in established iPSCs. In Atg3 null mouse ESCs, accumulation of aberrant mitochondria was accompanied by enhanced ROS generation, defective ATP production and attenuated pluripotency gene expression, leading to abnormal self-renewal and differentiation. These defects were rescued by reacquisition of wild-type but not lipidation-deficient Atg3 expression. Taken together, our findings highlight a critical role of ATG3-dependent autophagy for mitochondrial homeostasis regulation in both pluripotency acquirement and maintenance.

KEYWORDS: ATG3, mitochondria, mitophagy, pluripotent stem cell, reprogramming

Introduction

Pluripotent stem cells (PSCs), including induced pluripotent and embryonic stem cells (ESCs), hold great promise for regenerative medicine due to their unlimited self-renewal capability and their ability to differentiate into any cell types of 3 germ layers in our bodies. PSCs are characterized by their ability to self-renew, pluripotency, immortalization in culture, high proliferation rate, and short G1 phase.4,5 The maintenance of intracellular homeostasis in ESCs requires timely clearance of damaged organelles and toxic proteins, as well as quick synthesis of total biomass associated with their high proliferation rate. Autophagy is a conserved degradation pathway by which organelles and cytoplasm are sequestered and subsequently delivered to lysosomes for hydrolytic digestion.6,7 Autophagy serves as a protective strategy to eliminate toxic cytoplasmic contents, preventing cellular damage in response to stress.8 In addition, autophagy also serves as a dynamic recycling system that produces new building blocks and bioenergy to fuel cellular renovation and homeostasis during normal development and differentiation.6,9

Mitochondria are dynamic double-membrane organelles that play critical roles in multiple biological processes, such as energy production, apoptosis, and signal transduction.10,11 To meet the different bioenergetic demands of distinct cell types, cells regulate mitochondrial homeostasis through either biogenesis and degradation or dynamic fusion and fission.12 Nuclear reprogramming induces both structural and functional remodeling of parental mitochondria in somatic cells to a state typical of ESCs.13,14 However, how mitochondrial remodeling matches the requirements of somatic reprogramming and how ESCs harness their mitochondria to regulate pluripotency and self-renewal have not been clearly defined. Herein, we have determined that ATG3-dependent autophagy is an executor for both mitochondrial remodeling during reprogramming and mitochondrial homeostasis regulation in ESCs, and is pivotal for pluripotency acquirement and maintenance.

Results

Mitochondria are removed during reprogramming

During the acquisition of pluripotency, specific mitochondrial remodeling must occur to meet the energetic and anabolic requirements of the pluripotent states. To examine how mitochondria are remodeled during reprogramming, we first monitored changes in mitochondrial morphology and number using transmission electron microscopy and mitochondrial mass using MitoTracker staining of mouse embryonic fibroblasts (MEFs) and PSCs. While mitochondria in MEFs possess well-developed morphology with dense cristae and an elongated shape, PSC mitochondria have an immature morphology with a globular shape and low-density cristae (Fig. 1A). Furthermore, the number of mitochondria in PSCs was significantly lower than that in MEFs (Fig. 1B). Accordingly, the total mitochondrial mass in PSCs was significantly lower than that of MEFs (Fig. 1C). Then, we isolated FUT4/SSEA-1-positive cells at reprogramming d 10 and iPSCs for mitochondrial mass and mtDNA copy number determination (Fig. 1D). The established iPSCs have normal karyotype, express pluripotent markers, form teratomas and contribute to chimeric mice, supporting their pluripotency (Fig. S1). The total mitochondrial mass of reprogramming cells gradually decreased in parallel with reprogramming progression (Fig. 1E), indicating mitochondria are removed during reprogramming. In support of these observations, a quantitative polymerase chain reaction (PCR) assay using mitochondrial DNA (mtDNA) as a template was performed to track mtDNA copy number changes during reprogramming. As expected, genomic mtDNA gradually decreased as reprogramming progressed, confirming removal of mitochondria during this process (Fig. 1F).

Figure 1.
Mitochondrial numbers gradually decrease during reprogramming. (A) Representative transmission electronic microscopy images of mitochondria in MEFs, ESCs, and iPSCs. Bar: 5 μm. (B) MEFs had more mitochondria than ESCs and iPSCs. Mean ± ...

Autophagy is an executor of mitochondria removal during reprogramming

Because autophagy degrades both proteins and organelles, we next assessed whether autophagy was involved in mitochondrial removal during reprogramming. The autophagy inhibitors 3-methyladenine (3-MA) and bafilomycin A1 (Baf-A1) were added at the beginning of reprogramming for 3 d to block autophagic flux. Interestingly, both 3-MA and Baf-A1 treatments significantly inhibited mitochondrial removal without affecting proliferation and apoptosis, indicating autophagy contributes to mitochondrial removal during reprogramming (Fig. 1G; Fig. S2A and S2B). As a result, reprogramming efficiency was significantly reduced by 3-MA and Baf-A1 treatments (Fig. 1H).

ATG3 is a membrane-curvature sensor and plays a key role in autophagosome formation.15,16 To further investigate whether autophagy directly contributes to mitochondrial remodeling during reprogramming, we isolated both Atg3+/+ and atg3−/− MEFs (Fig. 2A). Atg3 deletion did not affect MEF proliferation and apoptosis (Fig. S2C and S2D). Unlike wild-type Atg3+/+ MEFs, we did not observe autophagosomes containing mitochondria in atg3−/− knockouts upon treatment with carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP), indicating a lack of autophagy-mediated mitochondrial removal in atg3−/− MEFs (Fig. S3A). We then transfected Atg3+/+ and atg3−/− MEFs with Yamanaka factors and monitored mitochondrial removal during reprogramming. Upon reprogramming initiation, mitophagy activation in Atg3+/+ but not atg3−/− MEFs was observed (Fig. 2B, C; Fig. S3B). Although a decrease in the number of mitochondria occurred during reprogramming in both Atg3+/+ and atg3−/− MEFs, the relative decrease of mitochondria in wild-type MEFs was significantly higher than that of atg3−/− MEFs, indicating ATG3-dependent mitophagy contributes to mitochondrial remodeling during reprogramming (Fig. 2D; Fig. S3C). Accordingly, the reprogramming efficiency was significantly inhibited (Fig. 2G).

Figure 2.
Lack of ATG3-dependent mitochondrial autophagy leads to defective reprogramming. (A) Western blot analysis of whole cell extracts from Atg3+/+ and atg3−/− MEFs; ACTB served as a loading control. (B) Mitophagy in Atg3+/+ and atg3−/− ...

ATG3-dependent autophagy is required for mitochondrial remodeling and successful reprogramming

An amphipathic helix domain at the N-terminus of ATG3 is responsible for membrane-curvature sensing and is critical for lipidation of the MAP1LC3/GABARAP family of autophagy proteins.15 Mutation of this domain leads to failure of autophagosome formation.15 To further confirm that impaired reprogramming of atg3−/− MEFs results from a defect in autophagy, gain-of-function assays were performed by overexpression of both wild-type and lipidation-deficient Atg3V8D in atg3−/− MEFs (Fig. 2E). We found ectopic expression of wild-type Atg3 but not lipidation-deficient mutant Atg3V8D in atg3−/− MEFs can rescue both defective mitochondrial clearance and reprogramming efficiency, supporting the view that ATG3-dependent autophagy is essential for mitochondrial remodeling and reprogramming (Fig. 2F, G).

We next investigated whether lack of ATG3-dependent autophagy affected the established iPSCs. By transmission electronic microscopy, we found that the abnormal mitochondria were accumulated in Atg3-deficient iPSCs and the mitochondrial cristae showed a blurred morphology (i.e., lack of clear definition) compared to Atg3+/+ iPSCs (Fig. 2D, H). Further investigation identified significantly lower mitochondrial membrane potential and oxygen consumption, and compromised ATP production in atg3−/− versus Atg3+/+ iPSCs, indicating the unsuccessful metabolic reprogramming of atg3−/− knockouts (Fig. 2I, J, K). Taken together, these data support the conclusion that ATG3-dependent autophagy contributes to both reprogramming efficiency and reprogramming quality and is required for successful reprogramming.

ATG3-dependent autophagy maintains ESC mitochondrial homeostasis and self-renewal

To investigate whether autophagy is involved in the regulation of mitochondrial dynamics and/or affects ESC identity, we first designed specific small interfering RNA (siRNA) targeting the autophagy regulator Atg3 and found transient inhibition of this gene leads to ESC differentiation and loss of pluripotent gene expression (Fig. S4A-C). Then, we treated ESCs with 3-MA and found autophagy inhibition significantly affected ESC self-renewal (Fig. S4D). These data suggest the involvement of autophagy in ESC identity maintenance. To further investigate how autophagy regulates mitochondrial dynamics in ESC and thereby affects ESC identity, Atg3+/+ and atg3−/− ESCs were isolated from the blastocyst of mice at embryonic d 3.5 (Fig. 3A). Upon FCCP treatment, we detected mitochondria associated with autophagic structures in wild-type Atg3+/+ but not atg3−/− ESCs (Fig. 3B; Fig. S5B and S5C), indicating a lack of ATG3-dependent mitophagy in atg3−/− ESCs. Meanwhile, abnormal accumulation of mitochondria was detected in atg3−/− ESCs, as indicated by the existence of increased mtDNA copy number, blurred-cristae mitochondria accumulation, enhanced ROS production and decreased ATP generation in atg3−/− ESCs (Fig. 3C–G). These data suggest ATG3-dependent autophagy plays critical roles to maintain mitochondrial homeostasis in ESCs. As a result, the clonogenic survival of atg3−/− ESCs was significantly impaired, indicating ATG3-dependent autophagic mitochondria removal is pivotal to ESC self-renewal (Fig. 3H).

Figure 3.
ATG3 regulates ESC mitochondrial homeostasis and self-renewal. (A) Western blot analysis of whole cell extracts from Atg3+/+ and atg3−/− ESCs; ACTB served as a loading control. (B) Lack of FCCP-induced mitophagy in atg3−/− ...

To further confirm whether the abnormal accumulation of mitochondria and aberrant self-renewal ability of atg3−/− ESCs was directly caused by the loss of ATG3-mediated mitophagy, gain-of-function assays were performed by introducing Atg3 expression into atg3−/− ESCs. We established stable atg3−/− ESC lines carrying an empty vector, wild-type Atg3, and mutant Atg3V8D. As expected, MAP1LC3B-II was only detected in atg3−/− stable ESC lines carrying wild-type Atg3 but not the empty vector or Atg3V8D mutant upon starvation, demonstrating recovery of autophagy in atg3−/− ESCs by expression of wild-type Atg3 (Fig. S5A). Meanwhile, mitophagosomes were again detected in atg3−/− ESCs expressing wild-type Atg3 upon FCCP treatment (Fig. S5B). Consequently, the increased mtDNA copy number in atg3−/− ESCs was significantly reduced by gain of wild-type Atg3 expression, indicating accumulation of mitochondria in Atg3-deficient ESCs was directly caused by defects in ATG3-dependent mitochondrial autophagy (Fig. 3E). Furthermore, ROS production and ATP generation were also rescued by reacquisition expression of wild-type but not V8D mutant Atg3 (Fig. 3F, G). Accordingly, the abnormal self-renewal of Atg3-deficient ESCs was compensated by reintroducing wild-type Atg3 but not lipidation-deficient Atg3V8D mutant (Fig. 3H). Together, these data suggest that ATG3-dependent canonical autophagy maintains ESC mitochondrial homeostasis and self-renewal.

ATG3-dependent autophagy regulates ESC pluripotency and differentiation

Differentiation of PSCs requires a specific remodeling process involving an increase in mitochondrial numbers. To test whether autophagy is involved in this process, we performed an embryonic body (EB) differentiation assay. The number of mitochondria gradually increased during EB differentiation as expected (Fig. 4A). We next assessed EB differentiation using Atg3+/+ and atg3−/− ESCs. Surprisingly, contrary to the relatively larger number of mitochondria in atg3−/− ESCs, the mitochondrial increase was delayed in atg3−/− ESCs compared to the wild-type during EB differentiation, indicating atg3−/− ESC mitochondria are abnormal and ATG3-dependent autophagy is crucial for mitochondrial remodeling during EB differentiation (Fig. 4B, C; Fig. S6A and S6B). Furthermore, decreased pluripotent gene expression was detected in atg3−/− vs. wild-type ESCs, suggesting elimination of ATG3-dependent autophagy leads to the compromised pluripotency in ESCs (Fig. 4D). In support of this assumption, atg3−/− ESCs showed abnormal EB differentiation, characterized by delayed expression of certain endodermic and mesodermic marker genes (Fig. 4E). Furthermore, a teratoma formation assay was employed to investigate the autophagy contribution to ESC differentiation. While both Atg3+/+ and atg3−/− ESCs formed teratomas, the average weight of teratomas formed by Atg3+/+ ESCs is significantly larger than that of atg3−/− ESCs, supporting the view that ATG3-dependent autophagy is critical for differentiation of pluripotent stem cells (Fig. 4F). Together, these data suggest ATG3-dependent autophagy is involved in mitochondrial remodeling during ESC differentiation and regulates ESC pluripotency and differentiation.

Figure 4.
Loss of ATG3-mediated mitochondrial autophagy compromises ESC pluripotency and differentiation. (A) Mitochondrial mass (Mito-mass) increased during EB differentiation. Data presented as the mean ± error from 3 independent experiments. (B) Mitochondrial ...

Discussion

In somatic cells, damaged or superfluous mitochondria are selectively recognized and sequestered by phagophores, followed by mitophagosomal fusion with lysosomes and autophagic degradation, a process called mitophagy.17-19 However, how mitochondrial homeostasis is regulated in PSCs is largely unknown. By using an Atg3 knockout model system, we demonstrate that ATG3-dependent autophagy is required for mitochondrial homeostasis regulation in both pluripotency acquirement and maintenance, and plays pivotal roles for ESC differentiation.

Somatic cell reprogramming remodels cellular components and organelles, as well as metabolism patterns. Transition of somatic cell oxidative phosphorylation to glycolysis through downregulation of mitochondrial respiratory chain complexes and upregulation of glycolytic genes has been identified as a critical event during reprogramming.20 In agreement with this model, a PDPK1/phosphoinositide-dependent kinase-1 activator, which facilitates the metabolic conversion from oxidative phosphorylation to glycolysis, in combination with POU5F1/OTF3 and a couple of small molecules could successfully induce pluripotency.21 However, how mitochondria remodel themselves to facilitate the metabolic switching in somatic cell reprogramming is not clear.

Mitochondria number and structure in PSCs were dramatically changed compared to somatic fibroblasts, suggesting a total mitochondrial remodeling process must occur during reprogramming. Although mitochondrial removal has been proposed to be required for nuclear reprogramming, the detailed mechanisms driving this remodeling process have not been clearly defined. Mitochondrial fission and fusion have been proposed to be responsible for mitochondrial remodeling during reprogramming.22,23 Inhibition of mitochondrial fission regulator DNM1L (dynamin 1-like) expression significantly decreased reprogramming efficiency, indicating the potential involvement of mitophagy in somatic cell reprogramming.23 Conversely, a recent study has demonstrated an ATG5-independent noncanonical mitophagy involvement in somatic reprogramming through ULK1 (unc-51 like kinase 1) and RAB9 pathway.24 In contrast, we have demonstrated that ATG3-dependent canonical autophagy contributes to mitochondrial remodeling and regulates reprogramming. Although Atg3 deletion did not completely block mitochondrial clearance and iPSC colony formation, the abnormal mitochondria accumulated in established atg3−/− iPSCs compared to Atg3+/+ iPSCs, leading to enhanced ROS generation and defective metabolic reprogramming. These data support the hypothesis that ATG3-dependent canonical autophagy regulates both mitochondrial quantity and quality and is critical for successful reprogramming. Consistent with this notion, recent studies have shown that transient activation of autophagy through MTOR (mechanistic target of rapamycin [serine/threonine kinase]) suppression either by SOX2 (SRY [sex determing region Y]-box 2) or rapamycin is a critical early step for successful reprogramming.25

Mitochondrial dynamics (fusion and fission) and mitophagy have been proposed to be important players for mitochondrial homeostasis regulation in somatic cells.12 Recent studies have shown that the mitochondrial fusion protein GFER (growth factor, erv1 [S. cerevisiae)-like (augmenter of liver regeneration]), which is highly expressed in ESCs, modulates the mitochondrial fission GTPase DNM1L to preserve mouse ESC mitochondrial homeostasis and function, and maintain pluripotency in ESCs.26 Knockdown of Gfer in ESCs leads to decreased pluripotent marker gene expression accompanied by excessive mitochondrial fragmentation and mitochondrial autophagy, indicating the involvement of mitochondrial dynamics in mitochondrial homeostasis and pluripotency regulation in mouse ESCs.26 By using atg3−/− ESCs, we demonstrate here that ATG3-dependent autophagy is required for ESC mitochondrial homeostasis and ESC identity maintenance. ESCs are endowed with high rates of catabolic processes to rapidly generate multiple building blocks for cellular reconstruction to meet their high proliferation rates. Furthermore, to maintain their identity, rapidly dividing ESCs have to efficiently remove damaged mitochondria to avoid the oxidative damage to their genomes. Atg3 null mouse ESCs accumulate aberrant mitochondria accompanied by enhanced mitochondrial mass, and generate increased levels of ROS and decreased levels of ATP, leading to compromised self-renewal and pluripotency ability. Thus we propose ESCs employ autophagy, by which dysfunctional mitochondria and the resulting excessive ROS can be rapidly removed, to protect their genome from oxidative damage and thus maintain their self-renewal and pluripotency.

In somatic cells, mitochondria can be eliminated either through selective mitochondrial autophagy or nonselective autophagy.17 The selective autophagic elimination of mitochondria has been extensively reported to be regulated by PARK2 and PINK1 (PTEN induced putative kinase 1).17,27-34 We did not observe defective mitochondrial clearance and impaired reprogramming efficiency when we used park2−/− MEFs for the reprogramming assay, indicating PARK2-mediated selective mitochondrial autophagy was not involved in mitochondrial remodeling during reprogramming, and other unidentified mitophagy pathways may play a role in this process.

Our findings significantly contribute to the current understanding of reprogramming and/or ESC identity maintenance in terms of mitochondrial homeostasis regulation, and may provide new strategies for directed differentiation of ESCs by manipulating autophagy. Furthermore, our data highlight the contribution of mitochondria in ESC identity maintenance. In the future, it would be of great interest to investigate how genes that mediate mitochondrial homeostasis are regulated by core pluripotency transcription factors in PSCs upon differentiation or, conversely, during reprogramming.

Materials and methods

Animals, reagents, antibodies, and plasmids

B6D2-Tg (CAG/Su9-DsRed2, Acr3-EGFP) RBGS002Osb (RBRC03743),35 MAP1LC3B-GFP (RBRC00806),36 and Atg3+/− (RBRC02761)16 mice were purchased from Riken BioResource Center. All protocols used for animal manipulation were approved by the Institutional Animal Care Committee. JC-1 (40705ES03), MitoTracker Green (40742ES50), and MitoTracker Red (40743ES50) were purchased from Yeasen. The anti-MAP1LC3B antibody (Medical and Biological Laboratories Co., PM036) was used at 1:200; Alexa Fluor® 488 donkey anti-rabbit IgG (H+L) (Invitrogen Thermo Fisher Scientific, A21206) was used at 1:500; anti-ACTB (1:5000) was obtained from Sigma Aldrich (A5441); anti-SQSTM1/p62 (1:2000) were purchased from Abcam (ab56416); anti-ATG3 (1:500) was purchased from Cell Signaling Technology (3415S); SSEA-1 were bought from Santa Cruz Biotechnology (SC-21702AF488); 3-MA, chloroquine, Hoechst 33342, Baf-A1, and anti-MAP1LC3B (1:2000) were obtained from Sigma Aldrich (M9281, C6628, B2261, B1793, L7543). pMXs-Pou5f1, pMXs-Sox2, pMXs-Klf4, and pMXs-cMyc were purchased from Addgene (13366, 13367, 13370, 13375; Deposited by Shinya Yamanaka lab). Atg3 and its lipidation-deficient V8D mutant were cloned into pMXs and pCDH-CAG-RFP lenti-vectors as described previously.15

ESC isolation and iPSC induction

Atg3+/+ and atg3−/− ESCs were isolated at embryonic d 3.5 and cultured on feeder layers for 5 d using 2i medium (ESC medium with 1 μM PD0325901 [Stemgent, 040006], 3 μM CHIR99021 [Stemgent, 040004] and 1000 U/ml leukemia inhibitory factor [Merck Millipore, ESG1107]). Then, ESC colonies were selected and cultured for 3 to 5 passages. After, isolated ESCs were routinely maintained in ESC medium (knockout Dulbecco's modified Eagle's medium [Gibco Life Technologies,10829018] with 15% fetal bovine serum [GE Healthcare Life Sciences, sh30070.03], 2 mM glutamine [Gibco Life Technologies, 25030164], 1 mM sodium pyruvate [Gibco Life Technologies, 11360070], 0.1 mM nonessential amino acids [Gibco Life Technologies, 11140050], 100 μg/ml streptomycin and 100 U/ml penicillin [Gibco Life Technologies, 15140122], 0.055 mM β-mercaptoethanol [Gibco Life Technologies, 21985023], and 1000 U/ml leukemia inhibitory factor [Merck Millipore, ESG1107]). For iPSC generation, MEFs were seeded at 50,000 cells/well in a 6-well plate and infected with a retrovirus cocktail expressing Pou5f1/Oct4, Sox2, Klf4, and Myc; iPSC colonies were picked 14 d after infection as described previously.37

The mtDNA copy number and mitochondrial mass detection

For mtDNA copy number determination, total DNA was extracted by a TIANamp Genomic DNA Kit (Tiangen Biotech [Beijing] Co., DP304-03). The mtDNA copy number was detected by quantitative real-time PCR using genomic DNA as a loading control. The primers used were published previously.38 Primers for genomic DNA (H19) were 5′-GTCCACGAGACCAATGACTG-3′ (reverse) and 5′-GTACCCACCTGTCGTCC-3′ (forward); mtDNA (CytB) primers were 5′-ATTCCTTCATGTCGGACGAG-3′ (reverse) and 5′-ACTGAGAAGCCCCCTCAAAT-3′ (forward). For total mitochondrial mass detection, cells were digested into single cell suspensions by trypsin (Gibco Life Technologies, 25200072), stained with 100 nM MitoTracker Green/Red for 30 min at 37°C, then washed and analyzed by a FACS calibur flow cytometer (BD Biosciences, Shanghai).

Measurement of OCR, ROS and ATP

Measurement of intact cellular respiration was performed using a Seahorse XF24 analyzer (Seahorse Bioscience Asia, Shanghai). Briefly, ESC or MEF cells were seeded at 60,000 cells/well 6 h before measurements. Then respiration was measured and determined using a standard protocol in the manual. The cellular ATP content was determined using a CellTiter-GloLuminescent Cell Viability Assay kit (Promega Corporation, 0000092970). The cellular ROS was measured by flow cytometry using HDCF-DA from Sigma Aldrich (D6883).

Immunofluorescence microscopy

The immunostaining was performed as previously described.39 Briefly, MEFs or ESCs cultured on gelatin-coated glass slides were fixed with 4% paraformaldehyde for 20 min, washed with Dulbecco's PBS (Corning Inc., R21-031-CV), permeabilized by 0.2% Triton-X100 (Sigma Aldrich, X100) for 0.5 h, blocked with 2% bovine serum albumin (Sigma Aldrich, A1933) for 1 h, stained with appropriate primary antibodies overnight at 4°C, and then incubated with secondary antibodies for 2 h at room temperature. Cell nuclei were counterstained with Hoechst 33342.

Quantitative real-time PCR

Total RNA was extracted from MEFs, ESCs, iPSCs, and EBs with an RNeasy Total RNA Isolation Kit (Qiagen, 74104). Total RNA (1 µg) was reverse transcribed into cDNA using a SuperScript™ III First-Strand Synthesis System (Invitrogen Thermo Fisher Scientific, 18080051). The primers used were as reported previously.40 Pou5f1: 5′-AGAGGATCACCTTGGGGTACA-3′ (forward), 5′- CGAAGCGACAGATGGTGGTC-3′ (reverse); Nanog: 5′-TCTTCCTGGTCCCCACAGTTT-3′ (forward), 5′-GCAAGAATAGTTCTCGGGATGAA-3′ (reverse); Sox2: 5′-GCGGAGTGGAAA CTTTTGTCC-3′ (forward), 5′-CGGGAAGCGTGTACTTATCCTT-3′ (reverse); Klf4: 5′-GTGCCCC GACTAACCGTTG-3′ (forward), 5′-GTCGTTGAACTCCTCGGTCT-3′ (reverse); Esrrb: 5′-CAG GCAAGGATGACAGACG-3′ (forward), 5′-GAGACAGCACGAAGGACTGC-3′ (reverse); Tbx3: 5′-TTGCAAAGGGTTTTCGAGAC-3′ (forward), 5′-TGGAGGACTCATCCGAAGTC-3′ (reverse); Tcl1: 5′-AAATTCCAGGTGATCTTGCG-3′ (forward), 5′-TGTCCTTGGGGTACAGTTGC-3′ (reverse); Fbxo15: 5′-TCGTGGGACTGAGCACAACTA-3′ (forward), 5′-TGACAGATGAGCCT CTAACAAAC-3′ (reverse); Rexo1: 5′-CCCTCGACAGACTGACCCTAA-3′ (forward), 5′-TCGGG GCTAATCTCACTTTCAT-3′ (reverse); Cdh1: 5′-CAGGTCTCCTCATGGCTTTGC-3′ (forward), 5′-CTTCCGAAAAGAAGGCTGTCC-3′ (reverse); Nes: 5′-CCCTGAAGTCGAGGAGCTG-3′ (forward), 5′-CTGCTGCACCTCTAAGCGA-3′ (reverse); Fgf5: 5′-CTGTATGGACCCAC AGGGAGTAAC-3′ (forward), 5′-ATTAAGCTCCTGGGTCGCAAG-3′ (reverse); Otx2: 5′-TATC TAAAGCAACCGCCTTACG-3′ (forward), 5′- AAGTCCATACCCGAAGTGGTC-3′ (reverse); Tal1: 5′-CTGGCCTCCAGCTACATTTCT-3′ (forward), 5′-GTCACGGTCTTTGCTCAACTT-3′ (reverse); Gata2: 5′-CACCCCGCCGTATTGAATG-3′ (forward), 5′-CCTGCGAGTCGAGATGGTTG-3′ (reverse); T: 5′-GCTTCAAGGAGCTAACTAACGAG-3′ (forward), 5′-CCAGCAAGAAAGAG TACATGGC-3′ (reverse); Afp: 5′-CTTCCCTCATCCTCCTGCTAC-3′ (forward), 5′-ACAAA CTGGGTAAAGGTGATGG-3′ (reverse); Gata4: 5′-CCCTACCCAGCCTACATGG-3′ (forward), 5′-ACATATCGAGATTGGGGTGTCT-3′ (reverse); Sox17: 5′-GATGCGGGATACGCCAGTG-3′ (forward), 5′-CCACCACCTCGCCTTTCAC-3′ (reverse); Actb: 5′-GGCTGTATTCCCCTC CATCG-3′ (forward), 5′-CCAGTTGGTAACAATGCCATGT-3′ (reverse).

Supplementary Material

KAUP_A_1212786_Supplementary_material.zip:

Abbreviations

3-MA
3-methyladenine
ATG3
autophagy-related 3
ATP
adenosine triphosphate
Baf-A1
bafilomycin A1
EB
embryonic body
ESC
embryonic stem cells
FCCP
carbonyl cyanide 4-(trifluoromethoxy) phenylhy-drazone
iPSC
induced pluripotent stem cell
MEFs
mouse embryonic fibroblasts
mtDNA
mitochondrial DNA
PSC
pluripotent stem cell
ROS
reactive oxygen species

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank G.W., at our institute, T.Z., J.L. and L.C.at IM-CAS for technique supports.

Funding

This work was supported by grants from the Strategic Priority Research Program of the Chinese Academy of Sciences XDA01040108, the China National Basic Research Program 2012CB966901, the National Natural Science Foundation of China Program 31271592, 31570995, and National Thousand Young Talents Program to T.Z.

References

[1] Zhang J, Nuebel E, Daley GQ, Koehler CM, Teitell MA. Metabolic regulation in pluripotent stem cells during reprogramming and self-renewal. Cell Stem Cell 2012; 11:589-95; PMID:23122286; http://dx.doi.org/10.1016/j.stem.2012.10.005 [PMC free article] [PubMed] [Cross Ref]
[2] Vessoni AT, Muotri AR, Okamoto OK. Autophagy in stem cell maintenance and differentiation. Stem Cells Dev 2012; 21:513-20; PMID:22066548; http://dx.doi.org/10.1089/scd.2011.0526 [PubMed] [Cross Ref]
[3] Suhr ST, Chang EA, Tjong J, Alcasid N, Perkins GA, Goissis MD, Ellisman MH, Perez GI, Cibelli JB. Mitochondrial rejuvenation after induced pluripotency. PLoS One 2010; 5:e14095; PMID:21124794; http://dx.doi.org/10.1371/journal.pone.0014095 [PMC free article] [PubMed] [Cross Ref]
[4] Ruiz S, Panopoulos AD, Herrerias A, Bissig KD, Lutz M, Berggren WT, Verma IM, Izpisua Belmonte JC. A high proliferation rate is required for cell reprogramming and maintenance of human embryonic stem cell identity. Curr Biol 2011; 21:45-52; PMID:21167714; http://dx.doi.org/10.1016/j.cub.2010.11.049 [PMC free article] [PubMed] [Cross Ref]
[5] White J, Dalton S. Cell cycle control of embryonic stem cells. Stem Cell Rev 2005; 1:131-8; PMID:17142847; http://dx.doi.org/10.1385/SCR:1:2:131 [PubMed] [Cross Ref]
[6] Mizushima N, Levine B. Autophagy in mammalian development and differentiation. Nat Cell Biol 2010; 12:823-30; PMID:20811354; http://dx.doi.org/10.1038/ncb0910-823 [PMC free article] [PubMed] [Cross Ref]
[7] He C, Klionsky DJ. Regulation mechanisms and signaling pathways of autophagy. Annu Rev Genet 2009; 43:67-93; PMID:19653858; http://dx.doi.org/10.1146/annurev-genet-102808-114910 [PMC free article] [PubMed] [Cross Ref]
[8] Orrenius S, Kaminskyy VO, Zhivotovsky B. Autophagy in toxicology: cause or consequence? Annu Rev Pharmacol Toxicol 2012; 53:275-97; PMID:23072380; http://dx.doi.org/10.1146/annurev-pharmtox-011112-140210 [PubMed] [Cross Ref]
[9] Mizushima N, Komatsu M. Autophagy: renovation of cells and tissues. Cell 2011; 147:728-41; PMID:22078875; http://dx.doi.org/10.1016/j.cell.2011.10.026 [PubMed] [Cross Ref]
[10] Levine B, Klionsky DJ. Development by self-digestion: molecular mechanisms and biological functions of autophagy. Dev Cell 2004; 6:463-77; PMID:15068787; http://dx.doi.org/10.1016/S1534-5807(04)00099-1 [PubMed] [Cross Ref]
[11] Attardi G, Schatz G. Biogenesis of mitochondria. Annu Rev Cell Biol 1988; 4:289-333; PMID:2461720; http://dx.doi.org/10.1146/annurev.cb.04.110188.001445 [PubMed] [Cross Ref]
[12] Westermann B.. Mitochondrial fusion and fission in cell life and death. Nat Rev Mol Cell Biol 2010; 11:872-84; PMID:21102612; http://dx.doi.org/10.1038/nrm3013 [PubMed] [Cross Ref]
[13] Liu K, Song Y, Yu H, Zhao T. Understanding the roadmaps to induced pluripotency. Cell Death Dis 2014; 5:e1232; PMID:24832604; http://dx.doi.org/10.1038/cddis.2014.205 [PMC free article] [PubMed] [Cross Ref]
[14] De Los Angeles A, Ferrari F, Xi R, Fujiwara Y, Benvenisty N, Deng H, Hochedlinger K, Jaenisch R, Lee S, Leitch HG, et al. Hallmarks of pluripotency. Nature 2015; 525:469-78; PMID:26399828; http://dx.doi.org/10.1038/nature15515 [PubMed] [Cross Ref]
[15] Nath S, Dancourt J, Shteyn V, Puente G, Fong WM, Nag S, Bewersdorf J, Yamamoto A, Antonny B, Melia TJ. Lipidation of the LC3/GABARAP family of autophagy proteins relies on a membrane-curvature-sensing domain in Atg3. Nat Cell Biol 2014; 16:415-24; PMID:24747438; http://dx.doi.org/10.1038/ncb2940 [PMC free article] [PubMed] [Cross Ref]
[16] Sou YS, Waguri S, Iwata J, Ueno T, Fujimura T, Hara T, Sawada N, Yamada A, Mizushima N, Uchiyama Y, et al. The Atg8 conjugation system is indispensable for proper development of autophagic isolation membranes in mice. Mol Biol Cell 2008; 19:4762-75; PMID:18768753; http://dx.doi.org/10.1091/mbc.E08-03-0309 [PMC free article] [PubMed] [Cross Ref]
[17] Youle RJ, Narendra DP. Mechanisms of mitophagy. Nat Rev Mol Cell Biol 2011; 12:9-14; PMID:21179058; http://dx.doi.org/10.1038/nrm3028 [PMC free article] [PubMed] [Cross Ref]
[18] Tolkovsky AM.. Mitophagy. Biochim Biophys Acta 2009; 1793:1508-15; PMID:19289147; http://dx.doi.org/10.1016/j.bbamcr.2009.03.002 [PubMed] [Cross Ref]
[19] Lemasters JJ.. Selective mitochondrial autophagy, or mitophagy, as a targeted defense against oxidative stress, mitochondrial dysfunction, and aging. Rejuvenation Res 2005; 8:3-5; PMID:15798367; http://dx.doi.org/10.1089/rej.2005.8.3 [PubMed] [Cross Ref]
[20] Folmes CD, Nelson TJ, Martinez-Fernandez A, Arrell DK, Lindor JZ, Dzeja PP, Ikeda Y, Perez-Terzic C, Terzic A. Somatic oxidative bioenergetics transitions into pluripotency-dependent glycolysis to facilitate nuclear reprogramming. Cell Metab 2011; 14:264-71; PMID:21803296; http://dx.doi.org/10.1016/j.cmet.2011.06.011 [PMC free article] [PubMed] [Cross Ref]
[21] Zhu S, Li W, Zhou H, Wei W, Ambasudhan R, Lin T, Kim J, Zhang K, Ding S. Reprogramming of human primary somatic cells by OCT4 and chemical compounds. Cell Stem Cell 2010; 7:651-5; PMID:21112560; http://dx.doi.org/10.1016/j.stem.2010.11.015 [PMC free article] [PubMed] [Cross Ref]
[22] Prigione A, Fauler B, Lurz R, Lehrach H, Adjaye J. The senescence-related mitochondrial/oxidative stress pathway is repressed in human induced pluripotent stem cells. Stem Cells 2010; 28:721-33; PMID:20201066; http://dx.doi.org/10.1002/stem.404 [PubMed] [Cross Ref]
[23] Vazquez-Martin A, Cufi S, Corominas-Faja B, Oliveras-Ferraros C, Vellon L, Menendez JA. Mitochondrial fusion by pharmacological manipulation impedes somatic cell reprogramming to pluripotency: new insight into the role of mitophagy in cell stemness. Aging (Albany NY) 2012; 4:393-401; PMID:22713507 [PMC free article] [PubMed]
[24] Ma T, Li J, Xu Y, Yu C, Xu T, Wang H, Liu K, Cao N, Nie BM, Zhu SY, et al. Atg5-independent autophagy regulates mitochondrial clearance and is essential for iPSC reprogramming. Nat Cell Biol 2015; 17:1379-87; PMID:26502054; http://dx.doi.org/10.1038/ncb3256 [PubMed] [Cross Ref]
[25] Wang S, Xia P, Ye B, Huang G, Liu J, Fan Z. Transient activation of autophagy via Sox2-mediated suppression of mTOR is an important early step in reprogramming to pluripotency. Cell Stem Cell 2013; 13:617-25; PMID:24209762; http://dx.doi.org/10.1016/j.stem.2013.10.005 [PubMed] [Cross Ref]
[26] Todd LR, Damin MN, Gomathinayagam R, Horn SR, Means AR, Sankar U. Growth factor erv1-like modulates Drp1 to preserve mitochondrial dynamics and function in mouse embryonic stem cells. Mol Biol Cell 2010; 21:1225-36; PMID:20147447; http://dx.doi.org/10.1091/mbc.E09-11-0937 [PMC free article] [PubMed] [Cross Ref]
[27] Deng H, Dodson MW, Huang H, Guo M. The Parkinson disease genes pink1 and parkin promote mitochondrial fission and/or inhibit fusion in Drosophila. Proc Natl Acad Sci U S A 2008; 105:14503-8; PMID:18799731; http://dx.doi.org/10.1073/pnas.0803998105 [PubMed] [Cross Ref]
[28] Poole AC, Thomas RE, Andrews LA, McBride HM, Whitworth AJ, Pallanck LJ. The PINK1/Parkin pathway regulates mitochondrial morphology. Proc Natl Acad Sci U S A 2008; 105:1638-43; PMID:18230723; http://dx.doi.org/10.1073/pnas.0709336105 [PubMed] [Cross Ref]
[29] Yang Y, Ouyang Y, Yang L, Beal MF, McQuibban A, Vogel H, Lu B. Pink1 regulates mitochondrial dynamics through interaction with the fission/fusion machinery. Proc Natl Acad Sci U S A 2008; 105:7070-5; PMID:18443288; http://dx.doi.org/10.1073/pnas.0711845105 [PubMed] [Cross Ref]
[30] Geisler S, Holmstrom KM, Skujat D, Fiesel FC, Rothfuss OC, Kahle PJ, Springer W. PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1. Nat Cell Biol 2010; 12:119-31; PMID:20098416; http://dx.doi.org/10.1038/ncb2012 [PubMed] [Cross Ref]
[31] Geisler S, Holmstrom KM, Treis A, Skujat D, Weber SS, Fiesel FC, Kahle PJ, Springer W. The PINK1/Parkin-mediated mitophagy is compromised by PD-associated mutations. Autophagy 2010; 6:871-8; PMID:20798600; http://dx.doi.org/10.4161/auto.6.7.13286 [PubMed] [Cross Ref]
[32] Springer W, Kahle PJ. Regulation of PINK1-Parkin-mediated mitophagy. Autophagy 2011; 7:266-78; PMID:21187721; http://dx.doi.org/10.4161/auto.7.3.14348 [PubMed] [Cross Ref]
[33] Koyano F, Okatsu K, Kosako H, Tamura Y, Go E, Kimura M, Kimura Y, Tsuchiya H, Yoshihara H, Hirokawa T, et al. Ubiquitin is phosphorylated by PINK1 to activate parkin. Nature 2014; 510:162-6; PMID:24784582 [PubMed]
[34] Lazarou M, Sliter DA, Kane LA, Sarraf SA, Wang C, Burman JL, Sideris DP, Fogel AI, Youle RJ. The ubiquitin kinase PINK1 recruits autophagy receptors to induce mitophagy. Nature 2015; 524:309-14; PMID:26266977; http://dx.doi.org/10.1038/nature14893 [PMC free article] [PubMed] [Cross Ref]
[35] Hasuwa H, Muro Y, Ikawa M, Kato N, Tsujimoto Y, Okabe M. Transgenic mouse sperm that have green acrosome and red mitochondria allow visualization of sperm and their acrosome reaction in vivo. Exp Anim 2010; 59:105-7; PMID:20224175; http://dx.doi.org/10.1538/expanim.59.105 [PubMed] [Cross Ref]
[36] Mizushima N, Yamamoto A, Matsui M, Yoshimori T, Ohsumi Y. In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker. Mol Biol Cell 2004; 15:1101-11; PMID:14699058; http://dx.doi.org/10.1091/mbc.E03-09-0704 [PMC free article] [PubMed] [Cross Ref]
[37] Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006; 126:663-76; PMID:16904174; http://dx.doi.org/10.1016/j.cell.2006.07.024 [PubMed] [Cross Ref]
[38] Vernia S, Cavanagh-Kyros J, Garcia-Haro L, Sabio G, Barrett T, Jung DY, Kim JK, Xu J, Shulha HP, Garber M, et al. The PPARalpha-FGF21 hormone axis contributes to metabolic regulation by the hepatic JNK signaling pathway. Cell Metab 2014; 20:512-25; PMID:25043817; http://dx.doi.org/10.1016/j.cmet.2014.06.010 [PMC free article] [PubMed] [Cross Ref]
[39] Zhao T, Zhang ZN, Westenskow PD, Todorova D, Hu Z, Lin T, Rong Z, Kim J, He J, Wang M, et al. Humanized mice reveal differential immunogenicity of cells derived from autologous induced pluripotent stem cells. Cell Stem Cell 2015; 17:353-9; PMID:26299572; http://dx.doi.org/10.1016/j.stem.2015.07.021 [PubMed] [Cross Ref]
[40] Zhao T, Zhang ZN, Rong Z, Xu Y. Immunogenicity of induced pluripotent stem cells. Nature 2011; 474:212-5; PMID:21572395; http://dx.doi.org/10.1038/nature10135 [PubMed] [Cross Ref]

Articles from Autophagy are provided here courtesy of Taylor & Francis