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Senescence-associated ultrastructural features of long-term cultures of induced pluripotent stem cells (iPSCs)

Colasuonno, Fiorella, Borghi, Rossella, Niceforo, Alessia, Muzzi, Maurizio, Bertini, Enrico, Di Giulio, Andrea, Moreno, Sandra, Compagnucci, Claudia·2017·Aging
DOI 10.18632/aging.101309PMC PMC5680563
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Figure 1 Bright field images, acquired at the inverted microscope of y-iPSCs (a), ma-iPSCs (b,d) and a-iPSCs (c,e). Scale bars, 20 μm (a-c), 10 μm (d,e).
Figure 1 Bright field images, acquired at the inverted microscope of y-iPSCs (a), ma-iPSCs (b,d) and a-iPSCs (c,e). Scale bars, 20 μm (a-c), 10 μm (d,e).CC BY · PubMed Central
Figure 2 FIB/SEM micrographs of y-iPSCs (a,b), ma-iPSCs (c,d) and a-iPSCs (e,f). Images have been electronically colored to highlight nuclei (in blue), mitochondria (in pink), and autophagosomes (in green). Arrowheads, gap junctions; asterisks, tight junctions; G, Golgi apparatus; RER, rough endoplasmic reticulum.
Figure 2 FIB/SEM micrographs of y-iPSCs (a,b), ma-iPSCs (c,d) and a-iPSCs (e,f). Images have been electronically colored to highlight nuclei (in blue), mitochondria (in pink), and autophagosomes (in green). Arrowheads, gap junctions; asterisks, tight junctions; G, Golgi apparatus; RER, rough endoplasmic reticulum.CC BY · PubMed Central
Figure 3 FIB/SEM micrographs of y-, ma- and a-iPSCs showing changes in mitochondrial structure during culturing time. Mitochondria with disorganized cristae are indicated by the asterisks in ma- and a-iPSCs. N, nuclei Scale bars, 1μm.
Figure 3 FIB/SEM micrographs of y-, ma- and a-iPSCs showing changes in mitochondrial structure during culturing time. Mitochondria with disorganized cristae are indicated by the asterisks in ma- and a-iPSCs. N, nuclei Scale bars, 1μm.CC BY · PubMed Central
Figure 4 FIB/SEM images showing ultrastructural details of ma- (a-c) and a-iPSCs (d-f). Arrows in b,c,e indicate autophagosomes containing mitochondria (m) or other partially digested cytoplasmic material. E, exosomes; N, nuclei; (TGN, trans-Golgi network; RER, rough endoplasmatic reticulum).
Figure 4 FIB/SEM images showing ultrastructural details of ma- (a-c) and a-iPSCs (d-f). Arrows in b,c,e indicate autophagosomes containing mitochondria (m) or other partially digested cytoplasmic material. E, exosomes; N, nuclei; (TGN, trans-Golgi network; RER, rough endoplasmatic reticulum).CC BY · PubMed Central
Figure 5 Autophagy is increased in ma-iPSCs when compared with y- and a-iPSCs. Western blot analysis of LC3 I/II, and alpha-tubulin as loading control is shown. Bar graph shows the LC3–II/I ratio and represents the mean± SD of three experiments.
Figure 5 Autophagy is increased in ma-iPSCs when compared with y- and a-iPSCs. Western blot analysis of LC3 I/II, and alpha-tubulin as loading control is shown. Bar graph shows the LC3–II/I ratio and represents the mean± SD of three experiments.CC BY · PubMed Central
Figure 6 Confocal microscopic images of y-, ma- and a-iPSCs marked with p62 (in red) and Hoechst (in blue) showing abundant granular p62 immunopositivity in ma-iPSCs and in a-iPSCs when compared with y-iPSCs.
Figure 6 Confocal microscopic images of y-, ma- and a-iPSCs marked with p62 (in red) and Hoechst (in blue) showing abundant granular p62 immunopositivity in ma-iPSCs and in a-iPSCs when compared with y-iPSCs.CC BY · PubMed Central
Figure 7 Confocal microscopy of y-, ma- and a-iPSCs immunostained with Beclin1 (in green), Lamp2 (in red) and Hoechst (in blue). In ma-iPSCs the central cell shows a granular distribution of Lamp2, while the arrow indicates a cell with a perinuclear staining similar to those observed in a-iPSCs.
Figure 7 Confocal microscopy of y-, ma- and a-iPSCs immunostained with Beclin1 (in green), Lamp2 (in red) and Hoechst (in blue). In ma-iPSCs the central cell shows a granular distribution of Lamp2, while the arrow indicates a cell with a perinuclear staining similar to those observed in a-iPSCs.CC BY · PubMed Central
Figure 8 Confocal microscopy of y- and a-iPSCs stained with anti-p62 antibody (in red), anti-Beclin1 (in green), Lamp2 (in red) and Hoechst (in blue), treated with rapamycin alone or in combination with bafilomycin.
Figure 8 Confocal microscopy of y- and a-iPSCs stained with anti-p62 antibody (in red), anti-Beclin1 (in green), Lamp2 (in red) and Hoechst (in blue), treated with rapamycin alone or in combination with bafilomycin.CC BY · PubMed Central
Figure 9 Schematic diagram of biological features observed in y-iPSCs, ma- (following 6 months of culture) and a-iPSCs (In particular, we highlighted the mitochondrial disruption, the progressive loss of cell- cell contacts, the presence of autophagosomes and extracellular vesicles with aging. m, mitochondria; N, nucleus; E, extracellular vesicles; A, autophagosomes.
Figure 9 Schematic diagram of biological features observed in y-iPSCs, ma- (following 6 months of culture) and a-iPSCs (In particular, we highlighted the mitochondrial disruption, the progressive loss of cell- cell contacts, the presence of autophagosomes and extracellular vesicles with aging. m, mitochondria; N, nucleus; E, extracellular vesicles; A, autophagosomes.CC BY · PubMed Central

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Citations

Cites

  • Feedback between p21 and reactive oxygen production is necessary for cell senescence

    Passos, J. F., Nelson, G., Wang, C., Richter, T., Simillion, C., Proctor, C. J., Miwa, S., Olijslagers, S., Hallinan, J., Wipat, A., et al. (2010) · Molecular Systems Biology · doi:10.1038/msb.2010.5 · Cited by 1,019 · cited 1×

    “These results are in line with previous data from ours and other Authors’ groups reporting. increased mitochondrial biogenesis [7, 23], and are possibly due to adaptation of the cells to the culture environment (5% CO 2 and 21% O 2 ).”

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Metadata

Authors
Colasuonno, F., Borghi, R., Niceforo, A., Muzzi, M., Bertini, E., Di Giulio, A., Moreno, S., & Compagnucci, C.
Affiliations
  • Department of Science, LIME, University "Roma Tre", Rome 00146, Italy
  • Department of Neuroscience, Unit of Neuromuscular and Neurodegenerative Diseases, Laboratory of Molecular Medicine, Bambino Gesu' Children's Research Hospital, IRCCS, Rome 00146, Italy
Venue
Aging
Publisher
Impact Journals, LLC
Published
2017-10-23
Volume
9
Issue
10
Pages
2209-2222
ISSN
1945-4589
DOI
10.18632/aging.101309
PMC
PMC5680563
Cited by
13
References
1
Language
en

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