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

Abstract

Induced pluripotent stem cells (iPSCs) hold great promise for developing personalized regenerative medicine, however characterization of their biological features is still incomplete. Moreover, changes occurring in long-term cultured iPSCs have been reported, suggesting these as a model of cellular aging. For this reason, we addressed the ultrastructural characterization of iPSCs, with a focus on possible time-dependent changes, involving specific cell compartments. To this aim, we comparatively analysed cultures at different timepoints, by an innovative electron microscopic technology (FIB/SEM). We observed progressive loss of cell-to-cell contacts, associated with increased occurrence of exosomes. Mitochondria gradually increased, while acquiring an elongated shape, with well-developed cristae. Such mitochondrial maturation was accompanied by their turnover, as assessed by the presence of autophagomes (undetectable in young iPSCs), some containing recognizable mitochondria. This finding was especially frequent in middle-aged iPSCs, while being occasional in aged cells, suggesting early autophagic activation followed by a decreased efficiency of the process with culturing time. Accordingly, confocal microscopy showed age-dependent alterations to the expression and distribution of autophagic markers. Interestingly, responsivity to rapamycin, highest in young iPSCs, was almost lost in aged cells. Overall, our results strongly support long-term cultured iPSCs as a model for studying relevant aspects of cellular senescence, involving intercellular communication, energy metabolism, and autophagy.

Keywords: FIB/SEM; aging; autophagy; cell-cell contacts; iPSCs; mitochondria.

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 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 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 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 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 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 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 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 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.