Mitochondrial rejuvenation after induced pluripotency

Abstract

Background: As stem cells of the early embryo mature and differentiate into all tissues, the mitochondrial complement undergoes dramatic functional improvement. Mitochondrial activity is low to minimize generation of DNA-damaging reactive oxygen species during pre-implantation development and increases following implantation and differentiation to meet higher metabolic demands. It has recently been reported that when the stem cell type known as induced pluripotent stem cells (IPSCs) are re-differentiated for several weeks in vitro, the mitochondrial complement progressively re-acquires properties approximating input fibroblasts, suggesting that despite the observation that IPSC conversion "resets" some parameters of cellular aging such as telomere length, it may have little impact on other age-affected cellular systems such as mitochondria in IPSC-derived cells.

Methodology/principal findings: We have examined the properties of mitochondria in two fibroblast lines, corresponding IPSCs, and fibroblasts re-derived from IPSCs using biochemical methods and electron microscopy, and found a dramatic improvement in the quality and function of the mitochondrial complement of the re-derived fibroblasts compared to input fibroblasts. This observation likely stems from two aspects of our experimental design: 1) that the input cell lines used were of advanced cellular age and contained an inefficient mitochondrial complement, and 2) the re-derived fibroblasts were produced using an extensive differentiation regimen that may more closely mimic the degree of growth and maturation found in a developing mammal.

Conclusions/significance: These results - coupled with earlier data from our laboratory - suggest that IPSC conversion not only resets the "biological clock", but can also rejuvenate the energetic capacity of derived cells.

Figure 1. Morphological characteristics and marker expression of FIB, IPSC, and TER cells.

FIBA and derivatives are used as examples. A–C) Phase-contrast images showing the morphology of FIB, IPSC, and TER lines as labeled. Insets in A and C show reactivity of FIBA and TERA with human-specific nuclear (HuNu) antigen antibody (green). Blue is DAPI co-stained DNA. D) Graphical representation of the relative expression of pluripotency-related gene mRNAs in all cell lines (IPSC value set at 100%). Pluripotency marker genes were detected at low levels in FIB and TER cells compared to IPSCs. E–F) Immunocytochemical analysis of IPSCs (E) and TER cells (F), indicating that TER cells had not only lost high-level of expression of pluripotency markers such as Nanog, but also gained expression of fibroblasts markers such as fibronectin (FBN) (see also Ref. 7). Magnification: 800X, insets 250X.

Figure 2. Functional comparison of mitochondria in FIB, IPSC, TER and ESCs.

A) Total cellular ATP (fmoles/cell) in each cell type as labeled. B) ADP/ATP ratio in each cell type as labeled. C) Relative mitochondrial mass in tested cell types. Error bars indicate SEM. Letters above individual bars indicate values that differ statistically (P<0.05) from other cell type(s). (FIB = F, IPS = I, TER = T, ESC = E).

Figure 3. Analysis of mitochondrial membrane potential in FIB, IPSC, TER and ESCs.

A) Representative scatter plots from flow cytometry analysis showing red/green JC1 dye fluorescence in cell types as labeled. The outlined area R4 is red fluorescence and area R3 is green fluorescence. B) Bar graph showing the ratio of JC-1 red and green (R/G) fluorescence indicative of mitochondrial membrane potential for each cell type. Error bars indicate SEM. Letters above individual bars indicate values that differ statistically (P<0.05) from other cell type(s). (FIB = F, IPS = I, TER = T, ESC = E).

Figure 4. Structural comparison of mitochondria in FIB, IPS, TER, and ESC cell types.

A–C) Electron tomography of the mitochondrial configurations observed. Three primary configurations were observed: A) top panel: A central slice through a tomographic volume showing the orthodox configuration. bottom panel: Top and side views of the segmented and surface-rendered volume of the orthodox mitochondrion showing individual cristae in various colors (top view (upper) side view (lower)). B) As in A, for the condensed configuration. C) As in A,B for the ultracondensed configuration. Arrowheads point to greatly enlarged crista junctions. D–K) Representative TEMI of mitochondria in all cell lines. (Mag = 7500-10000X). FIB and IPSC lines displayed a mix of mitochondrial configuration, most of condensed configuration (FIBA (D), FIBB (E), IPSCA (F), IPSCB (G)). TER cells displayed a preponderance of orthodox mitochondria (TERA (H), TERB (I)), and native ESCs displayed a preponderance of condensed mitochondrial forms (ESCA (J), ESCB (K)). L) Bar graph of the ratio of mitochondria scored as orthodox or condensed configuration (O/C) in FIB, IPS, TER, and ESC cell types as labeled. Error bars  =  SEM. Letters above TER bar indicate statistical difference (P<0.03) from all three other cell types.