Mitochondrial Rejuvenation After Induced Pluripotency

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 maturatio

Video of time-lapse imaging of live FGFR2-eGFP embryos from E1.5 to E2.5.

Video of time-lapse imaging of live FGFR2-eGFP embryos from E1.5 to E2.5.</p

Pearson correlation values and respective two-tailed p-values of FGFR2-eGFP, NANOG and GATA6 fluorescence based on embryo cell number.

Pearson correlation values and respective two-tailed p-values of FGFR2-eGFP, NANOG and GATA6 fluorescence based on embryo cell number.</p

Mouse embryo development by embryonic day, morphological stage and approximate cell number, based on Mihajlovic and Bruce, 2017 [19].

Mouse embryo development by embryonic day, morphological stage and approximate cell number, based on Mihajlovic and Bruce, 2017 [19].</p

Treatment of FGFR2-eGFP embryos with BMP signaling inhibitors.

A) Representative images of embryos from each treatment group at E4.5 after immunostaining for eGFP (green), NANOG (grey) and SOX17 (red). Scale bar is equal to 47μm. B) Graphic representation of Pearson correlation values between eGFP, NANOG and SOX17 after BMP inhibitor treatments. Blue lozenges represent values from control group, red squares represent values from 7- oxo treated group and green triangles represent values from dorsomorphin treated embryos. C) Graphic representation of cell counts in E4.5 embryos after different treatments. D) Graphic representation of quantitative image analysis after immunostaining of eGFP, NANOG and SOX17. Different superscript letters (a,b) denote significant statistical differences between groups. Control n = 64 cells, 7-oxo n = 61 cells, Dorso n = 67 cells.</p

Correlation of FGFR2-eGFP, NANOG and GATA6 based on embryonic day.

A) Representative images of homozygous FGFR2-eGFP embryos at different embryonic days after immunostaining against GFP (green), NANOG (grey) and GATA6 (red). Scale bar is equal to 40μm. B) Graphic representation of Pearson correlation values observed from E3.5 to E4.5. Blue dots represent eGFP and NANOG correlation, red squares represent eGFP and GATA6 correlation and green triangles represent NANOG and GATA6 correlation. C) Graphic representation of linear regression analysis of fluorescence intensity levels. Blue dots and blue line represents NANOG cell measurements and regression analysis considering NANOG and eGFP levels respectively. Red dots and red line represents GATA6 cell measurements and regression analysis considering GATA6 and eGFP levels respectively. D) Dot plots depicting measured levels of NANOG and GATA6 in individual cells. E3.5 (n = 66 cells); E3.75 (n = 125); E4.0 (n = 119); E4.25 (n = 101) and E4.5 (n = 107).</p

Correlation of FGFR2-eGFP, NANOG and GATA6 based on cell number.

A) Graphic representation of Pearson correlation values observed in embryos with 32 to 64 cells (n = 111 cells), 65 to 90 cells (n = 196), 91–120 cells (n = 139) and 121–150 cells (n = 65). Blue dots represent eGFP and NANOG correlation, red squares represent eGFP and GATA6 correlation and green triangles represent NANOG and GATA6 correlation. B) Graphic representation of linear regression analysis of fluorescence intensity levels. Blue dots and blue line represent NANOG cell measurements and regression analysis considering NANOG and eGFP levels respectively. Red dots and red line represent GATA6 cell measurements and regression analysis considering GATA6 and eGFP levels respectively. C) Dot plots depicting measured levels of NANOG and GATA6 in individual cells.</p