Reprogramming of adult human dermal fibroblasts to induced dorsal forebrain precursor cells maintains aging signatures

Abstract

Introduction: With the increase in aging populations around the world, the development of in vitro human cell models to study neurodegenerative disease is crucial. A major limitation in using induced pluripotent stem cell (hiPSC) technology to model diseases of aging is that reprogramming fibroblasts to a pluripotent stem cell state erases age-associated features. The resulting cells show behaviors of an embryonic stage exhibiting longer telomeres, reduced oxidative stress, and mitochondrial rejuvenation, as well as epigenetic modifications, loss of abnormal nuclear morphologies, and age-associated features. Methods: We have developed a protocol utilizing stable, non-immunogenic chemically modified mRNA (cmRNA) to convert adult human dermal fibroblasts (HDFs) to human induced dorsal forebrain precursor (hiDFP) cells, which can subsequently be differentiated into cortical neurons. Analyzing an array of aging biomarkers, we demonstrate for the first time the effect of direct-to-hiDFP reprogramming on cellular age. Results: We confirm direct-to-hiDFP reprogramming does not affect telomere length or the expression of key aging markers. However, while direct-to-hiDFP reprogramming does not affect senescence-associated β-galactosidase activity, it enhances the level of mitochondrial reactive oxygen species and the amount of DNA methylation compared to HDFs. Interestingly, following neuronal differentiation of hiDFPs we observed an increase in cell soma size as well as neurite number, length, and branching with increasing donor age suggesting that neuronal morphology is altered with age. Discussion: We propose direct-to-hiDFP reprogramming provides a strategy for modeling age-associated neurodegenerative diseases allowing the persistence of age-associated signatures not seen in hiPSC-derived cultures, thereby facilitating our understanding of neurodegenerative disease and identification of therapeutic targets.

Keywords: DNA methylation; direct cell reprogramming; human induced dorsal forebrain precursors; induced pluripotent stem cell; neurodegenerative disease; oxidative stress; senescence; telomere length.

Figure 1

Phase contrast images of (A) HDFs and (B) resulting hiDFP colonies from 20/30-, 50- and 70-year-old subjects. Scale = 300 μm. HDF, human dermal fibroblast; hiDFP, human induced dorsal forebrain precursor.

Figure 2

Reprogrammed hiDFPs maintain aging-associated features. (A) SA-βgal activity assay of HDFs and hiDFPs. No statistical significance was determined by two-way mixed ANOVA. (B) Mitochondrial superoxide levels in HDFs and hiDFPs. Data represent mean ± SEM with n = 3. Simple main effect of reprogramming on mitochondrial superoxide levels determined by two-way mixed ANOVA with * for p < 0.05.

Figure 3

hiPSC reprogramming reverts aging-associated changes in gene expression, while reprogrammed hiDFPs maintain gene expression. (A) RANBP17; (B) PCDH10; (C) CDKN1A; (D) CDKN2A. Data represent mean ± SEM with n = 3. Significance was determined by one-way ANOVA with Tukey post-hoc test, with * for p < 0.05, ** for p ≤ 0.01, and *** for p ≤ 0.001.

Figure 4

hiPSC reprogramming reverts aging-associated changes in telomere length and gene expression, while reprogrammed hiDFPs maintain expression. (A) Relative telomere length; (B) TERC; (C) TERT. Data represent mean ± SEM with n = 3. Significance was determined by one-way ANOVA with Tukey post-hoc test with * for p < 0.05, ** for p ≤ 0.01.

Figure 5

Reprogrammed hiDFPs exhibit increased amounts of DNA methylation as measured by the production of 5-methylcytosine (5-mC). (A) Amount of methylated DNA in hiPSCs and hiDFPs relative to HDF. Significance was determined by one-way ANOVA with Tukey post-hoc test. (B) Amount of methylated DNA in HDFs and hiDFPs. A simple main effect of reprogramming irrespective of age group was determined by two-way mixed ANOVA. Data represent mean ± SEM with n = 3. * for p < 0.05, ** for p ≤ 0.01.

Figure 6

hiDFP-derived neurons exhibit complex neuronal morphologies displaying enhanced complexity with age. (A) Representative images of TUJ1+ hiDFP-derived neurons for each donor age group and at two differentiation time points Day 14 (D14) and Day 19 (D19). Scale: 50 μm. (B) Average cell soma size. (C) Average number of neurites per cell. (D) Average total neurite length per cell. (E) Average number of branch points per cell. (B–E) Data represent mean ± SEM with n = 30 cells per age group at each time point. Significance was determined by two-way ANOVA with pairwise comparisons following a significant interaction. A significant effect of age group within a given time point (D14 or D19) is denoted by * for p < 0.05, ** for p < 0.01, *** for p ≤ 0.001. A significant effect of differentiation time point (comparison of D14 to D19) within each age group is denoted by ^# for p < 0.05, ^## for p < 0.01, ^### for p ≤ 0.001.