Establishment of Induced Pluripotent Stem Cells from Centenarians for Neurodegenerative Disease Research

<div><p>Induced pluripotent stem cell (iPSC) technology can be used to model human disorders, create cell-based models of human diseases, including neurodegenerative diseases, and in establishing therapeutic strategies. To detect subtle cellular abnormalities associated with common late-onset disease in iPSCs, valid control iPSCs derived from healthy donors free of serious late-onset diseases are necessary. Here, we report the generation of iPSCs from fibroblasts obtained immediately postmortem from centenarian donors (106- and 109-years-old) who were extremely healthy until an advanced age. The iPSCs were generated using a conventional method involving OCT4, SOX2, KLF4, and c-MYC, and then differentiated into neuronal cells using a neurosphere method. The expression of molecules that play critical roles in late-onset neurodegenerative diseases by neurons differentiated from the centenarian-iPSCs was compared to that of neurons differentiated from iPSCs derived from familial Alzheimer's disease and familial Parkinson's disease (PARK4: triplication of the α synuclein gene) patients. The results indicated that our series of iPSCs would be useful in neurodegeneration research. The iPSCs we describe, which were derived from donors with exceptional longevity who were presumed to have no serious disease risk factors, would be useful in longevity research and as valid super-controls for use in studies of various late-onset diseases.</p> </div

Expression of α-synuclein and tau by neurons differentiated from iPSCs 100–1 #8 and 100–1 #16, and the neurodegenerative disease-specific iPSCs.

<p>(A) Western blotting of α-synuclein and tau protein in the seven lines of neurons differentiated from iPSCs. Note that the level of α-synuclein expression by neurons differentiated from the PARK4-4 iPSCs is markedly increased. (B and C) Immunoblots were scanned and analyzed using densitometry. The level of α-synuclein (B) and tau (C) expression was normalized to the internal control α-tubulin. The histograms show expression relative to expression by 201B7. Data are from three independent experiments and are expressed as the mean ± standard deviation. Asterisks indicate a significant difference versus wild type as determined by one-way analysis of variance followed by Tukey-Kramer's post hoc test (<i>P</i> = 0.05).</p

Characterization of the neurodegeneration-related molecules Aβ, α-synuclein, and tau protein in neurons differentiated from centenarian-iPSCs.

<p>(A) Secretion of Aβ40 and Aβ42 from neurons differentiated from iPSCs 100–1 #8 and 100–1 #16, and the neurodegenerative disease-specific iPSCs PARK4-4, PARK4-14, PS1-4, and PS2-2. (B) The ratio of Aβ42/Aβ40 secreted from neurons differentiated from 100–1 iPSCs and neurodegenerative disease-specific iPSCs. The ratio of Aβ42/Aβ40 secreted by neurons differentiated from both the PS1 and PS2 iPSCs was significantly higher than that of neurons differentiated from the other iPSCs. Significant differences among groups were examined using one-way analysis of variance followed by Tukey-Kramer's post hoc test (*<i>P</i><0.05).</p

Differentiation of centenarian-iPSCs into neurons.

<p>(A) Neural differentiation of iPSCs 100–1 #8 and 100–1 #16. Representative images of immunocytochemical staining for the early neuronal marker βIII-tubulin following neural differentiation. (B) Confocal images of co-staining with the mature neuron marker MAP-2 and the dopaminergic and noradrenergic neuronal marker tyrosine hydroxylase (TH). Inserts show high magnifications of dotted white squares. Bar  = 20 μm. Cells were counterstained with DAPI (blue).</p

Generation of 100–1 and 100–2 iPSCs from centenarian donor fibroblasts.

<p>(A) Both the 100–1 and 100–2 iPSC lines exhibit markers of pluripotency. All iPSCs expressed the pluripotency markers Tra-1–60, Tra-1–81, SSEA3, and SSEA4. Nuclei were stained with DAPI. Bar  = 200 μm. (B) RT-PCR analysis of the transgenes OCT3/4, SOX2, KLF4, c-MYC and the endogenous hESC marker genes. Donor fibroblasts examined 6 days after transduction with the retroviruses are positive for the transgenes. RNA was extracted from the 100–1 and 100–2 iPSCs at passage 10.</p

Analysis of centenarian-iPSC telomere length.

<p>Southern blot showing telomere length in 100–1 #8 (passage 10) and 100–1 #16 (passage 12) iPSCs compared to that of their parental fibroblast (passage 5). HT1080 served as a control. TRF lengths were calculated by comparing the electromobility of sample TRFs to that of makers. Values are displayed in kilobases (kb) below the figure.</p

Teratomas derived from SCID mice injected with 100–1 and 100–2 iPSCs.

<p>Gross morphology and hematoxylin and eosin staining of representative teratomas generated from iPSCs 100–1 #8, 100–1 #16, and 100–2 #1. Tissues representing all 3 embryonic germ layers, including pigmented epithelium (ectoderm), cartilage (mesoderm), and glandular structure (endoderm) are shown. Bar  = 50 μm.</p

Additional file 1: of Heterogeneous ribonuclear protein A3 (hnRNP A3) is present in dipeptide repeat protein containing inclusions in Frontotemporal Lobar Degeneration and Motor Neurone disease associated with expansions in C9orf72 gene

Selected clinical, pathological and genetic details on cases studied. (XLSX 17 kb