DNA damage responses in human pluripotent stem cells

Pluripotent stem cells have the capability to undergo unlimited self-renewal and differentiation into all somatic cell types. They have acquired specific adjustments in the cell cycle structure that allow them to rapidly proliferate, including cell cycle independent expression of cell cycle regulators and lax G1 to S phase transition. However, due to the developmental role of embryonic stem cells (ES) it is essential to maintain genomic integrity and prevent acquisition of mutations that would be transmitted to multiple cell lineages. Here we show that several modifications in DNA damage response of ES cells accommodate dynamic cycling and preservation of genetic information. ATM-dependent checkpoint signaling cascade is activated after irradiation of ES cells, and induces G2/M, but not G1/S cell cycle arrest. The absence of a G1/S cell cycle arrest promotes apoptotic response of damaged cells before DNA changes can be fixed in the form of mutation during the S phase, while G2/M cell cycle arrest allows repair of damaged DNA following replication. Human ES cells express higher level of DNA repair proteins, and rely on homologous recombination to repair double strand breaks. Radiation does not lead to long-term loss of pluripotency, since irradiated ES cells show transient decrease in the level of pluripotency factor transcripts, while protein levels remains stable. One week after irradiation, ES cells retain capacity to differentiate into three germ layers and form teratomas in immunocompromised mice.Similarly to ES cells, induced pluripotent stem (iPS) cells are poised to proliferate and exhibit extreme sensitivity to DNA damage, lack of G1/S cell cycle arrest, and express high level of DNA repair genes, suggesting that DNA damage responses are controlled by developmental state of the cell.Public health significance of this study originates in great promise that human ES and iPS cells hold in cell replacement therapies. Since human ES, and particularly iPS, cells represent potential source of cells for clinical and pharmaceutical applications, the DNA damage response pathways that maintain genomic integrity need to be studied in greater detail

Derivation, Characterization, and Neural Differentiation of Integration-Free Induced Pluripotent Stem Cell Lines from Parkinson's Disease Patients Carrying SNCA, LRRK2, PARK2, and GBA Mutations

We report generation of induced pluripotent stem cell (iPSC) lines from ten Parkinson's disease (PD) patients carrying SNCA, PARK2, LRRK2, and GBA mutations, and one age-matched control. After validation of pluripotency, long-term genome stability, and integration-free reprogramming, eight of these lines (one of each SNCA, LRRK2 and GBA, four PARK2 lines, and the control) were differentiated into neural stem cells (NSC) and subsequently to dopaminergic cultures. We did not observe significant differences in the timeline of neural induction and NSC derivation between the patient and control line, nor amongst the patient lines, although we report considerable variability in the efficiency of dopaminergic differentiation among patient lines. We performed whole genome expression analyses of the lines at each stage of differentiation (fibroblast, iPSC, NSC, and dopaminergic culture) in an attempt to identify alterations by large-scale evaluation. While gene expression profiling clearly distinguished cells at different stages of differentiation, no mutation-specific clustering or difference was observed, though consistent changes in patient lines were detected in genes associated mitochondrial biology. We further examined gene expression in a stress model (MPTP-induced dopaminergic neuronal death) using two clones from the SNCA triplication line, and detected changes in genes associated with mitophagy. Our data suggested that even a well-characterized line of a monogenic disease may not be sufficient to determine the cause or mechanism of the disease, and highlights the need to use more focused strategies for large-scale data analysis

DNA Damage Responses in Human Induced Pluripotent Stem Cells and Embryonic Stem Cells

BACKGROUND: Induced pluripotent stem (iPS) cells have the capability to undergo self-renewal and differentiation into all somatic cell types. Since they can be produced through somatic cell reprogramming, which uses a defined set of transcription factors, iPS cells represent important sources of patient-specific cells for clinical applications. However, before these cells can be used in therapeutic designs, it is essential to understand their genetic stability.\ud \ud METHODOLOGY/PRINCIPAL FINDINGS: Here, we describe DNA damage responses in human iPS cells. We observe hypersensitivity to DNA damaging agents resulting in rapid induction of apoptosis after γ-irradiation. Expression of pluripotency factors does not appear to be diminished after irradiation in iPS cells. Following irradiation, iPS cells activate checkpoint signaling, evidenced by phosphorylation of ATM, NBS1, CHEK2, and TP53, localization of ATM to the double strand breaks (DSB), and localization of TP53 to the nucleus of NANOG-positive cells. We demonstrate that iPS cells temporary arrest cell cycle progression in the G(2) phase of the cell cycle, displaying a lack of the G(1)/S cell cycle arrest similar to human embryonic stem (ES) cells. Furthermore, both cell types remove DSB within six hours of γ-irradiation, form RAD51 foci and exhibit sister chromatid exchanges suggesting homologous recombination repair. Finally, we report elevated expression of genes involved in DNA damage signaling, checkpoint function, and repair of various types of DNA lesions in ES and iPS cells relative to their differentiated counterparts.\ud \ud CONCLUSIONS/SIGNIFICANCE: High degrees of similarity in DNA damage responses between ES and iPS cells were found. Even though reprogramming did not alter checkpoint signaling following DNA damage, dramatic changes in cell cycle structure, including a high percentage of cells in the S phase, increased radiosensitivity and loss of DNA damage-induced G(1)/S cell cycle arrest, were observed in stem cells generated by induced pluripotency.\ud \u

Kinome rewiring reveals AURKA limits PI3K-pathway inhibitor efficacy in breast cancer.

Dysregulation of the PI3K-AKT-mTOR signaling network is a prominent feature of breast cancers. However, clinical responses to drugs targeting this pathway have been modest, possibly because of dynamic changes in cellular signaling that drive resistance and limit drug efficacy. Using a quantitative chemoproteomics approach, we mapped kinome dynamics in response to inhibitors of this pathway and identified signaling changes that correlate with drug sensitivity. Maintenance of AURKA after drug treatment was associated with resistance in breast cancer models. Incomplete inhibition of AURKA was a common source of therapy failure, and combinations of PI3K, AKT or mTOR inhibitors with the AURKA inhibitor MLN8237 were highly synergistic and durably suppressed mTOR signaling, resulting in apoptosis and tumor regression in vivo. This signaling map identifies survival factors whose presence limits the efficacy of targeted therapies and reveals new drug combinations that may unlock the full potential of PI3K-AKT-mTOR pathway inhibitors in breast cancer

Microarray gene expression data quality control.

<p>A: the number of genes detected at p-value < 0.05 (red line) and p-value < 0.01 (blue line). Detection p-value is a measurement of confidence that a given transcript is expressed above the background level. B: Sample quality assessment by comparison of 95^th signal intensity values (red line) and signal-to-noise ratio (blue line) across samples. Signal-to-noise ratio is calculated as a ration of 95^th and 5^th percentile (p95/p05) in non-normalized data. C: Hierarchical clustering of samples after normalization and averaging of biological replicates.</p

Pluripotency verification of PD patient iPSC.

<p>A: Pluripotency score for iPSC lines A6, B119, I3, K20, P1, S110, T101,L1 and Y09. ESC H9 line served as a positive control, whereas fibroblasts from the healthy subject (Y line) were used as a negative control. Pluripotency range is depicted in red, whereas non-pluripotent range is shown in blue. B: Novelty score for tested samples. Low novelty score (green bars) is characteristic for pluripotent cell lines, whereas high novelty sore (red) highlights sample that deviated from the pluripotent transcriptional signature. C: Hierarchical clustering of all samples. D: Combination of pluripotency and novelty scores illustrates that iPSC and ESC samples are grouped together (red background—high pluripotency and low novelty scores). Y fibroblast line had the opposite result (blue background—low pluripotency and high novelty scores).</p

Genes up or down regulated in all PD-patient specific dopaminergic cultures relative to control.

<p>Genes up or down regulated in all PD-patient specific dopaminergic cultures relative to control.</p

Neural and dopaminergic differentiation.

<p>A-B: Immunocytochemistry in <i>SNCA</i> triplication (A6) NSCs with antibodies against NSC markers SOX1, NESTIN, and PAX6. C-D: Immunocytochemistry for dopaminergic (TH and LMX1A), and midbrain (FOXA2) markers. Scale bar as marked.</p