Vladimír Rotrekl

Base excision repair, aging and health span

DNA damage and mutagenesis are suggested to contribute to aging through their ability to mediate cellular dysfunction. The base excision repair (BER) pathway ameliorates a large number of DNA lesions that arise spontaneously. Many of these lesions are reported to increase with age. Oxidized guanine, repaired largely via base excision repair, is particularly well studied and shown to increase with age. Spontaneous mutant frequencies also increase with age which suggests that mutagenesis may contribute to aging. It is widely accepted that genetic instability contributes to age-related occurrences of cancer and potentially other age-related pathologies. BER activity decreases with age in multiple tissues. The specific BER protein that appears to limit activity varies among tissues. DNA polymerase-beta is reduced in brain from aged mice and rats while AP endonuclease is reduced in spermatogenic cells obtained from old mice. The differences in proteins that appear to limit BER activity among tissues may represent true tissue-specific differences in activity or may be due to differences in techniques, environmental conditions or other unidentified differences among the experimental approaches. Much remains to be addressed concerning the potential role of BER in aging and age-related health span.

Human Pluripotent Stem Cell-Derived Cardiomyocytes as Research and Therapeutic Tools

Human pluripotent stem cells (hPSCs), namely, embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), with their ability of indefinite self-renewal and capability to differentiate into cell types derivatives of all three germ layers, represent a powerful research tool in developmental biology, for drug screening, disease modelling, and potentially cell replacement therapy. Efficient differentiation protocols that would result in the cell type of our interest are needed for maximal exploitation of these cells. In the present work, we aim at focusing on the protocols for differentiation of hPSCs into functional cardiomyocytes in vitro as well as achievements in the heart disease modelling and drug testing on the patient-specific iPSC-derived cardiomyocytes (iPSC-CMs).

Forced aggregation and defined factors allow highly uniform-sized embryoid bodies and functional cardiomyocytes from human embryonic and induced pluripotent stem cells.

In vitro human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) can differentiate into functional cardiomyocytes (CMs). Protocols for cardiac differentiation of hESCs and hiPSCs include formation of the three-dimensional cell aggregates called embryoid bodies (EBs). The traditional suspension method for EB formation from clumps of cells results in an EB population heterogeneous in size and shape. In this study we show that forced aggregation of a defined number of single cells on AggreWell plates gives a high number of homogeneous EBs that can be efficiently differentiated into functional CMs by application of defined growth factors in the media. For cardiac differentiation, we used three hESC lines and one hiPSC line. Our contracting EBs and the resulting CMs express cardiac markers, namely myosin heavy chain α and β, cardiac ryanodine receptor/calcium release channel, and cardiac troponin T, shown by real-time polymerase chain reaction and immunocytochemistry. Using Ca2+ imaging and atomic force microscopy, we demonstrate the functionality of RyR2 to release Ca2+ from the sarcoplasmic reticulum as well as reliability in contractile and beating properties of hESC-EBs and hiPSC-EBs upon the stimulation or inhibition of the β-adrenergic pathway.

Mutagenesis Is Elevated in Male Germ Cells Obtained from DNA Polymerase-beta Heterozygous Mice

Abstract Gametes carry the DNA that will direct the development of the next generation. By compromising genetic integrity, DNA damage and mutagenesis threaten the ability of gametes to fulfill their biological function. DNA repair pathways function in germ cells and serve to ameliorate much DNA damage and prevent mutagenesis. High base excision repair (BER) activity is documented for spermatogenic cells. DNA polymerase-beta (POLB) is required for the short-patch BER pathway. Because mice homozygous null for the Polb gene die soon after birth, mice heterozygous for Polb were used to examine the extent to which POLB contributes to maintaining spermatogenic genomic integrity in vivo. POLB protein levels were reduced only in mixed spermatogenic cells. In vitro short-patch BER activity assays revealed that spermatogenic cell nuclear extracts obtained from Polb heterozygous mice had one third the BER activity of age-matched control mice. Polb heterozygosity had no effect on the BER activities of somatic tissues tested. The Polb heterozygous mouse line was crossed with the lacI transgenic Big Blue mouse line to assess mutant frequency. The spontaneous mutant frequency for mixed spermatogenic cells prepared from Polb heterozygous mice was 2-fold greater than that of wild-type controls, but no significant effect was found among the somatic tissues tested. These results demonstrate that normal POLB abundance is necessary for normal BER activity, which is critical in maintaining a low germline mutant frequency. Notably, spermatogenic cells respond differently than somatic cells to Polb haploinsufficiency..

Decrease in Abundance of Apurinic/Apyrimidinic Endonuclease Causes Failure of Base Excision Repair in Culture-Adapted Human Embryonic Stem Cells

The inevitable accumulation of chromosomal abnormalities in human embryonic stem cells (hESCs) during in vitro expansion represents a considerable obstacle for cell replacement therapies. To determine the source of chromosomal abnormalities, we examined hESCs maintained in culture for over 55 months for defects in telomere maintenance and DNA repair. Although prolonged culture affected neither telomerase activity nor nonhomologous end joining, the efficiency of base excision repair (BER) was significantly decreased and correlated with reduced expression of apurinic/apyrimidinic endonuclease 1 (APE1), the major nuclease required for BER. Interestingly, the expression of other BER enzymes was unchanged. Addition of human recombinant APE1 protein to nuclear extracts from late passage hESCs increased BER efficiency to the level typical of early passage hESCs. The link between BER and double‐strand breaks (DSB) was demonstrated by decreased DSB release after downregulation of APE1 in early passage hESCs via siRNA. Correspondingly lower APE1 level in late passage hESC resulted in slower and less intensive but long lasting DSB release upon ionizing radiation (IR). Downregulation of APE1 in early passage hESCs also led to approximately 30% decrease in γ‐H2AX signaling following IR, similar to that in late passage hESCs. We suggest that downregulation of APE1 significantly contributes to the failure of BER during long‐term culture of hESCs, and further that BER failure is one of the factors affecting the genomic instability of hESCs by altering BER‐dependent DSB release and cell cycle/checkpoint signaling. STEM CELLS 2013;31:693–702

Atomic force microscopy combined with human pluripotent stem cell derived cardiomyocytes for biomechanical sensing.

Cardiomyocyte contraction and relaxation are important parameters of cardiac function altered in many heart pathologies. Biosensing of these parameters represents an important tool in drug development and disease modeling. Human embryonic stem cells and especially patient specific induced pluripotent stem cell-derived cardiomyocytes are well established as cardiac disease model.. Here, a live stem cell derived embryoid body (EB) based cardiac cell syncytium served as a biorecognition element coupled to the microcantilever probe from atomic force microscope thus providing reliable micromechanical cellular biosensor suitable for whole-day testing. The biosensor was optimized regarding the type of cantilever, temperature and exchange of media; in combination with standardized protocol, it allowed testing of compounds and conditions affecting the biomechanical properties of EB. The studied effectors included calcium , drugs modulating the catecholaminergic fight-or-flight stress response such as the beta-adrenergic blocker metoprolol and the beta-adrenergic agonist isoproterenol. Arrhythmogenic effects were studied using caffeine. Furthermore, with EBs originating from patients stem cells, this biosensor can help to characterize heart diseases such as dystrophies.

Mutation Frequency Dynamics in HPRT Locus in Culture-Adapted Human Embryonic Stem Cells and Induced Pluripotent Stem Cells Correspond to Their Differentiated Counterparts

The genomic destabilization associated with the adaptation of human embryonic stem cells (hESCs) to culture conditions or the reprogramming of induced pluripotent stem cells (iPSCs) increases the risk of tumorigenesis upon the clinical use of these cells and decreases their value as a model for cell biology studies. Base excision repair (BER), a major genomic integrity maintenance mechanism, has been shown to fail during hESC adaptation. Here, we show that the increase in the mutation frequency (MF) caused by the inhibition of BER was similar to that caused by the hESC adaptation process. The increase in MF reflected the failure of DNA maintenance mechanisms and the subsequent increase in MF rather than being due solely to the accumulation of mutants over a prolonged period, as was previously suggested. The increase in the ionizing-radiation-induced MF in adapted hESCs exceeded the induced MF in nonadapted hESCs and differentiated cells. Unlike hESCs, the overall DNA maintenance in iPSCs, which was reflected by the MF, was similar to that in differentiated cells regardless of the time spent in culture and despite the upregulation of several genes responsible for genome maintenance during the reprogramming process. Taken together, our results suggest that the changes in BER activity during the long-term cultivation of hESCs increase the mutagenic burden, whereas neither reprogramming nor long-term propagation in culture changes the MF in iPSCs.

The effect of the sphingosine-1-phosphate analogue FTY720 on atrioventricular nodal tissue

The sphingosine‐1‐phosphate (S1P) receptor modulator, fingolimod (FTY720), has been used for the treatment of patients with relapsing forms of multiple sclerosis, but atrioventricular (AV) conduction block have been reported in some patients after the first dose. The underlying mechanism of this AV node conduction blockade is still not well‐understood. In this study, we hypothesize that expression of this particular arrhythmia might be related to a direct effect of FTY720 on AV node rather than a parasympathetic mimetic action. We, therefore, investigated the effect of FTY720 on AV nodal, using in vitro rat model preparation, under both basal as well as ischaemia/reperfusion conditions. We first look at the expression pattern of S1P receptors on the AV node using real‐time PCR. Although all three S1P receptor isoforms were expressed in AVN tissues, S1P1 receptor isoform expression level was higher than S1P2 and S1P3. The effect of 25 nM FTY720 on cycle length (CL) was subsequently studied via extracellular potentials recordings. FTY720 caused a mild to moderate prolongation in CL by an average 9% in AVN (n = 10, P < 0.05) preparations. We also show that FTY720 attenuated both ischaemia and reperfusion induced AVN rhythmic disturbance. To our knowledge, these remarkable findings have not been previously reported in the literature, and stress the importance for extensive monitoring period in certain cases, especially in patients taking concurrently AV node blocker agents.

TEC controls pluripotency and early cell fate decisions of human pluripotent stem cells via regulation of FGF2 secretion

Human pluripotent stem cells (hPSC) require signaling provided by fibroblast growth factor (FGF) receptors. This can be initiated by the recombinant FGF2 ligand supplied exogenously, but hPSC further support their niche by secretion of endogenous FGF2. In this study, we describe a role of tyrosine kinase expressed in hepatocellular carcinoma (TEC) kinase in this process. We show that TEC‐mediated FGF2 secretion is essential for hPSC self‐renewal, and its lack mediates specific differentiation. Following both short hairpin RNA‐ and small interfering RNA‐mediated TEC knockdown, hPSC secretes less FGF2. This impairs hPSC proliferation that can be rescued by increasing amounts of recombinant FGF2. TEC downregulation further leads to a lower expression of the pluripotency markers, an improved priming towards neuroectodermal lineage, and a failure to develop cardiac mesoderm. Our data thus demonstrate that TEC is yet another regulator of FGF2‐mediated hPSC pluripotency and differentiation. Stem Cells 2017;35:2050–2059

Tyrosine Kinase Expressed in Hepatocellular Carcinoma, TEC, Controls Pluripotency and Early Cell Fate Decisions of Human Pluripotent Stem Cells via Regulation of Fibroblast Growth Factor-2 Secretion

Human pluripotent stem cells (hPSC) require signaling provided by fibroblast growth factor (FGF) receptors. This can be initiated by the recombinant FGF2 ligand supplied exogenously, but hPSC further support their niche by secretion of endogenous FGF2. In this study, we describe a role of tyrosine kinase expressed in hepatocellular carcinoma (TEC) kinase in this process. We show that TEC‐mediated FGF2 secretion is essential for hPSC self‐renewal, and its lack mediates specific differentiation. Following both short hairpin RNA‐ and small interfering RNA‐mediated TEC knockdown, hPSC secretes less FGF2. This impairs hPSC proliferation that can be rescued by increasing amounts of recombinant FGF2. TEC downregulation further leads to a lower expression of the pluripotency markers, an improved priming towards neuroectodermal lineage, and a failure to develop cardiac mesoderm. Our data thus demonstrate that TEC is yet another regulator of FGF2‐mediated hPSC pluripotency and differentiation. Stem Cells 2017;35:2050–2059