Publications by authors named "Aleš Hampl"

96 Publications

Direct and Indirect Biomimetic Peptide Modification of Alginate: Efficiency, Side Reactions, and Cell Response.

Int J Mol Sci 2021 May 27;22(11). Epub 2021 May 27.

Institute of Macromolecular Chemistry, Czech Academy of Sciences, Heyrovsky Sq. 2, 16206 Prague, Czech Republic.

In the fast-developing field of tissue engineering there is a constant demand for new materials as scaffolds for cell seeding, which can better mimic a natural extracellular matrix as well as control cell behavior. Among other materials, polysaccharides are widely used for this purpose. One of the main candidates for scaffold fabrication is alginate. However, it lacks sites for cell adhesion. That is why one of the steps toward the development of suitable scaffolds for cells is the introduction of the biofunctionality to the alginate structure. In this work we focused on bone-sialoprotein derived peptide (TYRAY) conjugation to the molecule of alginate. Here the comparison study on four different approaches of peptide conjugation was performed including traditional and novel modification methods, based on 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide/-hydroxy succinimide (EDC/NHS), 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (DMTMM), thiol-Michael addition and Cu-catalyzed azide-alkyne cycloaddition reactions. It was shown that the combination of the alginate amidation with the use of and subsequent Cu-catalyzed azide-alkyne cycloaddition led to efficient peptide conjugation, which was proven with both NMR and XPS methods. Moreover, the cell culture experiment proved the positive effect of peptide presence on the adhesion of human embryonic stem cells.
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http://dx.doi.org/10.3390/ijms22115731DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8198284PMC
May 2021

Aspects of embryo selection and their preparation for the formation of human embryonic stem cells intended for human therapy.

Ceska Gynekol 2021 ;86(1):5-10

Objective: The work deals with a clinical part of human embryonic stem cell (hESC) research. The aim of the project is the differentiation of somatic cell types, useful in drug development, regenerative medicine and cell therapy. The aim of this work is to enable targeted therapy of yet incurable diseases. The pluripotent hESCs have unlimited self-renewal capacity. This ability is used in therapy to create missing or damaged cells in the human body. It is of interest to develop clinical-grade hESC lines useful in preclinical and clinical studies.

Methods: The derivation of the hESC must respect the legislation of the Czech Republic and the EU. The aim was to develop an informed consent of both donors for donated discarded embryos that are not suitable for treatment by in vitro fertilization according to Directive 2004/23/EC. The FNBs Center for Assisted Reproduction (CAR) participates in oocyte collection, cultivation and cryopreservation of embryos, communication with clients and ensuring the informed consent of embryo donors. A transport protocol and a methodology for handing over the thawed embryos with the original numerical code were developed. Before the embryos are handed over to the ICRC co-authors workplace (CTEF), they are thawed and, if necessary, recultivated to the blastocyst stage; afterwards, assisted hatching is performed.

Results: In the period from January 2018 to July 2020, 138 selected suitable clients were asked for donations, with 52 not responding, 19 terminating and 29 extending the embryo storage. Only 38 clients, i.e. 27.5%, agreed with the usage of their embryos for the preparation of hESCs. In the same period, personal communication with suitable CAR clients took place and another 17 embryo donors were obtained. A total of 160 embryos were obtained from 55 donors aged 26 to 42 years. The embryos were most often frozen in the blastocyst (53 embryos - 33.1%) and morula (74 embryos - 46.3%) stages. Of the 29 genetically examined embryos, only 5 are euploid (17.2%), 2 are mosaic and 22 are aneuploid or with translocations or carriers with a monogenic defect.

Conclusion: We have an informed consent prepared and approved by the Ethics Committee of the Masaryk University and the University Hospital Brno; 160 donated embryos have been selected and secured. A transport protocol and handover methodology are developed. The plan for the transfer of thawed anonymized embryos in the first phase, October - December 2020, includes approximately 5 thawed blastocysts per week with assisted hatching. After their transfer to the CTEF, the embryoblast will be isolated with subsequent cultivation. The established hESCs must meet the specified criteria of safety, stability and pluripotency. We believe that, in accordance with the project plan, we will obtain at least 3 clinical-grade hESC lines, the first created in the Czech Republic, respecting the requirements for Advanced Medicinal Therapy Products   (AMTP).
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http://dx.doi.org/10.48095/cccg20215DOI Listing
March 2021

AC Pulsed Field Ablation Is Feasible and Safe in Atrial and Ventricular Settings: A Proof-of-Concept Chronic Animal Study.

Front Bioeng Biotechnol 2020 3;8:552357. Epub 2020 Dec 3.

International Clinical Research Center, St. Anne's University Hospital Brno, Brno, Czechia.

Introduction: Pulsed field ablation (PFA) exploits the delivery of short high-voltage shocks to induce cells death via irreversible electroporation. The therapy offers a potential paradigm shift for catheter ablation of cardiac arrhythmia. We designed an AC-burst generator and therapeutic strategy, based on the existing knowledge between efficacy and safety among different pulses. We performed a proof-of-concept chronic animal trial to test the feasibility and safety of our method and technology.

Methods: We employed 6 female swine - weight 53.75 ± 4.77 kg - in this study. With fluoroscopic and electroanatomical mapping assistance, we performed ECG-gated AC-PFA in the following settings: in the left atrium with a decapolar loop catheter with electrodes connected in bipolar fashion; across the interventricular septum applying energy between the distal electrodes of two tip catheters. After procedure and 4-week follow-up, the animals were euthanized, and the hearts were inspected for tissue changes and characterized. We perform finite element method simulation of our AC-PFA scenarios to corroborate our method and better interpret our findings.

Results: We applied square, 50% duty cycle, AC bursts of 100 μs duration, 100 kHz internal frequency, 900 V for 60 pulses in the atrium and 1500 V for 120 pulses in the septum. The inter-burst interval was determined by the native heart rhythm - 69 ± 9 bpm. Acute changes in the atrial and ventricular electrograms were immediately visible at the sites of AC-PFA - signals were elongated and reduced in amplitude ( < 0.0001) and tissue impedance dropped ( = 0.011). No adverse event (e.g., esophageal temperature rises or gas bubble streams) was observed - while twitching was avoided by addition of electrosurgical return electrodes. The implemented numerical simulations confirmed the non-thermal nature of our AC-PFA and provided specific information on the estimated treated area and need of pulse trains. The postmortem chest inspection showed no peripheral damage, but epicardial and endocardial discolorations at sites of ablation. T1-weighted scans revealed specific tissue changes in atria and ventricles, confirmed to be fibrotic scars via trichrome staining. We found isolated, transmural and continuous scars. A surviving cardiomyocyte core was visible in basal ventricular lesions.

Conclusion: We proved that our method and technology of AC-PFA is feasible and safe for atrial and ventricular myocardial ablation, supporting their systematic investigation into effectiveness evaluation for the treatment of cardiac arrhythmia. Further optimization, with energy titration or longer follow-up, is required for a robust atrial and ventricular AC-PFA.
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http://dx.doi.org/10.3389/fbioe.2020.552357DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7744788PMC
December 2020

Myeloperoxidase mediated alteration of endothelial function is dependent on its cationic charge.

Free Radic Biol Med 2021 01 30;162:14-26. Epub 2020 Nov 30.

Department of Biophysics of Immune System, Institute of Biophysics of the Czech Academy of Sciences, Kralovopolska 135, Brno, Czech Republic. Electronic address:

Endothelial cell (EC) glycocalyx (GLX) comprise a multicomponent layer of proteoglycans and glycoproteins. Alteration of its integrity contributes to chronic vascular inflammation and leads to the development of cardiovascular diseases. Myeloperoxidase (MPO), a highly abundant enzyme released by polymorphonuclear neutrophils, binds to the GLX and deleteriously affects vascular EC functions. The focus of this study was to elucidate the mechanisms of MPO-mediated alteration of GLX molecules, and to unravel subsequent changes in endothelial integrity and function. MPO binding to GLX of human ECs and subsequent internalization was mediated by cell surface heparan sulfate chains. Moreover, interaction of MPO, which is carrying a cationic charge, with anionic glycosaminoglycans (GAGs) resulted in reduction of their relative charge. By means of micro-viscometry and atomic force microscopy, we disclosed that MPO can crosslink GAG chains. MPO-dependent modulation of GLX structure was further supported by alteration of wheat germ agglutinin staining. Increased expression of ICAM-1 documented endothelial cell activation by both catalytically active and also inactive MPO. Furthermore, MPO increased vascular permeability connected with reorganization of intracellular junctions, however, this was dependent on MPO's catalytic activity. Novel proteins interacting with MPO during transcytosis were identified by proteomic analysis. Altogether, these findings provide evidence that MPO through interaction with GAGs modulates overall charge of the GLX, causing modification of its structure and thus affecting EC function. Importantly, our results also suggest a number of proteins interacting with MPO that possess a variety of cellular localizations and functions.
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http://dx.doi.org/10.1016/j.freeradbiomed.2020.11.008DOI Listing
January 2021

A Clearance Period after Soluble Lead Nanoparticle Inhalation Did Not Ameliorate the Negative Effects on Target Tissues Due to Decreased Immune Response.

Int J Mol Sci 2020 Nov 19;21(22). Epub 2020 Nov 19.

Laboratory of Molecular Morphogenesis, Institute of Animal Physiology and Genetics, v.v.i., Czech Academy of Sciences, 602 00 Brno, Czech Republic.

The inhalation of metal (including lead) nanoparticles poses a real health issue to people and animals living in polluted and/or industrial areas. In this study, we exposed mice to lead(II) nitrate nanoparticles [Pb(NO) NPs], which represent a highly soluble form of lead, by inhalation. We aimed to uncover the effects of their exposure on individual target organs and to reveal potential variability in the lead clearance. We examined (i) lead biodistribution in target organs using laser ablation and inductively coupled plasma mass spectrometry (LA-ICP-MS) and atomic absorption spectrometry (AAS), (ii) lead effect on histopathological changes and immune cells response in secondary target organs and (iii) the clearance ability of target organs. In the lungs and liver, Pb(NO) NP inhalation induced serious structural changes and their damage was present even after a 5-week clearance period despite the lead having been almost completely eliminated from the tissues. The numbers of macrophages significantly decreased after 11-week Pb(NO) NP inhalation; conversely, abundance of alpha-smooth muscle actin (α-SMA)-positive cells, which are responsible for augmented collagen production, increased in both tissues. Moreover, the expression of nuclear factor κB () and selected cytokines, such as tumor necrosis factor alpha (), transforming growth factor beta 1 (), interleukin 6(), and , displayed a tissue-specific response to lead exposure. In summary, diminished inflammatory response in tissues after Pb(NO) NPs inhalation was associated with prolonged negative effect of lead on tissues, as demonstrated by sustained pathological changes in target organs, even after long clearance period.
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http://dx.doi.org/10.3390/ijms21228738DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7699374PMC
November 2020

3D Cell Culture Models Demonstrate a Role for FGF and WNT Signaling in Regulation of Lung Epithelial Cell Fate and Morphogenesis.

Front Cell Dev Biol 2020 21;8:574. Epub 2020 Jul 21.

Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Brno, Czechia.

FGF signaling plays an essential role in lung development, homeostasis, and regeneration. We employed mouse 3D cell culture models and imaging to study the role of FGF ligands and the interplay of FGF signaling with epithelial growth factor (EGF) and WNT signaling pathways in lung epithelial morphogenesis and differentiation. In non-adherent conditions, FGF signaling promoted formation of lungospheres from lung epithelial stem/progenitor cells (LSPCs). Ultrastructural and immunohistochemical analyses showed that LSPCs produced more differentiated lung cell progeny. In a 3D extracellular matrix, FGF2, FGF7, FGF9, and FGF10 promoted lung organoid formation. FGF9 showed reduced capacity to promote lung organoid formation, suggesting that FGF9 has a reduced ability to sustain LSPC survival and/or initial divisions. FGF7 and FGF10 produced bigger organoids and induced organoid branching with higher frequency than FGF2 or FGF9. Higher FGF concentration and/or the use of FGF2 with increased stability and affinity to FGF receptors both increased lung organoid and lungosphere formation efficiency, respectively, suggesting that the level of FGF signaling is a crucial driver of LSPC survival and differentiation, and also lung epithelial morphogenesis. EGF signaling played a supportive but non-essential role in FGF-induced lung organoid formation. Analysis of tissue architecture and cell type composition confirmed that the lung organoids contained alveolar-like regions with cells expressing alveolar type I and type II cell markers, as well as airway-like structures with club cells and ciliated cells. FGF ligands showed differences in promoting distinct lung epithelial cell types. FGF9 was a potent inducer of more proximal cell types, including ciliated and basal cells. FGF7 and FGF10 directed the differentiation toward distal lung lineages. WNT signaling enhanced the efficiency of lung organoid formation, but in the absence of FGF10 signaling, the organoids displayed limited branching and less differentiated phenotype. In summary, we present lung 3D cell culture models as useful tools to study the role and interplay of signaling pathways in postnatal lung development and homeostasis, and we reveal distinct roles for FGF ligands in regulation of mouse lung morphogenesis and differentiation .
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http://dx.doi.org/10.3389/fcell.2020.00574DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7396690PMC
July 2020

TGF-β regulates Sca-1 expression and plasticity of pre-neoplastic mammary epithelial stem cells.

Sci Rep 2020 07 9;10(1):11396. Epub 2020 Jul 9.

Institute of Biophysics of the Czech Academy of Sciences, Královopolská 135, 612 65, Brno, Czech Republic.

The epithelial-mesenchymal plasticity, in tight association with stemness, contributes to the mammary gland homeostasis, evolution of early neoplastic lesions and cancer dissemination. Focused on cell surfaceome, we used mouse models of pre-neoplastic mammary epithelial and cancer stem cells to reveal the connection between cell surface markers and distinct cell phenotypes. We mechanistically dissected the TGF-β family-driven regulation of Sca-1, one of the most commonly used adult stem cell markers. We further provided evidence that TGF-β disrupts the lineage commitment and promotes the accumulation of tumor-initiating cells in pre-neoplastic cells.
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http://dx.doi.org/10.1038/s41598-020-67827-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7347574PMC
July 2020

Slug-expressing mouse prostate epithelial cells have increased stem cell potential.

Stem Cell Res 2020 07 12;46:101844. Epub 2020 May 12.

Institute of Biophysics of the Czech Academy of Sciences, Královopolská 135, 612 65 Brno, Czech Republic; Center of Biomolecular and Cellular Engineering, International Clinical Research Center, St. Anne's University Hospital Brno, Pekařská 53, 656 91 Brno, Czech Republic; Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic. Electronic address:

Deciphering the properties of adult stem cells is crucial for understanding of their role in healthy tissue and in cancer progression as well. Both stem cells and cancer stem cells have shown association with epithelial-to-mesenchymal transition (EMT) in various tissue types. Aiming to investigate the epithelial and mesenchymal phenotypic traits in adult mouse prostate, we sorted subpopulations of basal prostate stem cells (mPSCs) and assessed the expression levels of EMT regulators and markers with custom-designed gene expression array. The population of mPSCs defined by a Lin/Sca-1CD49f/Trop-2 (LSC Trop-2) surface phenotype was enriched in mesenchymal markers, especially EMT master regulator Slug, encoded by the Snai2 gene. To further dissect the role of Slug in mPSCs, we used transgenic Snai2 reporter mouse strain. Using this model, we confirmed the presence of mesenchymal traits and increase of organoid forming capacity in Slug population of mPSCs. The Slug-derived organoids comprised all prostate epithelial cell types - basal, luminal, and neuroendocrine. Collectively, these data uncover the important role of Slug expression in the physiology of mouse prostate stem cells.
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http://dx.doi.org/10.1016/j.scr.2020.101844DOI Listing
July 2020

The CHK1 inhibitor MU380 significantly increases the sensitivity of human docetaxel-resistant prostate cancer cells to gemcitabine through the induction of mitotic catastrophe.

Mol Oncol 2020 10 16;14(10):2487-2503. Epub 2020 Jul 16.

Department of Cytokinetics, Institute of Biophysics of the Czech Academy of Sciences, Brno, Czech Republic.

As treatment options for patients with incurable metastatic castration-resistant prostate cancer (mCRPC) are considerably limited, novel effective therapeutic options are needed. Checkpoint kinase 1 (CHK1) is a highly conserved protein kinase implicated in the DNA damage response (DDR) pathway that prevents the accumulation of DNA damage and controls regular genome duplication. CHK1 has been associated with prostate cancer (PCa) induction, progression, and lethality; hence, CHK1 inhibitors SCH900776 (also known as MK-8776) and the more effective SCH900776 analog MU380 may have clinical applications in the therapy of PCa. Synergistic induction of DNA damage with CHK1 inhibition represents a promising therapeutic approach that has been tested in many types of malignancies, but not in chemoresistant mCRPC. Here, we report that such therapeutic approach may be exploited using the synergistic action of the antimetabolite gemcitabine (GEM) and CHK1 inhibitors SCH900776 and MU380 in docetaxel-resistant (DR) mCRPC. Given the results, both CHK1 inhibitors significantly potentiated the sensitivity to GEM in a panel of chemo-naïve and matched DR PCa cell lines under 2D conditions. MU380 exhibited a stronger synergistic effect with GEM than clinical candidate SCH900776. MU380 alone or in combination with GEM significantly reduced spheroid size and increased apoptosis in all patient-derived xenograft 3D cultures, with a higher impact in DR models. Combined treatment induced premature mitosis from G1 phase resulting in the mitotic catastrophe as a prestage of apoptosis. Finally, treatment by MU380 alone, or in combination with GEM, significantly inhibited tumor growth of both PC339-DOC and PC346C-DOC xenograft models in mice. Taken together, our data suggest that metabolically robust and selective CHK1 inhibitor MU380 can bypass docetaxel resistance and improve the effectiveness of GEM in DR mCRPC models. This approach might allow for dose reduction of GEM and thereby minimize undesired toxicity and may represent a therapeutic option for patients with incurable DR mCRPC.
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http://dx.doi.org/10.1002/1878-0261.12756DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7530791PMC
October 2020

Variability in the Clearance of Lead Oxide Nanoparticles Is Associated with Alteration of Specific Membrane Transporters.

ACS Nano 2020 03 4;14(3):3096-3120. Epub 2020 Mar 4.

Laboratory of Molecular Morphogenesis, Institute of Animal Physiology and Genetics, v.v.i., Czech Academy of Sciences, Brno 602 00, Czech Republic.

Lead oxide nanoparticles (PbONPs), upon their entry into the lungs inhalation, induce structural changes in primary and secondary target organs. The fate and ultrastructural localization of PbONPs in organs is known to be dependent on the specific organ. Here, we focused on the differences in the ability to clear the inhaled PbONPs from secondary target organs and on molecular and cellular mechanisms contributing to nanoparticle removal. Mice were exposed to PbONPs in whole-body inhalation chambers. Clearance of ionic lead and PbONPs (Pb/PbONPs) from the lungs and liver was very effective, with the lead being almost completely eliminated from the lungs and the physiological state of the lung tissue conspicuously restored. Kidneys exposed to nanoparticles did not exhibit serious signs of damage; however, LA-ICP-MS uncovered a certain amount of lead located preferentially in the kidney cortex even after a clearance period. The concentration of lead in femurs, as representatives of the axial skeleton, was the highest among studied organs at all designated time points after PbONP exposure, and the clearance ability of lead from the femurs was very low in contrast to other organs. The organ-specific increase of ABC transporters expression (ABCG2 in lungs and ABCC3 in the liver) was observed in exposed animals, suggesting their involvement in removing Pb/PbONPs from tissues. Moreover, the expression of and displayed a tissue-specific response to lead exposure. Our results uncovered high variability among the organs in their ability to clear Pb/PbONPs and in the transporters involved in this process.
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http://dx.doi.org/10.1021/acsnano.9b08143DOI Listing
March 2020

The effects of nano-sized PbO on biomarkers of membrane disruption and DNA damage in a sub-chronic inhalation study on mice.

Nanotoxicology 2020 03 15;14(2):214-231. Epub 2019 Nov 15.

Faculty of Science, RECETOX, Masaryk University, Brno, Czech Republic.

Although the production of engineered nanoparticles increases our knowledge of toxicity and mechanisms of bioactivity during relevant exposures is lacking. In the present study mice were exposed to PbO nanoparticles (PbONP; 192.5 µg/m; 1.93 × 10 particles/cm) for 2, 5 and 13 weeks through continuous inhalation. The analyses addressed Pb and PbONP distribution in organs (lung, liver, kidney, brain) using electrothermal atomic absorption spectrometry and transmission electron microscopy, as well as histopathology and analyses of oxidative stress biomarkers. New LC-MS/MS methods were validated for biomarkers of lipid damage F2-isoprostanes (8-iso-prostaglandins F and E) and hydroxylated deoxoguanosine (8-OHdG, marker of DNA oxidation). Commonly studied malondialdehyde was also measured as TBARS by HPLC-DAD. The study revealed fast blood transport and distribution of Pb from the lung to the kidney and liver. A different Pb accumulation trend was observed in the brain, suggesting transfer of NP along the nasal nerve to the olfactory bulbs. Long-term inhalation of PbONP caused lipid peroxidation in animal brains (increased levels of TBARS and both isoprostanes). Membrane lipid damage was also detected in the kidney after shorter exposures, but not in the liver or lung. On the contrary, longer exposures to PbONP increased levels of 8-OHdG in the lung and temporarily increased lung weight after 2 and 5 weeks of exposure. The histopathological changes observed mainly in the lung and liver indicated inflammation and general toxicity responses. The present long-term inhalation study indicates risks of PbONP to both human health and the environment.
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http://dx.doi.org/10.1080/17435390.2019.1685696DOI Listing
March 2020

HMGB2 is a negative regulator of telomerase activity in human embryonic stem and progenitor cells.

FASEB J 2019 12 26;33(12):14307-14324. Epub 2019 Oct 26.

Laboratory of Analysis of Chromosomal Proteins, Department of Cell Biology and Radiobiology, Institute of Biophysics of the Czech Academy of Sciences, Brno, Czech Republic.

High-mobility group box (HMGB)1 and HMGB2 proteins are the subject of intensive research because of their involvement in DNA replication, repair, transcription, differentiation, proliferation, cell signaling, inflammation, and tumor migration. Using inducible, stably transfected human embryonic stem cells (hESCs) capable of the short hairpin RNA-mediated knockdown (KD) of and , we provide evidence that deregulation of or expression in hESCs and their differentiated derivatives (neuroectodermal cells) results in distinct modulation of telomere homeostasis. Whereas HMGB1 enhances telomerase activity, HMGB2 acts as a negative regulator of telomerase activity in the cell. Stimulation of telomerase activity in the HMGB2-deficient cells may be related to activation of the PI3K/protein kinase B/ glycogen synthase kinase-3β/β-catenin signaling pathways by HMGB1, augmented TERT/telomerase RNA subunit transcription, and possibly also because of changes in telomeric repeat-containing RNA (TERRA) and TERRA-polyA transcription. The impact of KD on telomerase transcriptional regulation observed in neuroectodermal cells is partially masked in hESCs by their pluripotent state. Our findings on differential roles of HMGB1 and HMGB2 proteins in regulation of telomerase activity may suggest another possible outcome of HMGB1 targeting in cells, which is currently a promising approach aiming at increasing the anticancer activity of cytotoxic agents.-Kučírek, M., Bagherpoor, A. J., Jaroš, J., Hampl, A., Štros, M. HMGB2 is a negative regulator of telomerase activity in human embryonic stem and progenitor cells.
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http://dx.doi.org/10.1096/fj.201901465RRRDOI Listing
December 2019

Blind deconvolution estimation of an arterial input function for small animal DCE-MRI.

Magn Reson Imaging 2019 10 28;62:46-56. Epub 2019 May 28.

Institute of Scientific Instruments of the Czech Academy of Sciences, Kralovopolska 147, 61264 Brno, Czech Republic.

Purpose: One of the main obstacles for reliable quantitative dynamic contrast-enhanced (DCE) MRI is the need for accurate knowledge of the arterial input function (AIF). This is a special challenge for preclinical small animal applications where it is very difficult to measure the AIF without partial volume and flow artifacts. Furthermore, using advanced pharmacokinetic models (allowing estimation of blood flow and permeability-surface area product in addition to the classical perfusion parameters) poses stricter requirements on the accuracy and precision of AIF estimation. This paper addresses small animal DCE-MRI with advanced pharmacokinetic models and presents a method for estimation of the AIF based on blind deconvolution.

Methods: A parametric AIF model designed for small animal physiology and use of advanced pharmacokinetic models is proposed. The parameters of the AIF are estimated using multichannel blind deconvolution.

Results: Evaluation on simulated data show that for realistic signal to noise ratios blind deconvolution AIF estimation leads to comparable results as the use of the true AIF. Evaluation on real data based on DCE-MRI with two contrast agents of different molecular weights showed a consistence with the known effects of the molecular weight.

Conclusion: Multi-channel blind deconvolution using the proposed AIF model specific for small animal DCE-MRI provides reliable perfusion parameter estimates under realistic signal to noise conditions.
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http://dx.doi.org/10.1016/j.mri.2019.05.024DOI Listing
October 2019

Options for modeling the respiratory system: inserts, scaffolds and microfluidic chips.

Drug Discov Today 2019 04 12;24(4):971-982. Epub 2019 Mar 12.

Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Kamenice 753/5, 625 00 Brno, Czech Republic; International Clinical Research Center, St Anne's University Hospital Brno, Pekařská 664/53, 656 91 Brno, Czech Republic.

The human respiratory system is continuously exposed to varying levels of hazardous substances ranging from environmental toxins to purposely administered drugs. If the noxious effects exceed the inherent regenerative capacity of the respiratory system, injured tissue undergoes complex remodeling that can significantly affect lung function and lead to various diseases. Advanced near-to-native in vitro lung models are required to understand the mechanisms involved in pulmonary damage and repair and to reliably test the toxicity of compounds to lung tissue. This review is an overview of the development of in vitro respiratory system models used for study of lung diseases. It includes discussion of using these models for environmental toxin assessment and pulmonary toxicity screening.
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http://dx.doi.org/10.1016/j.drudis.2019.03.006DOI Listing
April 2019

Ligase 3-mediated end-joining maintains genome stability of human embryonic stem cells.

FASEB J 2019 06 26;33(6):6778-6788. Epub 2019 Feb 26.

Department of Biology, Faculty of Medicine, Masaryk University, Brno, Czech Republic.

Maintenance of human embryonic stem cells (hESCs) with stable genome is important for their future use in cell replacement therapy and disease modeling. Our understanding of the mechanisms maintaining genomic stability of hESC and our ability to modulate them is essential in preventing unwanted mutation accumulation during their cultivation. In this study, we show the DNA damage response mechanism in hESCs is composed of known, yet unlikely components. Clustered oxidative base damage is converted into DNA double-strand breaks (DSBs) by base excision repair (BER) and then quickly repaired by ligase (Lig)3-mediated end-joining (EJ). If there is further induction of clustered oxidative base damage by irradiation, then BER-mediated DSBs become essential in triggering the checkpoint response in hESCs. hESCs limit the mutagenic potential of Lig3-mediated EJ by DNA break end protection involving p53 binding protein 1 (53BP1), which results in fast and error-free microhomology-mediated repair and a low mutant frequency in hESCs. DSBs in hESCs are also repaired homologous recombination (HR); however, DSB overload, together with massive end protection by 53BP1, triggers competition between error-free HR and mutagenic nonhomologous EJ.-Kohutova, A., Raška, J., Kruta, M., Seneklova, M., Barta, T., Fojtik, P., Jurakova, T., Walter, C. A., Hampl, A., Dvorak, P., Rotrekl, V. Ligase 3-mediated end-joining maintains genome stability of human embryonic stem cells.
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http://dx.doi.org/10.1096/fj.201801877RRDOI Listing
June 2019

Human Embryonic Stem Cells Acquire Responsiveness to TRAIL upon Exposure to Cisplatin.

Stem Cells Int 2019 21;2019:4279481. Epub 2019 Jan 21.

Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Kamenice 5, 62500 Brno, Czech Republic.

Tumor necrosis factor-related apoptosis-inducing ligand-TRAIL-is a protein operating as a ligand capable of inducing apoptosis particularly in cancerously transformed cells, while normal healthy cells are typically nonresponsive. We have previously demonstrated that pluripotent human embryonic stem cells (hESC) are also refractory to TRAIL, even though they express all canonical components of the death receptor-induced apoptosis pathway. In this study, we have examined a capacity of DNA damage to provoke sensitivity of hESC to TRAIL. The extent of DNA damage, behavior of molecules involved in apoptosis, and response of hESC to TRAIL were investigated. The exposure of hESC to 1 M and 2 M concentrations of cisplatin have led to the formation of 53BP1 and H2AX foci, indicating the presence of double-strand breaks in DNA, without affecting the expression of proteins contributing to mitochondrial membrane integrity. Interestingly, cisplatin upregulated critical components of the extrinsic apoptotic pathway-initiator caspase 8, effector caspase 3, and the cell death receptors. The observed increase of expression of the extrinsic apoptotic pathway components was sufficient to sensitize hESC to TRAIL-induced apoptosis; immense cell dying accompanied by enhanced PARP cleavage, processing of caspase 8, and full activation of caspase 3 were all observed after the treatment combining cisplatin and TRAIL. Finally, we have demonstrated the central role of caspase 8 in this process, since its downregulation abrogated the sensitizing effect of cisplatin.
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http://dx.doi.org/10.1155/2019/4279481DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6360567PMC
January 2019

Fibroblast growth factor receptor influences primary cilium length through an interaction with intestinal cell kinase.

Proc Natl Acad Sci U S A 2019 03 19;116(10):4316-4325. Epub 2019 Feb 19.

Department of Biology, Faculty of Medicine, Masaryk University, 62500 Brno, Czech Republic;

Vertebrate primary cilium is a Hedgehog signaling center but the extent of its involvement in other signaling systems is less well understood. This report delineates a mechanism by which fibroblast growth factor (FGF) controls primary cilia. Employing proteomic approaches to characterize proteins associated with the FGF-receptor, FGFR3, we identified the serine/threonine kinase intestinal cell kinase (ICK) as an FGFR interactor. ICK is involved in ciliogenesis and participates in control of ciliary length. FGF signaling partially abolished ICK's kinase activity, through FGFR-mediated ICK phosphorylation at conserved residue Tyr15, which interfered with optimal ATP binding. Activation of the FGF signaling pathway affected both primary cilia length and function in a manner consistent with cilia effects caused by inhibition of ICK activity. Moreover, knockdown and knockout of ICK rescued the FGF-mediated effect on cilia. We provide conclusive evidence that FGF signaling controls cilia via interaction with ICK.
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http://dx.doi.org/10.1073/pnas.1800338116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6410813PMC
March 2019

Generation of human iPSCs from fetal prostate fibroblasts HPrF.

Stem Cell Res 2019 03 7;35:101405. Epub 2019 Feb 7.

Department of Cytokinetics, Institute of Biophysics of the Czech Academy of Sciences, Brno, Czech Republic; International Clinical Research Center, St. Anne's University Hospital Brno, Brno, Czech Republic. Electronic address:

Human induced pluripotent stem cell line was generated from commercially available primary human prostate fibroblasts HPrF derived from a fetus, aged 18-24 weeks of gestation. The fibroblast cell line was reprogrammed with Yamanaka factors (OCT4, SOX2, c-MYC, KLF4) using CytoTune™-iPS 2.0 Sendai Reprogramming Kit. Pluripotency of the derived transgene-free iPS cell line was confirmed both in vitro by detecting the expression of factors of pluripotency on a single-cell level, and in vivo using teratoma formation assay. This iPS cell line will be a useful tool for studying both normal prostate development and prostate cancer disease.
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http://dx.doi.org/10.1016/j.scr.2019.101405DOI Listing
March 2019

TEM ExosomeAnalyzer: a computer-assisted software tool for quantitative evaluation of extracellular vesicles in transmission electron microscopy images.

J Extracell Vesicles 2019 21;8(1):1560808. Epub 2019 Jan 21.

Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic.

Extracellular vesicles (EVs) function as important conveyers of information between cells and thus can be exploited as drug delivery systems or disease biomarkers. Transmission electron microscopy (TEM) remains the gold standard method for visualisation of EVs, however the analysis of individual EVs in TEM images is time-consuming if performed manually. Therefore, we present here a software tool for computer-assisted evaluation of EVs in TEM images. TEM ExosomeAnalyzer detects EVs based on their shape and edge contrast criteria and subsequently analyses their size and roundness. The software tool is compatible with common negative staining protocols and isolation methods used in the field of EV research; even with challenging TEM images (EVs both lighter and darker than the background, images containing artefacts or precipitated stain, .). If the fully-automatic analysis fails to produce correct results, users can promptly adjust the detected seeds of EVs as well as their boundaries manually. The performance of our tool was evaluated for three different modes with variable levels of human interaction, using two datasets with various heterogeneity. The semi-automatic mode analyses EVs with high success rate in the homogenous dataset (F1 score 0.9094, Jaccard coefficient 0.8218) as well as in the highly heterogeneous dataset containing EVs isolated from cell culture medium and patient samples (F1 score 0.7619, Jaccard coefficient 0.7553). Moreover, the extracted size distribution profiles of EVs isolated from malignant ascites of ovarian cancer patients overlap with those derived by cryo-EM and are comparable to NTA- and TRPS-derived data. In summary, TEM ExosomeAnalyzer is an easy-to-use software tool for evaluation of many types of vesicular microparticles and is available at http://cbia.fi.muni.cz/exosome-analyzer free of charge for non-commercial and research purposes. The web page contains also detailed description how to use the software tool including a video tutorial.
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http://dx.doi.org/10.1080/20013078.2018.1560808DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6346710PMC
January 2019

Egg maturity assessment prior to ICSI prevents premature fertilization of late-maturing oocytes.

J Assist Reprod Genet 2019 Mar 12;36(3):445-452. Epub 2019 Jan 12.

Faculty of Medicine, Department of Histology and Embryology, Masaryk University, Kamenice 3, 625 00, Brno, Czech Republic.

Propose: The presence of metaphase II (MII) spindle together with the polar body (PB) indicates completion of oocyte maturation. This study was designed to explore if spindle imaging can be used to optimize timing of intracytoplasmic sperm injection (ICSI).

Methods: The study involved 916 oocytes from 234 conventionally stimulated ICSI cycles with an unexpectedly poor ovarian response. All PB-displaying oocytes were subjected to polarized light microscopy (PLM) prior to ICSI. When MII spindle was absent in the majority of oocytes, ICSI was postponed and performed after additional spindle imaging. Fertilization, embryo development, and clinical outcome were evaluated with respect to the observed spindle pattern.

Results: The visible spindle was absent in 32.64% of PB-displaying oocytes. The late-maturing oocytes extruding PB in vitro were less likely to exhibit a spindle signal than in vivo matured MII oocytes (38.86% vs. 89.84%). When fertilization was postponed, 59.39% of initially spindle-negative oocytes developed detectable MII spindle. Spindled eggs had significantly higher developmental potential, and the presence of the spindle has been identified as an independent measure for predicting the formation of the blastocyst. Embryos derived from spindle-positive oocytes also showed a higher chance to implant and develop to term. Notably, 11 children were conceived by finely timed fertilization of late-maturing oocytes which are normally discarded.

Conclusions: The study confirms the prognostic value of spindle imaging and demonstrates that immature oocytes can be clinically utilized and give rise to live births when the timing of ICSI is adjusted to their developmental stage.
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http://dx.doi.org/10.1007/s10815-018-1393-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6439061PMC
March 2019

Biphasic calcium phosphate scaffolds with controlled pore size distribution prepared by in-situ foaming.

Mater Sci Eng C Mater Biol Appl 2019 Feb 23;95:363-370. Epub 2018 Mar 23.

CEITEC - Central European Institute of Technology, Brno University of Technology, Purkynova 123, 612 00 Brno, Czech Republic.

In this study, a reproducible method of fabricating hierarchically 3D porous scaffolds with high porosity and pore interconnectivity is reported. The method is based on in-situ foaming of a dispersion of diisocyanate, polyol, water and hydroxyapatite (HA) to form a hard foamed HA/polyurethane composite which after heat treatment provided a bi-phase calcium phosphate scaffold. This technique, combining the advantages of polymer sponge and direct foaming methods, provides a better control over the macrostructure of the scaffold. A modification of the multi-scaled porous macrostructure of scaffolds produced by changing the ratio of input reactants and by sintering temperature was studied. The pore morphology, size, and distribution were characterized using a scanning electron microscope and mercury porosimetry. The pores were open and interconnected with multi-scale (from several nanometres to millimetres) sizes convenient for using in tissue engineering applications. The bioactivity was confirmed by growing an apatite layer on the surfaces after immersion in simulated body fluid. The material was biocompatible, as shown by using normal human adipose tissue-derived stem cells (ASC). When seeded onto the scaffolds, the ASC adhered and remained healthy while maintaining their typical morphology.
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http://dx.doi.org/10.1016/j.msec.2018.03.022DOI Listing
February 2019

Generation of human iPSCs from human prostate cancer-associated fibroblasts IBPi002-A.

Stem Cell Res 2018 12 16;33:255-259. Epub 2018 Nov 16.

Department of Cytokinetics, Institute of Biophysics of the Czech Academy of Sciences, Brno, Czech Republic; International Clinical Research Center, St. Anne's University Hospital Brno, Brno, Czech Republic. Electronic address:

A human induced pluripotent stem cell line was generated from cancer-associated fibroblasts of a 68-years old patient with diagnosed prostate adenocarcinoma (PCa). The fibroblast cell line was reprogrammed with Epi5™ Episomal iPSC Reprogramming Kit. Pluripotency of the derived transgene-free iPS cell line was confirmed both in vitro by detecting expression of factors of pluripotency on a single-cell level, and also in vivo using teratoma formation assay. This new iPS cell line may be used for differentiation into different prostate-specific cell types in differentiation studies.
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http://dx.doi.org/10.1016/j.scr.2018.11.006DOI Listing
December 2018

Inactivation of PLK4-STIL Module Prevents Self-Renewal and Triggers p53-Dependent Differentiation in Human Pluripotent Stem Cells.

Stem Cell Reports 2018 10 6;11(4):959-972. Epub 2018 Sep 6.

Department of Histology and Embryology, Masaryk University, Faculty of Medicine, Brno 625 00, Czech Republic. Electronic address:

Centrioles account for centrosomes and cilia formation. Recently, a link between centrosomal components and human developmental disorders has been established. However, the exact mechanisms how centrosome abnormalities influence embryogenesis and cell fate are not understood. PLK4-STIL module represents a key element of centrosome duplication cycle. We analyzed consequences of inactivation of the module for early events of embryogenesis in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). We demonstrate that blocking of PLK4 or STIL functions leads to centrosome loss followed by both p53-dependent and -independent defects, including prolonged cell divisions, upregulation of p53, chromosome instability, and, importantly, reduction of pluripotency markers and induction of differentiation. We show that the observed loss of key stem cells properties is connected to alterations in mitotic timing and protein turnover. In sum, our data define a link between centrosome, its regulators, and the control of pluripotency and differentiation in PSCs.
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http://dx.doi.org/10.1016/j.stemcr.2018.08.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6178195PMC
October 2018

Presence of growth/differentiation factor-15 cytokine in human follicular fluid, granulosa cells, and oocytes.

J Assist Reprod Genet 2018 Aug 13;35(8):1407-1417. Epub 2018 Jun 13.

International Clinical Research Center, St. Anne's University Hospital Brno, Brno, Czech Republic.

Purpose: The purpose of the study was to determine whether the GDF-15 is present in follicular fluid; to evaluate if there is a relation between follicular and serum levels of GDF-15 and fertility status of study subjects; and to test whether granulosa cells, oocytes, or both produce GDF-15.

Methods: This study used follicular fluid (FF, serum, and oocytes obtained under informed consent from women undergoing oocyte retrieval for in vitro fertilization. It also used ovaries from deceased preterm newborns. Collection of FF and blood at the time of oocyte retrieval, ELISA and western blot were performed to determine levels and forms of GDF-15. Concentrations of GDF-15 in FF and serum, its expression in ovarian tissue, and secretion from granulosa cells were analyzed.

Results: GDF-15 concentration in FF ranged from 35 to 572 ng/ml, as determined by ELISA. Western blot analysis revealed the GDF-15 pro-dimer only in FF. Both normal healthy and cancerous granulosa cells secreted GDF-15 into culture media. Primary oocytes displayed cytoplasmic GDF-15 positivity in immunostained newborn ovaries, and its expression was also observed in fully grown human oocytes.

Conclusions: To the best of our knowledge, this is the first documentation of cytokine GDF-15 presence in follicular fluid. Its concentration was not associated with donor/patient fertility status. Our data also show that GDF-15 is expressed and inducible in both normal healthy and cancerous granulosa cells, as well as in oocytes.
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http://dx.doi.org/10.1007/s10815-018-1230-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6086784PMC
August 2018

Soluble Cripto-1 Induces Accumulation of Supernumerary Centrosomes and Formation of Aberrant Mitoses in Human Embryonic Stem Cells.

Stem Cells Dev 2018 08 17;27(16):1077-1084. Epub 2018 Jul 17.

1 Department of Histology and Embryology, Faculty of Medicine, Masaryk University , Brno, Czech Republic .

Chromosomal instability evoked by abnormalities in centrosome numbers has been traditionally considered as a hallmark of aberrant, typically cancerous or senescent cells. We have reported previously that pristine human embryonic stem cells (hESC) suffer from high frequency of supernumerary centrosomes and hence may be prone to undergo abnormal mitotic divisions. We have also unraveled that this phenomenon of multicentrosomal mitoses vanishes with prolonged time in culture and with initiation of differentiation, and it is strongly affected by the culture substratum. In this study, we report for the first time that Cripto-1 protein (teratocarcinoma-derived growth factor 1, epidermal growth factor-Cripto/FRL-1/Cryptic) produced by hESC represents a factor capable of inducing formation of supernumerary centrosomes in cultured hESC. Elimination of Cripto-1 signaling on the other hand restores the normal number of centrosomes in hESC. Linking the secretory phenotype of hESC to the centrosomal metabolism may help to develop better strategies for propagation of stable and safe bioindustrial and clinical grade cultures of hESC. From a broader point of view, it may lead to unravelling Cripto-1 as a micro-environmental factor contributing to adverse cell behaviors in vivo.
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http://dx.doi.org/10.1089/scd.2018.0017DOI Listing
August 2018

Intact Cell Mass Spectrometry as a Quality Control Tool for Revealing Minute Phenotypic Changes of Cultured Human Embryonic Stem Cells.

Stem Cells Transl Med 2018 01 16;7(1):109-114. Epub 2017 Dec 16.

Faculty of Medicine, Department of Histology and Embryology, Masaryk University, Brno, Czech Republic.

The stability of in vitro cell cultures is an important issue for any clinical, bio-industrial, or pharmacological use. Embryonic stem cells are pluripotent; consequently, they possess the ability to differentiate into all three germ layers and are inherently prone to respond to differentiation stimuli. However, long-term culture inevitably yields clones that are best adapted to the culture conditions, passaging regimes, or differentiation sensitivity. This cellular plasticity is a major obstacle in the development of bio-industrial or clinical-grade cultures. At present, the quality control of cell cultures is limited by the lack of reliable (epi)genetic or molecular markers or by the focus on a particular type of instability such as karyotype abnormalities or adverse phenotypic traits. Therefore, there is an ongoing need for robust, feasible, and sensitive methods of determining or confirming cell status and for revealing potential divergences from the optimal state. We modeled both intrinsic and extrinsic changes in human embryonic stem cell (hESC) states using different experimental strategies and addressed the changes in cell status by intact cell mass spectrometry fingerprinting. The analysis of spectral fingerprints by methods routinely used in analytical chemistry clearly distinguished the morphologically and biochemically similar populations of hESCs and provided a biomarker-independent tool for the quality control of cell culture. Stem Cells Translational Medicine 2018;7:109-114.
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http://dx.doi.org/10.1002/sctm.17-0107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5746154PMC
January 2018

Generation of a Close-to-Native In Vitro System to Study Lung Cells-Extracellular Matrix Crosstalk.

Tissue Eng Part C Methods 2018 01 23;24(1):1-13. Epub 2017 Oct 23.

3 i3S-Instituto de Investigação e Inovação em Saúde , Porto, Portugal .

Extracellular matrix (ECM) is an essential component of the tissue microenvironment, actively shaping cellular behavior. In vitro culture systems are often poor in ECM constituents, thus not allowing for naturally occurring cell-ECM interactions. This study reports on a straightforward and efficient method for the generation of ECM scaffolds from lung tissue and its subsequent in vitro application using primary lung cells. Mouse lung tissue was subjected to decellularization with 0.2% sodium dodecyl sulfate, hypotonic solutions, and DNase. Resultant ECM scaffolds were devoid of cells and DNA, whereas lung ECM architecture of alveolar region and blood and airway networks were preserved. Scaffolds were predominantly composed of core ECM and ECM-associated proteins such as collagens I-IV, nephronectin, heparan sulfate proteoglycan core protein, and lysyl oxidase homolog 1, among others. When homogenized and applied as coating substrate, ECM supported the attachment of lung fibroblasts (LFs) in a dose-dependent manner. After ECM characterization and biocompatibility tests, a novel in vitro platform for three-dimensional (3D) matrix repopulation that permits live imaging of cell-ECM interactions was established. Using this system, LFs colonized the ECM scaffolds, displaying a close-to-native morphology in intimate interaction with the ECM fibers, and showed nuclear translocation of the mechanosensor yes-associated protein (YAP), when compared with cells cultured in two dimensions. In conclusion, we developed a 3D-like culture system, by combining an efficient decellularization method with a live-imaging culture platform, to replicate in vitro native lung cell-ECM crosstalk. This is a valuable system that can be easily applied to other organs for ECM-related drug screening, disease modeling, and basic mechanistic studies.
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http://dx.doi.org/10.1089/ten.tec.2017.0283DOI Listing
January 2018

An Efficient Method for Generation of Knockout Human Embryonic Stem Cells Using CRISPR/Cas9 System.

Stem Cells Dev 2017 11 2;26(21):1521-1527. Epub 2017 Oct 2.

1 Department of Histology and Embryology, Masaryk University , Brno, Czech Republic .

Human embryonic stem cells (hESCs) represent a promising tool to study functions of genes during development, to model diseases, and to even develop therapies when combined with gene editing techniques such as CRISPR/CRISPR-associated protein-9 nuclease (Cas9) system. However, the process of disruption of gene expression by generation of null alleles is often inefficient and tedious. To circumvent these limitations, we developed a simple and efficient protocol to permanently downregulate expression of a gene of interest in hESCs using CRISPR/Cas9. We selected p53 for our proof of concept experiments. The methodology is based on series of hESC transfection, which leads to efficient downregulation of p53 expression even in polyclonal population (p53 Low cells), here proven by a loss of regulation of the expression of p53 target gene, microRNA miR-34a. We demonstrate that our approach achieves over 80% efficiency in generating hESC clonal sublines that do not express p53 protein. Importantly, we document by a set of functional experiments that such genetically modified hESCs do retain typical stem cells characteristics. In summary, we provide a simple and robust protocol to efficiently target expression of gene of interest in hESCs that can be useful for laboratories aiming to employ gene editing in their hESC applications/protocols.
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http://dx.doi.org/10.1089/scd.2017.0058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6328014PMC
November 2017

Revealing 3D Ultrastructure and Morphology of Stem Cell Spheroids by Electron Microscopy.

Methods Mol Biol 2017 ;1612:417-431

Faculty of Medicine, Department of Histology and Embryology, Masaryk University, Kamenice 3/A1, 625 00, Brno, Czech Republic.

Cell culture methods have been developed in efforts to produce biologically relevant systems for developmental and disease modeling, and appropriate analytical tools are essential. Knowledge of ultrastructural characteristics represents the basis to reveal in situ the cellular morphology, cell-cell interactions, organelle distribution, niches in which cells reside, and many more. The traditional method for 3D visualization of ultrastructural components, serial sectioning using transmission electron microscopy (TEM), is very labor-intensive due to contentious TEM slice preparation and subsequent image processing of the whole collection. In this chapter, we present serial block-face scanning electron microscopy, together with complex methodology for spheroid formation, contrasting of cellular compartments, image processing, and 3D visualization. The described technique is effective for detailed morphological analysis of stem cell spheroids, organoids, as well as organotypic cell cultures.
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http://dx.doi.org/10.1007/978-1-4939-7021-6_30DOI Listing
March 2018

Lungosphere Assay: 3D Culture of Lung Epithelial Stem/Progenitor Cells.

Methods Mol Biol 2017 ;1612:149-165

Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Kamenice 753/5, 625 00, Brno, Czech Republic.

Lung epithelium contains distinctive subpopulations of lung stem/progenitor cells (LSPCs) that are essential for lung epithelial maintenance and repair in vivo. Hence, LSPCs are in the center of interest of lung biology due to their promising therapeutic applications. To reach this goal, proper characterization of LSPCs, understanding of their proliferation and differentiation potentials and elucidation of mechanisms that control them are necessary. Therefore, development of reliable in vitro clonogenic assays has been needed. We established lungosphere assay, an in vitro sphere-forming 3D culture assay that enables to evaluate stem/progenitor cell activity, self-renewal and differentiation capacity of LSPCs and to conveniently test the effect of various treatments on LSPCs. Here we provide a detailed description of procedures for isolation of adult mouse lung epithelial cells, their culture in non-adherent conditions to form LSPC-derived spheroids (lungospheres) and for embedding of lungospheres into 3D extracellular matrix to model processes of lung tissue maintenance in a physiologically relevant microenvironment.
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http://dx.doi.org/10.1007/978-1-4939-7021-6_11DOI Listing
March 2018
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