Mesenchymal stem cells (MSCs) represent a population of adult stem cells that have potent immunoregulatory, anti-inflammatory, and antiapoptotic properties. In addition, they have ability to migrate to the site of inflammation or injury, where they contribute to the regeneration and healing process. For these properties, MSCs have been used as therapeutic cells in several models, including treatment of damages or disorders of the ocular surface. If the damage of the ocular surface is extensive and involves a limbal region where limbal stem cell reside, MSC therapy has been proved as the effective treatment approach. Although the anti-inflammatory properties of MSCs have been well characterized, mechanisms of antiapoptotic action of MSCs are not well recognized. Using a chemically damaged cornea in a mouse model, we showed that the injury decreases expression of the gene for antiapoptotic molecule Bcl-2 and increases the expression of proapoptotic genes Bax and p53. These changes were attenuated by local transplantation of MSCs after corneal damage. The antiapoptotic effect of MSCs was tested in an in vitro model of co-cultivation of corneal explants with MSCs. The apoptosis of corneal cells in the explants was induced by proinflammatory cytokines and was significantly inhibited in the presence of MSCs. The antiapoptotic effect of MSCs was mediated by paracrine action, as confirmed by separation of the explants in inserts or by supernatants from MSCs. In addition, MSCs decreased the expression of genes for the molecules associated with endoplasmic reticulum stress Atf4, Bip, and p21, which are associated with apoptosis. The results show that MSCs inhibit the expression of proapoptotic genes and decrease the number of apoptotic cells in the damaged corneas, and this action might be one of the mechanisms of the therapeutic action of MSCs.
- Klíčová slova
- Bax, Bcl-2, antiapoptotic properties, cornea, mesenchymal stem cells, mouse model,
- MeSH
- apoptóza genetika MeSH
- cyklooxygenasa 2 genetika metabolismus MeSH
- cytokiny genetika metabolismus MeSH
- hepatocytární růstový faktor genetika metabolismus MeSH
- keratitida genetika metabolismus patologie MeSH
- kultivované buňky MeSH
- lidé MeSH
- mezenchymální kmenové buňky cytologie metabolismus MeSH
- modely nemocí na zvířatech * MeSH
- myši inbrední BALB C MeSH
- myši MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- poranění rohovky genetika metabolismus terapie MeSH
- regulace genové exprese * MeSH
- rohovka metabolismus MeSH
- transplantace mezenchymálních kmenových buněk metody MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- cyklooxygenasa 2 MeSH
- cytokiny MeSH
- hepatocytární růstový faktor MeSH
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.
- Klíčová slova
- Cripto-1, centrosomes, culture adaptation, embryonic stem cells, multipolar mitoses,
- MeSH
- buněčná diferenciace genetika MeSH
- centrozom * MeSH
- GPI-vázané proteiny antagonisté a inhibitory genetika MeSH
- lidé MeSH
- lidské embryonální kmenové buňky cytologie metabolismus MeSH
- mezibuněčné signální peptidy a proteiny genetika MeSH
- mitóza genetika MeSH
- nádorové proteiny antagonisté a inhibitory genetika MeSH
- signální transdukce genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- GPI-vázané proteiny MeSH
- mezibuněčné signální peptidy a proteiny MeSH
- nádorové proteiny MeSH
- TDGF1 protein, human MeSH Prohlížeč
New approaches in regenerative medicine and vasculogenesis have generated a demand for sufficient numbers of human endothelial cells (ECs). ECs and their progenitors reside on the interior surface of blood and lymphatic vessels or circulate in peripheral blood; however, their numbers are limited, and they are difficult to expand after isolation. Recent advances in human induced pluripotent stem cell (hiPSC) research have opened possible avenues to generate unlimited numbers of ECs from easily accessible cell sources, such as the peripheral blood. In this study, we reprogrammed peripheral blood mononuclear cells, human umbilical vein endothelial cells (HUVECs), and human saphenous vein endothelial cells (HSVECs) into hiPSCs and differentiated them into ECs. The phenotype profiles, functionality, and genome stability of all hiPSC-derived ECs were assessed and compared with HUVECs and HSVECs. hiPSC-derived ECs resembled their natural EC counterparts, as shown by the expression of the endothelial surface markers CD31 and CD144 and the results of the functional analysis. Higher expression of endothelial progenitor markers CD34 and kinase insert domain receptor (KDR) was measured in hiPSC-derived ECs. An analysis of phosphorylated histone H2AX (γH2AX) foci revealed that an increased number of DNA double-strand breaks upon reprogramming into pluripotent cells. However, differentiation into ECs restored a normal number of γH2AX foci. Our hiPSCs retained a normal karyotype, with the exception of the HSVEC-derived hiPSC line, which displayed mosaicism due to a gain of chromosome 1. Peripheral blood from adult donors is a suitable source for the unlimited production of patient-specific ECs through the hiPSC interstage. hiPSC-derived ECs are fully functional and comparable to natural ECs. The protocol is eligible for clinical applications in regenerative medicine, if the genomic stability of the pluripotent cell stage is closely monitored.
- Klíčová slova
- endothelial differentiation, induced pluripotent stem cells, peripheral blood mononuclear cells,
- MeSH
- biologické markery metabolismus MeSH
- buněčná diferenciace fyziologie MeSH
- endoteliální buňky pupečníkové žíly (lidské) cytologie metabolismus MeSH
- endoteliální buňky cytologie metabolismus MeSH
- fibroblasty cytologie metabolismus MeSH
- fyziologická neovaskularizace fyziologie MeSH
- indukované pluripotentní kmenové buňky cytologie metabolismus MeSH
- kultivované buňky MeSH
- leukocyty mononukleární cytologie metabolismus MeSH
- lidé MeSH
- regenerativní lékařství metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- biologické markery MeSH
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.
- Klíčová slova
- CRISPR/Cas9, gene engineering, human embryonic stem cells, knockout,
- MeSH
- buněčné linie MeSH
- CRISPR-Cas systémy * MeSH
- down regulace MeSH
- embryonální kmenové buňky cytologie metabolismus MeSH
- genový knockout metody MeSH
- kultivované buňky MeSH
- lidé MeSH
- mikro RNA genetika MeSH
- myši MeSH
- nádorový supresorový protein p53 genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- mikro RNA MeSH
- MIRN34 microRNA, human MeSH Prohlížeč
- nádorový supresorový protein p53 MeSH
- TP53 protein, human MeSH Prohlížeč
Retinal disorders represent the main cause of decreased quality of vision and even blindness worldwide. The loss of retinal cells causes irreversible damage of the retina, and there are currently no effective treatment protocols for most retinal degenerative diseases. A promising approach for the treatment of retinal disorders is represented by stem cell-based therapy. The perspective candidates are mesenchymal stem cells (MSCs), which can differentiate into multiple cell types and produce a number of trophic and growth factors. In this study, we show the potential of murine bone marrow-derived MSCs to differentiate into cells expressing retinal markers and we identify the key supportive role of interferon-γ (IFN-γ) in the differentiation process. MSCs were cultured for 7 days with retinal extract and supernatant from T-cell mitogen concanavalin A-stimulated splenocytes, simulating the inflammatory site of retinal damage. MSCs cultured in such conditions differentiated to the cells expressing retinal cell markers such as rhodopsin, S antigen, retinaldehyde-binding protein, calbindin 2, recoverin, and retinal pigment epithelium 65. To identify a supportive molecule in the supernatants from activated spleen cells, MSCs were cultured with retinal extract in the presence of various T-cell cytokines. The expression of retinal markers was enhanced only in the presence of IFN-γ, and the supportive role of spleen cell supernatants was abrogated with the neutralization antibody anti-IFN-γ. In addition, differentiated MSCs were able to express a number of neurotrophic factors, which are important for retinal regeneration. Taken together, the results show that MSCs can differentiate into cells expressing retinal markers and that this differentiation process is supported by IFN-γ.
- Klíčová slova
- differentiation, interferon-γ, mesenchymal stem cell, neurotrophic factor, retina, rhodopsin,
- MeSH
- buněčná diferenciace * MeSH
- cis-trans-isomerasy genetika metabolismus MeSH
- interferon gama farmakologie MeSH
- kalbindin 2 genetika metabolismus MeSH
- kultivované buňky MeSH
- mezenchymální kmenové buňky cytologie účinky léků metabolismus MeSH
- myši inbrední BALB C MeSH
- myši MeSH
- rekoverin genetika metabolismus MeSH
- retina cytologie metabolismus MeSH
- rodopsin genetika metabolismus MeSH
- transportní proteiny genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- 11-cis-retinal-binding protein MeSH Prohlížeč
- cis-trans-isomerasy MeSH
- interferon gama MeSH
- kalbindin 2 MeSH
- rekoverin MeSH
- retinoid isomerohydrolase MeSH Prohlížeč
- rodopsin MeSH
- transportní proteiny MeSH
HMGB1 and HMGB2 proteins have been implicated in numerous cellular processes, including proliferation, differentiation, apoptosis, and tumor growth. It is unknown whether they are involved in regulating the typical functions of pluripotent human embryonic stem cells (hESCs) and/or those of the differentiated derivatives of hESCs. Using inducible, stably transfected hESCs capable of shRNA-mediated knockdown of HMGB1 and HMGB2, we provide evidence that downregulation of HMGB1 and/or HMGB2 in undifferentiated hESCs does not affect the stemness of cells and induces only minor changes to the proliferation rate, cell-cycle profile, and apoptosis. After differentiation is induced, however, the downregulation of those proteins has important effects on proliferation, apoptosis, telomerase activity, and the efficiency of differentiation toward the neuroectodermal lineage. Furthermore, those processes are affected only when one, but not both, of the two proteins is downregulated; the knockdown of both HMGB1 and HMGB2 results in a normal phenotype. Those results advance our knowledge of regulation of hESC and human neuroectodermal cell differentiation and illustrate the distinct roles of HMGB1 and HMGB2 during early human development.
- Klíčová slova
- HMGB1, HMGB2, differentiation, human embryonic stem cells, neuroectodermal cells,
- MeSH
- apoptóza genetika MeSH
- buněčná diferenciace * MeSH
- buněčná sebeobnova genetika MeSH
- buněčné linie MeSH
- buněčný cyklus genetika MeSH
- buněčný rodokmen genetika MeSH
- down regulace genetika MeSH
- histony metabolismus MeSH
- lidé MeSH
- lidské embryonální kmenové buňky cytologie metabolismus MeSH
- neurální ploténka cytologie MeSH
- proliferace buněk genetika MeSH
- protein HMGB1 metabolismus MeSH
- protein HMGB2 metabolismus MeSH
- telomerasa metabolismus MeSH
- transfekce MeSH
- tvar buňky genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- histony MeSH
- protein HMGB1 MeSH
- protein HMGB2 MeSH
- telomerasa MeSH
Regenerative endodontic procedures of immature teeth with necrotic pulp have become a part of therapeutic endodontic spectrum and are considered as an alternative to calcium hydroxide or mineral trioxide aggregate apexification. In last decade, numerous case reports and series with usage of cell-free approaches known as revascularization, revitalization, or maturogenesis have been published. This cell-free approach prevails in clinical regenerative endodontics because of its relative ease of performance, lower financial demands, and absence of complications such as tumorigenesis of used stem cells. In this article, the integral steps of cell-free treatment approaches such as source of stem cells, possible endogenous scaffolds, sources of growth factors, and width of apical foramen in the context of sufficient disinfection of root canal system and outcome of treatment are discussed. Despite not being a fully established treatment protocol, the achieved outcomes are promising regardless of it having a reparative character than a regenerative one.
- Klíčová slova
- blood clot, dental pulp stem cell, regenerative endodontic procedure, revascularization, revitalization, stem cells from apical papilla,
- MeSH
- bakteriální infekce terapie MeSH
- bezbuněčný systém MeSH
- endodoncie metody MeSH
- kmenové buňky cytologie MeSH
- lidé MeSH
- regenerace fyziologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
This study was focused on characterizing the differentiation of bone marrow-derived mesenchymal stem cells (MSCs) into corneal-like cells. Mouse MSCs were isolated from the bone marrow, grown in cell culture for 3 weeks, and purified using a magnetic activated cell sorter. Purified MSCs were cultured with an extract prepared from excised corneas and in the presence or absence of insulin-like growth factor-I (IGF-I). Analysis by quantitative real-time polymerase chain reaction showed that the expression of corneal specific markers, such as cytokeratin 12 (K12), keratocan, and lumican, was already induced after a 3-day cultivation and gradually increased during the 10-day incubation of MSCs with the extract. The presence of IGF-I significantly increased differentiation. Immunofluorescence analysis of differentiated MSCs showed positive results for the K12 protein. The morphology of the differentiated cells and the expression of cell surface markers CD45, CD11b, CD73, CD44, and CD105 were comparable in the control and differentiated MSCs. Proliferative activity was even higher in differentiated cells than in untreated MSCs. Both untreated and differentiated MSCs inhibited the production of interleukin-2 and interferon-γ in spleen cells stimulated with Concanavalin A. The results thus show that MSCs cultured in the presence of corneal extract and IGF-I efficiently differentiate into corneal-like cells. The differentiated cells possess characteristics of corneal epithelial cells and keratocytes, while at the same time maintaining MSC properties.
- MeSH
- biologické markery metabolismus MeSH
- buněčná diferenciace účinky léků genetika MeSH
- imunosupresivní léčba MeSH
- insulinu podobný růstový faktor I farmakologie MeSH
- mezenchymální kmenové buňky cytologie účinky léků metabolismus MeSH
- myši inbrední BALB C MeSH
- proliferace buněk účinky léků genetika MeSH
- regulace genové exprese účinky léků MeSH
- rohovka cytologie MeSH
- tvar buňky účinky léků genetika MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- biologické markery MeSH
- insulinu podobný růstový faktor I MeSH
The aim of this study was to investigate the effects of systemically administered bone-marrow-derived mesenchymal stromal cells (MSCs) on the early acute phase of inflammation in the alkali-burned eye. Mice with damaged eyes were either untreated or treated 24 h after the injury with an intravenous administration of fluorescent-dye-labeled MSCs that were unstimulated or pretreated with interleukin-1α (IL-1α), transforming growth factor-β (TGF-β), or interferon-γ (IFN-γ). Analysis of cell suspensions prepared from the eyes of treated mice on day 3 after the alkali burn revealed that MSCs specifically migrated to the damaged eye and that the number of labeled MSCs was more than 30-times higher in damaged eyes compared with control eyes. The study of the composition of the leukocyte populations within the damaged eyes showed that all types of tested MSCs slightly decreased the number of infiltrating lymphoid and myeloid cells, but only MSCs pretreated with IFN-γ significantly decreased the percentage of eye-infiltrating cells with a more profound effect on myeloid cells. Determining cytokine and NO production in the damaged eyes confirmed that the most effective immunomodulation was achieved with MSCs pretreated with IFN-γ, which significantly decreased the levels of the proinflammatory molecules IL-1α, IL-6, and NO. Taken together, the results show that systemically administered MSCs specifically migrate to the damaged eye and that IFN-γ-pretreated MSCs are superior in inhibiting the acute phase of inflammation, decreasing leukocyte infiltration, and attenuating the early inflammatory environment.
- MeSH
- alkálie toxicita MeSH
- alografty MeSH
- antivirové látky farmakologie MeSH
- chemické popálení patologie terapie MeSH
- interferon gama farmakologie MeSH
- interleukin-1alfa metabolismus MeSH
- myši inbrední BALB C MeSH
- myši MeSH
- nika kmenových buněk * MeSH
- popálení oka chemicky indukované metabolismus patologie terapie MeSH
- transformující růstový faktor beta metabolismus MeSH
- transplantace mezenchymálních kmenových buněk * MeSH
- zánět chemicky indukované metabolismus terapie MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- alkálie MeSH
- antivirové látky MeSH
- interferon gama MeSH
- interleukin-1alfa MeSH
- transformující růstový faktor beta MeSH
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.
- MeSH
- buněčná diferenciace účinky záření MeSH
- buněčné linie MeSH
- genetické lokusy * MeSH
- hypoxanthinfosforibosyltransferasa genetika metabolismus MeSH
- indukované pluripotentní kmenové buňky cytologie metabolismus MeSH
- lidé MeSH
- mutační rychlost * MeSH
- záření gama MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- hypoxanthinfosforibosyltransferasa MeSH
