The generation of haematopoietic progenitors from human pluripotent stem cells (hPSCs) presents great promise for cell-replacement therapies. However, current protocols for haematopoietic differentiation of hPSCs suffer from low efficiency and functional defects in the derived cells. The technology is also limited by variable ability of hPSC lines to generate blood cells in vitro. To address this issue, methodologies for haematopoietic differentiation in feeder-free conditions were applied to available human embryonic stem cell (hESC) and human induced pluripotent stem cell (hiPSC) lines in this study. It was found that these cell lines did not generate haematopoietic progenitors to such an extent as did H1 and H9 hESC lines that were used for this purpose in the vast majority of relevant studies. These results suggest that for clinical application of blood cells derived from hPSCs, possibly from autologous hiPSCs, it is necessary to overcome the variability in the haematopoietic developmental potential of individual hPSC lines.

• MeSH
• buněčná diferenciace MeSH
• buněčné linie MeSH
• hematopoetické kmenové buňky cytologie   MeSH
• indukované pluripotentní kmenové buňky cytologie   MeSH
• lidé MeSH
• pluripotentní kmenové buňky cytologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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).

• MeSH
• kardiomyocyty * MeSH
• lidé MeSH
• pluripotentní kmenové buňky * MeSH
• výzkum kmenových buněk * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

The generation of human pluripotent stem cells (hPSCs) of sufficient quantity and quality remains a major challenge for biomedical application. Here we present an efficient feeder-free, high-density monolayer system in which hPSCs become SSEA-3-high and gradually more viable than their feeder-dependent counterparts without changes attributed to culture adaptation. As a consequence, monolayer hPSCs possess advantages over their counterparts in embryoid body development, teratoma formation, freezing as a single-cell suspension, and colony-forming efficiency. Importantly, this monolayer culture system is reversible, preserving the competence of hPSCs to gradually reacquire features of colony growth, if necessary. Therefore, the monolayer culture system is highly suitable for long-term, large-scale propagation of hPSCs, which is necessary in drug development and pluripotent stem cell-based therapies.

• MeSH
• analýza kolonii tvořících jednotek MeSH
• biologické markery metabolismus   MeSH
• buněčné kultury metody   MeSH
• buněčné linie MeSH
• indukované pluripotentní kmenové buňky cytologie   metabolismus   MeSH
• lidé MeSH
• myši MeSH
• pluripotentní kmenové buňky cytologie   metabolismus   MeSH
• podkladové buňky cytologie   MeSH
• proliferace buněk MeSH
• teratom patologie   MeSH
• viabilita buněk MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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

For human embryonic stem cells (ESC) to be used in cell replacement therapies, they must be grown under good manufacturing conditions in a chemically defined medium that lacks animal proteins. This study examined the ability of a newly designed medium containing the plant-derived serum replacement VegetaCell and other reagents of human origin to support undifferentiated growth and pluripotency of human ESC. This medium was tested in several culture systems, using human fibroblasts as a feeder layer or Matrigel in a feeder-free culture. Even under the most stringent feeder-free conditions without conditioned medium, human ESC exhibited an undifferentiated morphology, expressed markers of undifferentiated cells, demonstrated high alkaline phosphatase activity and multilineage differentiation and retained a normal karyotype. Compared with human ESC grown in standard culture conditions, human ESC maintained in humanized VegetaCell medium show longer cell cycles and decreased cell death. The availability of an animal protein-free medium supplemented with the low-cost VegetaCell reagent expands the repertoire of media for culturing human ESC as well as induced pluripotent stem cells for drug testing and cell replacement therapy.

• MeSH
• apoptóza účinky léků   MeSH
• buněčná diferenciace účinky léků   MeSH
• buněčné kultury metody   MeSH
• buněčný cyklus účinky léků   MeSH
• embryonální kmenové buňky cytologie   MeSH
• fibroblasty účinky léků   MeSH
• fixní kombinace léků MeSH
• indukované pluripotentní kmenové buňky MeSH
• kolagen MeSH
• kultivační média bez séra farmakologie   MeSH
• kultivační média speciální farmakologie   MeSH
• kultivační média MeSH
• laminin MeSH
• lidé MeSH
• pluripotentní kmenové buňky cytologie   MeSH
• proliferace buněk účinky léků   MeSH
• proteoglykany MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Ectopic expression of defined sets of genetic factors can reprogramme somatic cells to induced pluripotent stem cells (iPSCs) that closely resemble embryonic stem cells. However, the low reprogramming efficiency is a significant handicap for mechanistic studies and potential clinical application. In this study, we used human bone marrow-derived mesenchymal stem cells (hBMMSCs) as target cells for reprogramming and investigated efficient iPSC generation from hBMMSCs using the compounds of p53 siRNA, valproic acid (VPA) and vitamin C (Vc) with four transcription factors OCT4, SOX2, KLF4, and c-MYC (compound induction system). The synergetic mechanism of the compounds was studied. Our results showed that the compound induction system could efficiently reprogramme hBMMSCs to iPSCs. hBMMSC-derived iPSC populations expressed pluripotent markers and had multi-potential to differentiate into three germ layer-derived cells. p53 siRNA, VPA and Vc had a synergetic effect on cell reprogramming and the combinatorial use of these substances greatly improved the efficiency of iPSC generation by suppressing the expression of p53, decreasing cell apoptosis, up-regulating the expression of the pluripotent gene OCT4 and modifying the cell cycle. Therefore, our study highlights a straightforward method for improving the speed and efficiency of iPSC generation and provides versatile tools for investigating early developmental processes such as haemopoiesis and relevant diseases. In addition, this study provides a paradigm for the combinatorial use of genetic factors and molecules to improve the efficiency of iPSC generation.

• MeSH
• biologické markery metabolismus   MeSH
• buněčné kultury metody   MeSH
• buněčný cyklus účinky léků   genetika   MeSH
• buňky kostní dřeně cytologie   účinky léků   metabolismus   MeSH
• down regulace genetika   účinky záření   MeSH
• indukované pluripotentní kmenové buňky cytologie   účinky léků   metabolismus   MeSH
• kyselina askorbová farmakologie   MeSH
• kyselina valproová farmakologie   MeSH
• lidé MeSH
• malá interferující RNA metabolismus   MeSH
• mezenchymální kmenové buňky cytologie   účinky léků   metabolismus   MeSH
• multipotentní kmenové buňky cytologie   metabolismus   MeSH
• myši inbrední ICR MeSH
• myši MeSH
• nádorový supresorový protein p53 metabolismus   MeSH
• oktamerní transkripční faktor 3 genetika   metabolismus   MeSH
• přeprogramování buněk účinky léků   genetika   MeSH
• upregulace účinky léků   genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Stem cells are unique pools of cells that are crucial for embryonic development and maintenance of adult tissue homeostasis. The landmark Nobel Prize winning research by Yamanaka and colleagues to induce pluripotency in somatic cells has reshaped the field of stem cell research. The complications related to the usage of pluripotent embryonic stem cells (ESCs) in human medicine, particularly ESC isolation and histoincompatibility were bypassed with induced pluripotent stem cell (iPSC) technology. The human iPSCs can be used for studying embryogenesis, disease modeling, drug testing and regenerative medicine. iPSCs can be diverted to different cell lineages using small molecules and growth factors. In this review we have focused on iPSC differentiation towards cardiac and neuronal lineages. Moreover, we deal with the use of iPSCs in regenerative medicine and modeling diseases like myocardial infarction, Timothy syndrome, dilated cardiomyopathy, Parkinson's, Alzheimer's and Huntington's disease. Despite the promising potential of iPSCs, genome contamination and low efficacy of cell reprogramming remain significant challenges.

• MeSH
• buněčná diferenciace MeSH
• indukované pluripotentní kmenové buňky cytologie   metabolismus   transplantace   MeSH
• kardiomyocyty cytologie   MeSH
• lidé MeSH
• neurodegenerativní nemoci terapie   MeSH
• neurony cytologie   MeSH
• přeprogramování buněk MeSH
• regenerativní lékařství * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

Neural differentiation of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) can produce a valuable and robust source of human neural cell subtypes, holding great promise for the study of neurogenesis and development, and for treating neurological diseases. However, current hESCs and hiPSCs neural differentiation protocols require either animal factors or embryoid body formation, which decreases efficiency and yield, and strongly limits medical applications. Here we develop a simple, animal-free protocol for neural conversion of both hESCs and hiPSCs in adherent culture conditions. A simple medium formula including insulin induces the direct conversion of >98% of hESCs and hiPSCs into expandable, transplantable, and functional neural progenitors with neural rosette characteristics. Further differentiation of neural progenitors into dopaminergic and spinal motoneurons as well as astrocytes and oligodendrocytes indicates that these neural progenitors retain responsiveness to instructive cues revealing the robust applicability of the protocol in the treatment of different neurodegenerative diseases. The fact that this protocol includes animal-free medium and human extracellular matrix components avoiding embryoid bodies makes this protocol suitable for the use in clinic. Stem Cells Translational Medicine 2017;6:1217-1226.

• MeSH
• buněčná a tkáňová terapie MeSH
• buněčná diferenciace fyziologie   MeSH
• embryonální kmenové buňky fyziologie   MeSH
• indukované pluripotentní kmenové buňky cytologie   MeSH
• kultivované buňky MeSH
• lidé MeSH
• pluripotentní kmenové buňky cytologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Human pluripotent stem cells have the potential to change the way in which human diseases are cured. Clinical-grade human embryonic stem cells and human induced pluripotent stem cells have to be created according to current good manufacturing practices and regulations. Quality and safety must be of the highest importance when humans' lives are at stake. With the rising number of clinical trials, there is a need for a consensus on hPSCs characterization. Here, we summarize mandatory and 'for information only' characterization methods with release criteria for the establishment of clinical-grade hPSC lines.

• MeSH
• Bacteria MeSH
• buněčná a tkáňová terapie metody   MeSH
• endotoxiny MeSH
• indukované pluripotentní kmenové buňky MeSH
• lidé MeSH
• lidské embryonální kmenové buňky MeSH
• Mycoplasma MeSH
• pluripotentní kmenové buňky * MeSH
• viry MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Preclinical studies have demonstrated the promising potential of human induced pluripotent stem cells (hiPSCs) for clinical application. To fulfil this goal, efficient and safe methods to generate them must be established. Various reprogramming techniques were presented during seven years of hiPSCs research. Genome non-integrating and completely xeno-free protocols from the first biopsy to stable hiPSC clones are highly preferable in terms of future clinical application. In this short communication, we summarize the reprogramming experiments performed in our laboratories. We successfully generated hiPSCs using STEMCCA lentivirus, Sendai virus or episomal vectors. Human neonatal fibroblasts and CD34(+) blood progenitors were used as cell sources and were maintained either on mouse embryonic feeder cells or in feeder-free conditions. The reprogramming efficiency was comparable for all three methods and both cell types, while the best results were obtained in feeder-free conditions.

• MeSH
• antigeny CD34 metabolismus   MeSH
• biologické markery metabolismus   MeSH
• buněčné kultury metody   MeSH
• buněčné linie MeSH
• genom lidský genetika   MeSH
• imunohistochemie MeSH
• indukované pluripotentní kmenové buňky cytologie   metabolismus   MeSH
• lidé MeSH
• myši MeSH
• pluripotentní kmenové buňky cytologie   metabolismus   MeSH
• přeprogramování buněk genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH