iPS cells Dotaz Zobrazit nápovědu
Alexander disease (AxD) is a rare and severe neurodegenerative disorder caused by mutations in glial fibrillary acidic protein (GFAP). While the exact disease mechanism remains unknown, previous studies suggest that mutant GFAP influences many cellular processes, including cytoskeleton stability, mechanosensing, metabolism, and proteasome function. While most studies have primarily focused on GFAP-expressing astrocytes, GFAP is also expressed by radial glia and neural progenitor cells, prompting questions about the impact of GFAP mutations on central nervous system (CNS) development. In this study, we observed impaired differentiation of astrocytes and neurons in co-cultures of astrocytes and neurons, as well as in neural organoids, both generated from AxD patient-derived induced pluripotent stem (iPS) cells with a GFAPR239C mutation. Leveraging single-cell RNA sequencing (scRNA-seq), we identified distinct cell populations and transcriptomic differences between the mutant GFAP cultures and a corrected isogenic control. These findings were supported by results obtained with immunocytochemistry and proteomics. In co-cultures, the GFAPR239C mutation resulted in an increased abundance of immature cells, while in unguided neural organoids and cortical organoids, we observed altered lineage commitment and reduced abundance of astrocytes. Gene expression analysis revealed increased stress susceptibility, cytoskeletal abnormalities, and altered extracellular matrix and cell-cell communication patterns in the AxD cultures, which also exhibited higher cell death after stress. Overall, our results point to altered cell differentiation in AxD patient-derived iPS-cell models, opening new avenues for AxD research.
- Klíčová slova
- Alexander disease, GFAP, iPS cells, neural organoids,
- MeSH
- Alexanderova nemoc * genetika patologie metabolismus MeSH
- astrocyty * metabolismus patologie MeSH
- buněčná diferenciace * fyziologie MeSH
- gliový fibrilární kyselý protein * metabolismus genetika MeSH
- indukované pluripotentní kmenové buňky * metabolismus MeSH
- kokultivační techniky MeSH
- kultivované buňky MeSH
- lidé MeSH
- mutace MeSH
- nervové kmenové buňky metabolismus MeSH
- neurony metabolismus patologie MeSH
- organoidy metabolismus patologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- GFAP protein, human MeSH Prohlížeč
- gliový fibrilární kyselý protein * MeSH
Induced pluripotent stem (iPS) cells are derived from differentiated cells by different reprogramming techniques, by introducing specific transcription factors responsible for pluripotency. Induced pluripotent stem cells can serve as an excellent source for differentiated neural stem/progenitor cells (NSCs/NPs). Several methods and protocols are utilized to create a robust number of NSCs/NPs without jeopardizing the safety issues required for in vivo applications. A variety of disease-specific iPS cells have been used to study nervous system diseases. In this chapter, we will focus on some of the derivation and differentiation approaches and the application of iPS-NPs in the treatment of spinal cord injury and stroke.
- Klíčová slova
- Induced pluripotent stem cells, Neural stem cells, Neuronal differentiation, Spinal cord injury, Stroke,
- MeSH
- buněčná diferenciace * MeSH
- cévní mozková příhoda patologie terapie MeSH
- indukované pluripotentní kmenové buňky cytologie MeSH
- lidé MeSH
- modely neurologické * MeSH
- nervové kmenové buňky cytologie MeSH
- poranění míchy patologie terapie MeSH
- přeprogramování buněk MeSH
- transkripční faktory metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- transkripční faktory 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.
- Klíčová slova
- Cellular therapy, Clinical translation, Differentiation, Embryonic stem cells, Induced pluripotent stem cells, Neural differentiation, Pluripotent stem cells,
- 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
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
Spinal cord injury (SCI), is a devastating condition leading to the loss of locomotor and sensory function below the injured segment. Despite some progress in acute SCI treatment using stem cells and biomaterials, chronic SCI remains to be addressed. We have assessed the use of laminin-coated hydrogel with dual porosity, seeded with induced pluripotent stem cell-derived neural progenitors (iPSC-NPs), in a rat model of chronic SCI. iPSC-NPs cultured for 3 weeks in hydrogel in vitro were positive for nestin, glial fibrillary acidic protein (GFAP) and microtubule-associated protein 2 (MAP2). These cell-polymer constructs were implanted into a balloon compression lesion, 5 weeks after lesion induction. Animals were behaviorally tested, and spinal cord tissue was immunohistochemically analyzed 28 weeks after SCI. The implanted iPSC-NPs survived in the scaffold for the entire experimental period. Host axons, astrocytes and blood vessels grew into the implant and an increased sprouting of host TH+ fibers was observed in the lesion vicinity. The implantation of iPSC-NP-LHM cell-polymer construct into the chronic SCI led to the integration of material into the injured spinal cord, reduced cavitation and supported the iPSC-NPs survival, but did not result in a statistically significant improvement of locomotor recovery.
- Klíčová slova
- Chronic spinal cord injury, HEMA hydrogel, human induced pluripotent stem cells, laminin, neural progenitors, surface charge,
- MeSH
- buněčná diferenciace MeSH
- chronická nemoc MeSH
- hydrogely MeSH
- indukované pluripotentní kmenové buňky metabolismus MeSH
- krysa rodu Rattus MeSH
- nervové kmenové buňky transplantace MeSH
- poranění míchy terapie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu Rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- hydrogely MeSH
A promising therapeutic strategy for amyotrophic lateral sclerosis (ALS) treatment is stem cell therapy. Neural progenitors derived from induced pluripotent cells (NP-iPS) might rescue or replace dying motoneurons (MNs). However, the mechanisms responsible for the beneficial effect are not fully understood. The aim here was to investigate the mechanism by studying the effect of intraspinally injected NP-iPS into asymptomatic and early symptomatic superoxide dismutase (SOD)1G93A transgenic rats. Prior to transplantation, NP-iPS were characterized in vitro for their ability to differentiate into a neuronal phenotype. Motor functions were tested in all animals, and the tissue was analyzed by immunohistochemistry, qPCR, and Western blot. NP-iPS transplantation significantly preserved MNs, slowed disease progression, and extended the survival of all treated animals. The dysregulation of spinal chondroitin sulfate proteoglycans was observed in SOD1G93A rats at the terminal stage. NP-iPS application led to normalized host genes expression (versican, has-1, tenascin-R, ngf, igf-1, bdnf, bax, bcl-2, and casp-3) and the protection of perineuronal nets around the preserved MNs. In the host spinal cord, transplanted cells remained as progenitors, many in contact with MNs, but they did not differentiate. The findings suggest that NP-iPS demonstrate neuroprotective properties by regulating local gene expression and regulate plasticity by modulating the central nervous system (CNS) extracellular matrix such as perineuronal nets (PNNs).
- Klíčová slova
- ALS, iPS, motoneuron death, neurodegeneration, plasticity, proteoglycans, stem cells, transplantation,
- MeSH
- amyotrofická laterální skleróza terapie MeSH
- indukované pluripotentní kmenové buňky cytologie MeSH
- krysa rodu Rattus MeSH
- kultivované buňky MeSH
- lidé MeSH
- nervové kmenové buňky cytologie metabolismus transplantace MeSH
- neuroplasticita * MeSH
- neurotrofní faktory genetika metabolismus MeSH
- periferní nervy fyziologie MeSH
- potkani Sprague-Dawley MeSH
- proteiny regulující apoptózu genetika metabolismus MeSH
- regenerace nervu MeSH
- tenascin genetika metabolismus MeSH
- transplantace kmenových buněk metody MeSH
- versikany genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu Rattus MeSH
- lidé MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- neurotrofní faktory MeSH
- proteiny regulující apoptózu MeSH
- tenascin MeSH
- versikany 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.
- Klíčová slova
- cGMP, cell therapy, characterization, clinical, hESC, hPSCs, hiPSC, human embryonic stem cells, human induced pluripotent stem cells, human pluripotent stem cells,
- 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
- Názvy látek
- endotoxiny 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
The successful development and characterization of human induced pluripotent stem cells (iPSCs) provides a powerful tool to study the molecular mechanisms that control cell fate decisions and differentiation toward distinct lineages. Here we focus on the ability of donors derived iPSCs to differentiate toward hematopoietic progenitor cells and on the analysis of their telomere length. The ability to screen telomere length in individual donors is important for defining cellular senescence, which correlates with their differentiation potential toward hematopoietic lineages. We have modified iPSC culture protocol and telomere length analysis to suit for high throughput screening of telomere length in large number of individual donors. This approach can be used to demonstrate the heterogeneity or changes of telomere length and its shortening as an exclusion criterion for selection of suitable donors for future stem cell therapies.
- Klíčová slova
- Cellular senescence, Hematopoiesis, Hematopoietic stem cells, Induced pluripotent stem cells, Telomere length,
- MeSH
- biologické markery * MeSH
- buněčné kultury * MeSH
- čipová analýza tkání metody MeSH
- hematopoetické kmenové buňky cytologie metabolismus MeSH
- indukované pluripotentní kmenové buňky cytologie metabolismus MeSH
- lidé MeSH
- rychlé screeningové testy * MeSH
- stárnutí buněk * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- biologické markery * MeSH
In vitro gametogenesis, the process of generating gametes from pluripotent cells in culture, is a powerful tool for improving our understanding of germ cell development and an alternative source of gametes. Here, we induced primordial germ cell-like cells (PGCLCs) from pluripotent stem cells of the northern white rhinoceros (NWR), a species for which only two females remain, and southern white rhinoceros (SWR), the closest species to the NWR. PGCLC differentiation from SWR embryonic stem cells is highly reliant on bone morphogenetic protein and WNT signals. Genetic analysis revealed that SRY-box transcription factor 17 (SOX17) is essential for SWR-PGCLC induction. Under the defined condition, NWR induced pluripotent stem cells differentiated into PGCLCs. We also identified cell surface markers, CD9 and Integrin subunit alpha 6 (ITGA6), that enabled us to isolate PGCLCs without genetic alteration in pluripotent stem cells. This study provides a first step toward the production of NWR gametes in culture and understanding of the basic mechanism of primordial germ cell specification in a large animal.
