BACKGROUND: Human embryonic stem cells (hESCs) have the unique ability to differentiate into any cell type in the human body and to proliferate indefinitely. Cell therapies involving hESC have shown very promising results for the treatment of certain diseases and confirmed the safety of hESC-derived cells for humans. They are used in cell therapy, mainly in targeted therapy of diseases that are currently incurable. OBJECTIVES: The aim of this study was the derivation of clinical-grade hESCs usable in drug development, non-native medicine and cell therapy. MATERIAL AND METHODS: Embryos were thawed, cultivated to the blastocyst stage if necessary, and assisted hatching was subsequently performed. Embryoblasts were mechanically isolated using narrow needles. Each line was kept as a separate batch. The derived hESCs were cultured under hypoxic culture conditions (5% O2, 5% CO2, 37°C) in a NutriStem® hPSC XF Medium with a daily medium change. RESULTS: From January 2018 to July 2020, 138 selected clients were asked for consent to donate embryos, of whom 52 did not respond, 19 terminated the storage of their embryos and 29 extended the storage. Only 38 clients (27.5%) agreed to donate embryos for the derivation of hESCs. At the same time, personal communication with clients took place and another 17 embryo donors were recruited. A total of 160 embryos from 55 donors aged 26-42 years were collected. The embryos were frozen at the blastocyst (33.1%) or morula (46.3%) stage. After the preparation of 64 embryos, embryoblasts were isolated and cultured. Finally, 7 hESC lines were obtained, 4 research-grade and 3 clinical-grade, the first in the Czech Republic. CONCLUSIONS: We established a current good manufacturing practice (cGMP)-defined xeno-free and feeder-free system for the derivation, culture and banking of clinical-grade hESC lines that are suitable for preclinical and clinical trials. The quality control testing with criteria concerning sterility, safety and characterization according to cGMP ensured the clinical-grade quality of hESC lines.
- MeSH
- buněčné linie MeSH
- embryo savčí MeSH
- embryonální kmenové buňky * MeSH
- kvalita života * MeSH
- lidé MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Česká republika MeSH
Human embryonic stem cells (hESCs) have unique abilities that enable their use in cell therapy, disease modeling, and drug development. Their derivation is usually performed using a feeder layer, which is undefined and can potentially cause a contamination by xeno components, therefore there is a tendency to replace feeders with xeno-free defined substrates in recent years. Three hESC lines were successfully derived on the vitronectin with a truncated N-terminus (VTN-N) in combination with E-cadherin in xeno-free conditions for the first time, and their undifferentiated state, hESC morphology, and standard karyotypes together with their potential to differentiate into three germ layers were confirmed. These results support the conclusion that the VTN-N/E-cadherin is a suitable substrate for the xeno-free derivation of hESCs and can be used for the derivation of hESCs according to good manufacturing practices.
- MeSH
- buněčná a tkáňová terapie MeSH
- kadheriny genetika MeSH
- lidé MeSH
- lidské embryonální kmenové buňky * MeSH
- obchod MeSH
- vitronektin MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Diabetes mellitus je chronické metabolické onemocnění způsobené buď imunitně zprostředkovanou ztrátou beta buněk produkujících inzulin (diabetes 1. typu), nebo jejich nedostatečnou funkcí a vyčerpáním v důsledku nefyziologických metabolických podmínek (diabetes 2. typu). Přestože v léčbě diabetu bylo v poslední době dosaženo významného pokroku, současné terapeutické možnosti nejsou u většiny pacientů schopny zajistit dlouhodobou optimální kontrolu jejich glykemie. Proto je třeba vyvinout pokročilé terapeutické postupy schopné nahradit ztracené nebo poškozené beta buňky tak, aby u diabetických pacientů mohlo být dosaženo fyziologické kontroly hladiny glukózy v krvi. V tomto přehledném článku jsou prezentovány potenciálně slibné přístupy, jejichž cílem je náhrada beta buněk pomocí buněčné terapie. Zahrnují jednak diferenciaci embryonálních/pluripotentních kmenových buněk, přeprogramování různých typů pankreatických buněk na buňky produkující inzulin a také stimulaci proliferace samotných beta buněk. Kromě toho jsou zde také uvedeny informace o dokončených, probíhajících a plánovaných klinických studiích využívajících buňky produkující inzulin získaných diferenciací kmenových buněk.
Diabetes mellitus is a chronic metabolic disease caused either by the immune-mediated loss of insulin-producing beta cells (Type 1 diabetes) or their insufficient function and exhaustion due to hostile metabolic conditions (Type 2 diabetes). Although significant advances have been achieved in diabetes treatment, current therapeutic options are still unable to maintain optimal control of glycemic levels in most patients. Therefore, advanced therapies able to restore lost or impaired beta cells need to be developed in order to achieve physiological control of blood glucose levels in diabetic patients. Promising approaches aiming to replace beta cells are presented in this review. These include the differentiation of embryonic/pluripotent stem cells, reprogramming of various pancreatic cell types into insulin-producing cells, and induction of beta cell proliferation. Moreover, information about completed, ongoing and proposed clinical trials utilizing stem cell derived insulin-producing cells are also presented here.
The involvement of microRNAs (miRNAs) in orchestrating self-renewal and differentiation of stem cells has been revealed in a number of recent studies. And while in human pluripotent stem cells, miRNAs have been directly linked to the core pluripotency network, including the cell cycle regulation and the maintenance of the self-renewing capacity, their role in the onset of differentiation in other contexts, such as determination of neural cell fate, remains poorly described. To bridge this gap, we used three model cell types to study miRNA expression patterns: human embryonic stem cells (hESCs), hESCs-derived self-renewing neural stem cells (NSCs), and differentiating NSCs. The comprehensive miRNA profiling presented here reveals novel sets of miRNAs differentially expressed during human neural cell fate determination in vitro. Furthermore, we report a miRNA expression profile of self-renewing human NSCs, which has been lacking to this date. Our data also indicates that miRNA clusters enriched in NSCs share the target-determining seed sequence with cell cycle regulatory miRNAs expressed in pluripotent hESCs. Lastly, our mechanistic experiments confirmed that cluster miR-17-92, one of the NSCs-enriched clusters, is directly transcriptionally regulated by transcription factor c-MYC.
Human embryonic stem cells (hESCs) are increasingly used in clinical trials as they can change the outcome of treatment for many human diseases. They are used as a starting material for further differentiation into specific cell types and to achieve the desirable result of the cell therapy; thus, the quality of hESCs has to be taken into account. Therefore, current good manufacturing practice (cGMP) has to be implemented in the transport of embryos, derivation of inner cell mass to xeno-free, feeder-free and defined hESC culture, and cell freezing. The in-depth characterization of hESC lines focused on safety, pluripotency, differentiation potential and genetic background has to complement this process. In this paper, we show the derivation of three clinical-grade hESC lines, MUCG01, MUCG02, and MUCG03, following these criteria. We developed and validated the system for the manufacture of xeno-free and feeder-free clinical-grade hESC lines that present high-quality starting material suitable for cell therapy according to cGMP.
Závěrečná zpráva o řešení grantu Agentury pro zdravotnický výzkum MZ ČR
nestr.
Because of their unprecedented self-renewing and differentiation capacity, pluripotent stem cells (PSCs), both embryonic and induced, represent an unmatched cell source for advanced cell-based therapeutic scenarios and products, which are currently being devised and clinically tested at many respected institutions around globe. The key element in moving the Advanced Therapy Medicinal Products into clinical practice is their adherence to regulations ensuring ethical standards, safety, pharmaceutical quality, and efficacy. In the Czech Republic, clinically applicable lines of PSCs have not yet been produced and/or manipulated that imposes a major barrier to any clinical development. Therefore, here we propose to abrogate this hurdle by creating new platform, which will involve 1) set of clinical grade lines of embryonic SCs produced from ethically acceptable discarded human embryos under Good Manufacturing Practice (GMP)-compliant principles, and also 2) all corresponding manufacturing and regulatory elements such as Standard Operating Procedures (SOPs) and Quality Control procedures.
Pluripotentní kmenové buňky (PSC), embryonální i indukované, mají neomezenou reprodukční a diferenciační kapacitu a představují proto téměř dokonalý zdroj pro přípravu léčivých přípravků pro pokročilé terapie, které jsou v současnosti vyvíjeny a klinicky testovány na mnoha významných institucích na celém světě. Jedním z prvků, které jsou klíčové pro posun tohoto druhu léčivých přípravků do kliniky, je jejich soulad s regulačními požadavky, zajišťujícími etický původ, bezpečnost, farmakologickou kvalitu a účinnost. V České republice dosud žádné takové klinicky aplikovatelné PSC nebyly vytvořeny, což je zásadní bariéra pro jakýkoli další vývoj směrem k jejich využití v klinické medicíně. V tomto projektu proto navrhujeme odstranit toto omezení vybudováním nové platformy, která bude zahrnovat 1) set linií lidských embryonálních kmenových buněk derivovaných z eticky přijatelných vyřazených embryí za respektování principů dobré výrobní praxe (GMP) a také 2) všechny související výrobní a regulační prvky, jako jsou zejména standardní operační postupy (SOP) a postupy pro hodnocení kvality.
Embryonic stem cells and induced pluripotent stem cells provided us with fascinating new knowledge in recent years. Mechanistic insight into intricate regulatory circuitry governing pluripotency stemness and disclosing parallels between pluripotency stemness and cancer instigated numerous studies focusing on roles of pluripotency transcription factors, including Oct4, Sox2, Klf4, Nanog, Sall4 and Tfcp2L1, in cancer. Although generally well substantiated as tumour-promoting factors, oncogenic roles of pluripotency transcription factors and their clinical impacts are revealing themselves as increasingly complex. In certain tumours, both Oct4 and Sox2 behave as genuine oncogenes, and reporter genes driven by composite regulatory elements jointly recognized by both the factors can identify stem-like cells in a proportion of tumours. On the other hand, cancer stem cells seem to be biologically very heterogeneous both among different tumour types and among and even within individual tumours. Pluripotency transcription factors are certainly implicated in cancer stemness, but do not seem to encompass its entire spectrum. Certain cancer stem cells maintain their stemness by biological mechanisms completely different from pluripotency stemness, sometimes even by engaging signalling pathways that promote differentiation of pluripotent stem cells. Moreover, while these signalling pathways may well be antithetical to stemness in pluripotent stem cells, they may cooperate with pluripotency factors in cancer stem cells - a paradigmatic example is provided by the MAPK-AP-1 pathway. Unexpectedly, forced expression of pluripotency transcription factors in cancer cells frequently results in loss of their tumour-initiating ability, their phenotypic reversion and partial epigenetic normalization. Besides the very different signalling contexts operating in pluripotent and cancer stem cells, respectively, the pronounced dose dependency of reprogramming pluripotency factors may also contribute to the frequent loss of tumorigenicity observed in induced pluripotent cancer cells. Finally, contradictory cell-autonomous and non-cell-autonomous effects of various signalling molecules operate during pluripotency (cancer) reprogramming. The effects of pluripotency transcription factors in cancer are thus best explained within the concept of cancer stem cell heterogeneity.
- MeSH
- buněčná diferenciace genetika MeSH
- embryonální kmenové buňky MeSH
- indukované pluripotentní kmenové buňky * metabolismus MeSH
- lidé MeSH
- nádory * genetika metabolismus MeSH
- oktamerní transkripční faktor 3 genetika metabolismus MeSH
- pluripotentní kmenové buňky * MeSH
- přeprogramování buněk genetika MeSH
- transkripční faktory genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: The progenitors to lung airway epithelium that are capable of long-term propagation may represent an attractive source of cells for cell-based therapies, disease modeling, toxicity testing, and others. Principally, there are two main options for obtaining lung epithelial progenitors: (i) direct isolation of endogenous progenitors from human lungs and (ii) in vitro differentiation from some other cell type. The prime candidates for the second approach are pluripotent stem cells, which may provide autologous and/or allogeneic cell resource in clinically relevant quality and quantity. METHODS: By exploiting the differentiation potential of human embryonic stem cells (hESC), here we derived expandable lung epithelium (ELEP) and established culture conditions for their long-term propagation (more than 6 months) in a monolayer culture without a need of 3D culture conditions and/or cell sorting steps, which minimizes potential variability of the outcome. RESULTS: These hESC-derived ELEP express NK2 Homeobox 1 (NKX2.1), a marker of early lung epithelial lineage, display properties of cells in early stages of surfactant production and are able to differentiate to cells exhibitting molecular and morphological characteristics of both respiratory epithelium of airway and alveolar regions. CONCLUSION: Expandable lung epithelium thus offer a stable, convenient, easily scalable and high-yielding cell source for applications in biomedicine.
In mammals, the conserved telomere binding protein Rap1 serves a diverse set of nontelomeric functions, including activation of the NF-kB signaling pathway, maintenance of metabolic function in vivo, and transcriptional regulation. Here, we uncover the mechanism by which Rap1 modulates gene expression. Using a separation-of-function allele, we show that Rap1 transcriptional regulation is largely independent of TRF2-mediated binding to telomeres and does not involve direct binding to genomic loci. Instead, Rap1 interacts with the TIP60/p400 complex and modulates its histone acetyltransferase activity. Notably, we show that deletion of Rap1 in mouse embryonic stem cells increases the fraction of two-cell-like cells. Specifically, Rap1 enhances the repressive activity of Tip60/p400 across a subset of two-cell-stage genes, including Zscan4 and the endogenous retrovirus MERVL. Preferential up-regulation of genes proximal to MERVL elements in Rap1-deficient settings implicates these endogenous retroviral elements in the derepression of proximal genes. Altogether, our study reveals an unprecedented link between Rap1 and the TIP60/p400 complex in the regulation of pluripotency.
- MeSH
- genom MeSH
- myší embryonální kmenové buňky metabolismus MeSH
- myši MeSH
- proteiny vázající telomery * genetika metabolismus MeSH
- regulace genové exprese MeSH
- savci genetika MeSH
- telomery * metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Intact (whole) cell matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) is an established method for biotyping in clinical microbiology as well as for revealing phenotypic shifts in cultured eukaryotic cells. Intact cell MALDI-TOF MS has recently been introduced as a quality control tool for long-term cultures of pluripotent stem cells. Despite the potential this method holds for revealing minute changes in cells, there is still a need for improving the ionization efficiency or peak reproducibility. Here we report for the first time that supplementation by fine particles of black phosphorus to the standard MALDI matrices, such as sinapinic and α-cyano-4-hydroxycinnamic acids enhance intensities of mass spectra of particular amino acids and peptides, presumably by interactions with aromatic groups within the molecules. In addition, the particles of black phosphorus induce the formation of small and regularly dispersed crystals of sinapinic acid and α-cyano-4-hydroxycinnamic acid with the analyte on a steel MALDI target plate. Patterns of mass spectra recorded from intact cells using black phosphorus-enriched matrix were more reproducible and contained peaks of higher intensities when compared to matrix without black phosphorus supplementation. In summary, enrichment of common organic matrices by black phosphorus can improve discrimination data analysis by enhancing peak intensity and reproducibility of mass spectra acquired from intact cells.
- MeSH
- aminokyseliny analýza chemie MeSH
- buněčné kultury metody MeSH
- buněčné linie MeSH
- fosfor chemie MeSH
- lidé MeSH
- lidské embryonální kmenové buňky MeSH
- peptidy analýza chemie MeSH
- reprodukovatelnost výsledků MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice metody normy MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
