hPSCs
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The combination of aminophylline and salbutamol is frequently used in clinical practice in the treatment of obstructive lung diseases. While the side effects (including arrhythmias) of the individual bronchodilator drugs were well described previously, the side effects of combined treatment are almost unknown. We aimed to study the arrhythmogenic potential of combined aminophylline and salbutamol treatment in vitro. For this purpose, we used the established atomic force microscopy (AFM) model coupled with cardiac organoids derived from human pluripotent stem cells (hPSC-CMs). We focused on the chronotropic, inotropic, and arrhythmogenic effects of salbutamol alone and aminophylline and salbutamol combined treatment. We used a method based on heart rate/beat rate variability (HRV/BRV) analysis to detect arrhythmic events in the hPSC-CM based AFM recordings. Salbutamol and aminophylline had a synergistic chronotropic and inotropic effect compared to the effects of monotherapy. Our main finding was that salbutamol reduced the arrhythmogenic effect of aminophylline, most likely mediated by endothelial nitric oxide synthase activated by beta-2 adrenergic receptors. These findings were replicated and confirmed using hPSC-CM derived from two cell lines (CCTL4 and CCTL12). Data suggest that salbutamol as an add-on therapy may not only deliver a bronchodilator effect but also increase the cardiovascular safety of aminophylline, as salbutamol reduces its arrhythmogenic potential.
- MeSH
- albuterol * farmakologie MeSH
- aminofylin * farmakologie MeSH
- bronchodilatancia farmakologie MeSH
- buněčné linie MeSH
- kardiomyocyty účinky léků metabolismus MeSH
- lidé MeSH
- mikroskopie atomárních sil MeSH
- pluripotentní kmenové buňky účinky léků cytologie MeSH
- srdeční arytmie * farmakoterapie MeSH
- srdeční frekvence účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Despite therapeutic advances, neurodegenerative diseases and disorders remain some of the leading causes of mortality and morbidity in the United States. Therefore, cell-based therapies to replace lost or damaged neurons and supporting cells of the central nervous system (CNS) are of great therapeutic interest. To that end, human pluripotent stem cell (hPSC) derived neural progenitor cells (hNPCs) and their neuronal derivatives could provide the cellular 'raw material' needed for regenerative medicine therapies for a variety of CNS disorders. In addition, hNPCs derived from patient-specific hPSCs could be used to elucidate the underlying mechanisms of neurodegenerative diseases and identify potential drug candidates. However, the scientific and clinical application of hNPCs requires the development of robust, defined, and scalable substrates for their long-term expansion and neuronal differentiation. In this study, we rationally designed a vitronectin-derived peptide (VDP) that served as an adhesive growth substrate for the long-term expansion of several hNPC lines. Moreover, VDP-coated surfaces allowed for the directed neuronal differentiation of hNPC at levels similar to cells differentiated on traditional extracellular matrix protein-based substrates. Overall, the ability of VDP to support the long-term expansion and directed neuronal differentiation of hNPCs will significantly advance the future translational application of these cells in treating injuries, disorders, and diseases of the CNS.
- MeSH
- biokompatibilní potahované materiály farmakologie MeSH
- buněčná adheze účinky léků MeSH
- buněčná diferenciace účinky léků MeSH
- extracelulární matrix - proteiny metabolismus MeSH
- lidé MeSH
- molekuly buněčné adheze metabolismus MeSH
- myši MeSH
- nervové kmenové buňky cytologie účinky léků metabolismus MeSH
- neurony cytologie účinky léků metabolismus MeSH
- peptidy farmakologie MeSH
- pluripotentní kmenové buňky cytologie účinky léků metabolismus MeSH
- proliferace buněk účinky léků MeSH
- vitronektin farmakologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural 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
Primary cilia are key regulators of embryo development and tissue homeostasis. However, their mechanisms and functions, particularly in the context of human cells, are still unclear. Here, we analyzed the consequences of primary cilia modulation for human pluripotent stem cells (hPSCs) proliferation and differentiation. We report that neither activation of the cilia-associated Hedgehog signaling pathway nor ablation of primary cilia by CRISPR gene editing to knockout Tau Tubulin Kinase 2 (TTBK2), a crucial ciliogenesis regulator, affects the self-renewal of hPSCs. Further, we show that TTBK1, a related kinase without previous links to ciliogenesis, is upregulated during hPSCs-derived neural rosette differentiation. Importantly, we demonstrate that while TTBK1 fails to localize to the mother centriole, it regulates primary cilia formation in the differentiated, but not the undifferentiated hPSCs. Finally, we show that TTBK1/2 and primary cilia are implicated in the regulation of the size of hPSCs-derived neural rosettes.
- MeSH
- centrioly metabolismus MeSH
- cilie metabolismus MeSH
- lidé MeSH
- pluripotentní kmenové buňky * metabolismus MeSH
- protein-serin-threoninkinasy genetika metabolismus MeSH
- proteiny hedgehog * genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
Human pluripotent stem cells (hPSCs) are a promising source of autologous endothelial progenitor cells (EPCs) that can be used for the treatment of vascular diseases. However, this kind of treatment requires a large amount of EPCs. Therefore, a highly efficient, robust, and easily reproducible differentiation protocol is necessary. We present a novel serum-free differentiation protocol that exploits the synergy of multiple powerful differentiation effectors. Our protocol follows the proper physiological pathway by differentiating EPCs from hPSCs in three phases that mimic in vivo embryonic vascular development. Specifically, hPSCs are differentiated into (i) primitive streak, which is subsequently turned into (ii) mesoderm, which finally differentiates into (iii) EPCs. This differentiation process yields up to 15 differentiated cells per seeded hPSC in 5 days. Endothelial progenitor cells constitute up to 97% of these derived cells. The experiments were performed on the human embryonic stem cell line H9 and six human induced pluripotent stem cell lines generated in our laboratory. Therefore, robustness was verified using many hPSC lines. Two previously established protocols were also adapted and compared to our synergistic three-phase protocol. Increased efficiency and decreased variability were observed for our differentiation protocol in comparison to the other tested protocols. Furthermore, EPCs derived from hPSCs by our protocol expressed the high-proliferative-potential EPC marker CD157 on their surface in addition to the standard EPC surface markers CD31, CD144, CD34, KDR, and CXCR4. Our protocol enables efficient fully defined production of autologous endothelial progenitors for research and clinical applications.
- Publikační typ
- časopisecké články MeSH
The potential clinical applications of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs) are limited by the difficulty of recapitulating embryoid hematopoiesis and by the unknown differentiation potential of hPSC lines. To evaluate their hematopoietic developmental potential, available hPSC lines were differentiated by an embryoid body (EB) suspension culture in serum-free medium supplemented with three different cytokine mixes (CMs). The hPSC differentiation status was investigated by the flow cytometry expression profiles of cell surface molecules, and the gene expression of pluripotency and differentiation markers over time was evaluated by real-time reverse transcription polymerase chain reaction (qRT-PCR). hPSC lines differed in several aspects of the differentiation process, including the absolute yield of hematopoietic progenitors, the proportion of hematopoietic progenitor populations, and the effect of various CMs. The ability to generate hematopoietic progenitors was then associated with the morphology of the developing EBs, the expression of the endodermal markers AFP and SOX17, and the hematopoietic transcription factor RUNX1. These findings deepen the knowledge about the hematopoietic propensity of hPSCs and identify its variability as an aspect that must be taken into account before the usage of hPSC-derived HSCs in downstream applications.
- MeSH
- buněčné linie MeSH
- diferenciační antigeny biosyntéza MeSH
- embryoidní tělíska cytologie metabolismus MeSH
- endoderm cytologie metabolismus MeSH
- hematopoéza * MeSH
- lidé MeSH
- lidské embryonální kmenové buňky cytologie metabolismus MeSH
- regulace genové exprese * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Mild hypoxia (5% O2) as well as FGFR1-induced activation of phosphatidylinositol-4,5-bisphosphate 3-kinase/protein kinase B (PI3K/AKT) and MAPK signaling pathways markedly support pluripotency in human pluripotent stem cells (hPSCs). This study demonstrates that the pluripotency-promoting PI3K/AKT signaling pathway is surprisingly attenuated in mild hypoxia compared to the 21% O2 environment. Hypoxia is known to be associated with lower levels of reactive oxygen species (ROS), which are recognized as intracellular second messengers capable of upregulating the PI3K/AKT signaling pathway. Our data denote that ROS downregulation results in pluripotency upregulation and PI3K/AKT attenuation in a hypoxia-inducible factor 1 (HIF-1)-dependent manner in hPSCs. Using specific MAPK inhibitors, we show that the MAPK pathway also downregulates ROS and therefore attenuates the PI3K/AKT signaling-this represents a novel interaction between these signaling pathways. This inhibition of ROS initiated by MEK1/2-ERK1/2 may serve as a negative feedback loop from the MAPK pathway toward FGFR1 and PI3K/AKT activation. We further describe the molecular mechanism resulting in PI3K/AKT upregulation in hPSCs-ROS inhibit the PI3K's primary antagonist PTEN and upregulate FGFR1 phosphorylation. These novel regulatory circuits utilizing ROS as second messengers may contribute to the development of enhanced cultivation and differentiation protocols for hPSCs. Since the PI3K/AKT pathway often undergoes an oncogenic transformation, our data could also provide new insights into the regulation of cancer stem cell signaling.
- Publikační typ
- časopisecké články MeSH
Human pluripotent stem cells (hPSCs) have gained a solid foothold in basic research and drug industry as they can be used in vitro to study human development and have potential to offer limitless supply of various somatic cell types needed in drug development. Although the hepatic differentiation of hPSCs has been extensively studied, only a little attention has been paid to the role of the extracellular matrix. In this study we used laminin-511, laminin-521, and fibronectin, found in human liver progenitor cells, as culture matrices for hPSC-derived definitive endoderm cells. We observed that laminin-511 and laminin-521 either alone or in combination support the hepatic specification and that fibronectin is not a vital matrix protein for the hPSC-derived definitive endoderm cells. The expression of the laminin-511/521-specific integrins increased during the definitive endoderm induction and hepatic specification. The hepatic cells differentiated on laminin matrices showed the upregulation of liver-specific markers both at mRNA and protein levels, secreted human albumin, stored glycogen, and exhibited cytochrome P450 enzyme activity and inducibility. Altogether, we found that laminin-511 and laminin-521 can be used as stage-specific matrices to guide the hepatic specification of hPSC-derived definitive endoderm cells.
- MeSH
- biomimetické materiály chemie MeSH
- buněčná diferenciace fyziologie MeSH
- buněčné linie MeSH
- extracelulární matrix - proteiny metabolismus MeSH
- extracelulární matrix metabolismus MeSH
- hepatocyty cytologie fyziologie MeSH
- laminin metabolismus MeSH
- lidé MeSH
- pluripotentní kmenové buňky cytologie metabolismus MeSH
- techniky vsádkové kultivace metody MeSH
- tkáňové inženýrství metody 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
