scholarly journals Cardiac Safety of Kinase Inhibitors – Improving Understanding and Prediction of Liabilities in Drug Discovery Using Human Stem Cell-Derived Models

2021 ◽  
Vol 8 ◽  
Author(s):  
Ricarda Ziegler ◽  
Fabian Häusermann ◽  
Stephan Kirchner ◽  
Liudmila Polonchuk
Keyword(s):  
Stem Cell ◽  
Cardiac Function ◽  
Kinase Inhibitors ◽  
Cell Number ◽  
Human Stem Cell ◽  
Biologically Relevant ◽  
Parameter Evaluation ◽  
Integrative Assessment ◽  
Beating Rate

Many small molecule kinase inhibitors (SMKIs) used to fight cancer have been associated with cardiotoxicity in the clinic. Therefore, preventing their failure in clinical development is a priority for preclinical discovery. Our study focused on the integration and concurrent measurement of ATP, apoptosis dynamics and functional cardiac indexes in human stem cell-derived cardiomyocytes (hSC-CMs) to provide further insights into molecular determinants of compromised cardiac function. Ten out of the fourteen tested SMKIs resulted in a biologically relevant decrease in either beating rate or base impedance (cell number index), illustrating cardiotoxicity as one of the major safety liabilities of SMKIs, in particular of those involved in the PI3K–AKT pathway. Pearson's correlation analysis indicated a good correlation between the different read-outs of functional importance. Therefore, measurement of ATP concentrations and apoptosis in vitro could provide important insight into mechanisms of cardiotoxicity. Detailed investigation of the cellular signals facilitated multi-parameter evaluation allowing integrative assessment of cardiomyocyte behavior. The resulting correlation can be used as a tool to highlight changes in cardiac function and potentially to categorize drugs based on their mechanisms of action.

scholarly journals A hydrogel platform for in vitro three dimensional assembly of human stem cell-derived islet cells and endothelial cells

2019 ◽  
Vol 97 ◽  
pp. 272-280 ◽  
Author(s):  
Punn Augsornworawat ◽  
Leonardo Velazco-Cruz ◽  
Jiwon Song ◽  
Jeffrey R. Millman
Keyword(s):  
Endothelial Cells ◽  
Stem Cell ◽  
Islet Cells ◽  
Three Dimensional ◽  
Human Stem Cell

Current Progress in the Creation, Characterization, and Application of Human Stem Cell-derived in Vitro Neuromuscular Junction Models

2021 ◽  
Author(s):  
Eileen Lynch ◽  
Emma Peek ◽  
Megan Reilly ◽  
Claire FitzGibbons ◽  
Samantha Robertson ◽  
...  
Keyword(s):  
Stem Cell ◽  
Neuromuscular Junction ◽  
Human Stem Cell ◽  
The Creation

scholarly journals Assessing Drug-Induced Long QT and Proarrhythmic Risk Using Human Stem-Cell-Derived Cardiomyocytes in a Ca2+ Imaging Assay: Evaluation of 28 CiPA Compounds at Three Test Sites

2019 ◽  
Vol 170 (2) ◽  
pp. 345-356 ◽  
Author(s):  
Hua Rong Lu ◽  
Haoyu Zeng ◽  
Ralf Kettenhofen ◽  
Liang Guo ◽  
Ivan Kopljar ◽  
...  
Keyword(s):  
Stem Cell ◽  
Microelectrode Array ◽  
Torsade De Pointes ◽  
Transient Assay ◽  
Potential Drug ◽  
Human Stem Cell ◽  
Long Qt ◽  
Drug Induced ◽  
Vitro Assay

Abstract The goal of this research consortium including Janssen, MSD, Ncardia, FNCR/LBR, and Health and Environmental Sciences Institute (HESI) was to evaluate the utility of an additional in vitro assay technology to detect potential drug-induced long QT and torsade de pointes (TdP) risk by monitoring cytosolic free Ca2+ transients in human stem-cell-derived cardiomyocytes (hSC-CMs). The potential proarrhythmic risks of the 28 comprehensive in vitro proarrhythmia assay (CiPA) drugs linked to low, intermediate, and high clinical TdP risk were evaluated in a blinded manner using Ca2+-sensitive fluorescent dye assay recorded from a kinetic plate reader system (Hamamatsu FDSS/µCell and FDSS7000) in 2D cultures of 2 commercially available hSC-CM lines (Cor.4U and CDI iCell Cardiomyocytes) at 3 different test sites. The Ca2+ transient assay, performed at the 3 sites using the 2 different hSC-CMs lines, correctly detected potential drug-induced QT prolongation among the 28 CiPA drugs and detected cellular arrhythmias-like/early afterdepolarization in 7 of 8 high TdP-risk drugs (87.5%), 6 of 11 intermediate TdP-risk drugs (54.5%), and 0 of 9 low/no TdP-risk drugs (0%). The results were comparable among the 3 sites and from 2 hSC-CM cell lines. The Ca2+ transient assay can serve as a user-friendly and higher throughput alternative to complement the microelectrode array and voltage-sensing optical action potential recording assays used in the HESI-CiPA study for in vitro assessment of drug-induced long QT and TdP risk.

Validation of a novel human stem cell-based gene expression assay for in vitro DART assessment

2019 ◽  
Vol 88 ◽  
pp. 18-19
Author(s):  
Peter I. Racz ◽  
Inger Brandsma ◽  
Sabine Hartvelt ◽  
Tom Zwetsloot ◽  
Giel Hendriks
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Human Stem Cell ◽  
Gene Expression Assay

scholarly journals An In Vitro Model of Latency and Reactivation of Varicella Zoster Virus in Human Stem Cell-Derived Neurons

2015 ◽  
Vol 11 (6) ◽  
pp. e1004885 ◽  
Author(s):  
Amos Markus ◽  
Ilana Lebenthal-Loinger ◽  
In Hong Yang ◽  
Paul R. Kinchington ◽  
Ronald S. Goldstein
Keyword(s):  
Stem Cell ◽  
Varicella Zoster Virus ◽  
In Vitro Model ◽  
Human Stem Cell ◽  
Varicella Zoster ◽  
Vitro Model

scholarly journals In Vitro Differentiated Human Stem Cell-Derived Neurons Reproduce Synaptic Synchronicity Arising during Neurodevelopment

2020 ◽  
Vol 15 (1) ◽  
pp. 22-37 ◽  
Author(s):  
Filip Rosa ◽  
Ashutosh Dhingra ◽  
Betül Uysal ◽  
G. Dulini C. Mendis ◽  
Heidi Loeffler ◽  
...  
Keyword(s):  
Stem Cell ◽  
Human Stem Cell

Normal Hematopoietic Stem Cell Functioning in p21−/− Mice.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1701-1701
Author(s):  
Leonie M. Kamminga ◽  
Kyrjon van Pelt ◽  
Bert Dontje ◽  
Gerald de Haan
Keyword(s):  
Stem Cell ◽  
Kinase Inhibitors ◽  
Cell Function ◽  
Cell Activity ◽  
Homozygous Deletion ◽  
Hematopoietic Stem ◽  
Mixed Genetic Background ◽  
Senescent Phenotype

Abstract Recently, several studies have suggested that the family of cyclin-dependent kinase inhibitors plays a crucial role in regulating hematopoietic stem and progenitor pool size. However, due to a lack of appropriate transplantation models, competitive repopulation assays have not been performed. In the present study we have backcrossed a p21 null allele from mice with a mixed genetic background to inbred C57BL/6 mice. As expected, mouse embryonic fibroblasts (MEFs) derived from B6p21−/− mice failed to undergo senescence, whereas B6p21+/+ MEFs show a normal senescent phenotype. Moreover, B6p21−/− CFU-GM were more resistant to radiation compared to B6p21+/+. In contrast, homozygous deletion of the p21 allele did not affect the percentage of Lin− Sca-1+ c-kit+ cells in S-phase when measured by 7-AAD staining, and did not result in any alterations of in vitro cobblestone area forming cell activity. In a competitive repopulating assay different ratios of Ly5.2 BM cells from B6p21−/− or B6p21+/+ littermates were competed with 2 x 106 Ly5.1 B6 BM cells. Assuming similar repopulating capacity of both cell populations, expected chimerism was calculated. Surprisingly, observed and expected chimerism were identical, strongly suggesting that B6p21−/− stem cells had completely normal competitive repopulating activity for up to 1 year after transplant. Our data argue against an important role of p21 in maintaining stem cell function during steady-state hematopoiesis.

Ex-Vivo Generation and Expansion of Both Lymphoid and Myeloid Lineages from Human Cord Blood (CB) HSC Using a Serum-Free Human Mesenchymal Stem Cell Based Culture System.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2888-2888
Author(s):  
Ana Frias ◽  
Christopher D. Porada ◽  
Kirsten B. Crapnell ◽  
Joaquim M.S. Cabral ◽  
Esmail D. Zanjani ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cord Blood ◽  
Culture System ◽  
Ex Vivo ◽  
Cell Number ◽  
Human Cord Blood ◽  
Serum Free ◽  
Gm Csf ◽  
Cd34 Cells

Abstract The in vitro culture of a hematopoietic stem cell (HSC) graft with either media containing animal-derived components or a feeder layer with ill-defined pathogenic potential such as xenogeneic cell lines or cells modified by viral transformation poses risks that concern scientists and regulatory agencies. In the present studies, we avoided these risks by evaluating the ability of a human stromal-based serum free culture system (hu-ST) to support the ex-vivo expansion/maintenance of human CB HSC. CB CD34+ enriched cells were cultured in serum free medium in the presence of hu-ST with SCF, bFGF, LIF and Flt-3, and the cultures were analyzed for expansion, phenotype and clonogenic ability. We have previously reported the ability of this culture system to allow the successful expansion/maintenance of HSC along the myeloid pathway. In the present study, we investigated whether we could further develop this culture system to simultaneously expand myelopoiesis and lymphopoiesis in vitro. To this end, cord blood CD34+ cells were cultured for a total of 28 days and analyzed every 3 days for expansion and phenotype. There was a progressive increase in CD34 cell number with time in culture. The differentiative profile was primarily shifted towards the myeloid lineage with the presence of CD33, CD15, and CD14. However, a significant number of CD7+ cells were also generated. At week 2 of culture, we observed that 30% of the cells in the culture were CD7 positive. These CD7+CD2-CD3-CD5-CD56-CD16-CD34- cells were then sorted and either plated on top of new irradiated hu-ST layers in the presence of SCF, FLT-3, IL-7, IL-2, and IL-15, or cultured with IL-4, GM-CSF, and FLT-3 in the absence of stroma. Both of these cultures were maintained for an additional 2 weeks. In both sets of cultures, further expansion in the total cell number occurred with the time in culture, and by the end of the week 2, we observed that 25.3±4.18% of the cells had become CD56+ CD3-, a phenotype consistent with that of NK cells. Furthermore, cytotoxicity assays were performed and showed cytotoxic activity that increased in an E:T ratio-dependent fashion. 38.6% of the CD7+ cells grown in the presence of IL-4, GM-CSF, and FLT-3 became CD123+CD11c-, a phenotype characteristic of nonactivated dendritic cells, while 7.3–12.1% adopted an activitated dendritic cell phenotype CD83+CD1a+. In summary, we developed an in vitro culture system that reproducibly allows the effective ex vivo expansion of human cord blood HSCs while maintaining the capability of generating both myeloid and lymphoid hematopoiesis in vitro.

AMN107 Appears Equipotent and May Synergise with Imatinib at the CML Stem Cell Level through Interaction with ABCG2.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1080-1080 ◽  
Author(s):  
H. Jorgensen ◽  
E. Allan ◽  
N. Jordanides ◽  
A. Hamilton ◽  
J. Mountford ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Stem Cell ◽  
Single Agent ◽  
Cell Number ◽  
Total Cell ◽  
Dependent Manner ◽  
Cell Level ◽  
Clinical Resistance

Abstract AMN107 (Novartis) is a novel Abl tyrosine kinase inhibitor specifically developed to be more selective for BcrAbl. AMN107 also maintains activity against the most common mutations associated with clinical resistance to imatinib mesylate (IM). In preclinical studies in cell lines and animal models, AMN107 was found to have greater potency than IM. By 3H-thymidine proliferation assays, the IC50 for AMN107 in K562 cells was 30 +/− 10nM compared with 600 +/− 60nM for IM. AMN107 and IM reduced K562 output cell number to 25% of input at 50 and 1000nM respectively, at 72h. These data are in keeping with the reported 20-fold increase in potency of AMN107 over IM. In addition, we have tested AMN107 for in vitro activity against primary CD34+Ph+ CML cells during 72h of culture in 5 growth factors. In CML cells (n=5), AMN107 and IM failed to reduce input cell number although the total cell output was restricted to 50% of PBS treated control at 2 +/− 1μM for AMN107 and to 31 +/− 7% of PBS treated control for 5μM IM suggesting the drugs were equipotent. The ability of the drugs to inhibit BcrAbl activity was then measured indirectly via the phosphorylation status of CrkL using a specific antiphospho-CrkL antibody and flow cytometry. Once again AMN107 and IM appeared equipotent in CML cells with 5μM of each compound leading to equal de-phosphorylation of CrkL. We next tested the efficacy of AMN107 as a single agent and in combination with IM against quiescent CML cells using in vitro dye (CFSE) tracking experiments. We evaluated by flow cytometry the proportion of input cells remaining alive, CD34+ and undivided (CFSEmax) or in first division. Compared to PBS treated control, 1.7, 2.5, 3.8 and 4.7-fold increases were found in the proportion of input CD34+ cells recovered in divisions 0 and 1 after 3 days exposure to 0.005, 0.05, 0.5 and 5μM AMN107, respectively. This was less accumulation than observed in the IM (5μM)-treated cells (11.0-fold). The combination of IM and AMN107, each at 5μM, was more effective in terms of total cell kill (54 and 74% fewer total cells remaining than with IM and AMN107 alone, respectively) and resulted in fewer viable cells recovered in divisions 0 and 1 than with either agent alone (for the combination, 1.9-fold on PBS treated recovery). We finally assessed the role of ABCG2 in modulating AMN107’s access to its intracellular BcrAbl target. We have previously shown ABCG2 to be over-expressed on CML stem cells and to interact with IM (Blood (2004); 104: 205a). We hypothesised that AMN107 and IM may co-operate as ABCG2 substrates or inhibitors to increase the intracellular levels of either or both drugs thus amplifying their efficacy against target protein specifically in CML stem cells. In competition assays with a known fluorescent substrate of ABCG2 (ie BODIPY-prazosin, BP), a specific inhibitor of the ABCG2 pump (fumitremorgin C, FTC) and an ABCG2 stably transfected AML cell line (AML6.2), the sample treated with BP plus FTC is taken to have greatest retention (100%). AMN107 inhibited efflux in a dose dependent manner to a maximum of 88% at 5μM, similarly to IM. Thus, AMN107 was equipotent with IM in primary CML stem cells in terms of restricting cell growth, inhibiting BcrAbl activity and interacting with ABCG2. However, AMN107 alone lead to less accumulation of quiescent CML cells in vitro as compared to IM, with the combination even more effective in this regard. The apparent co-operative effect of AMN107 and IM at the stem cell level would be predicted to improve clinical responses if tolerated in patients.

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