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Stem Cells Translational Medicine Jun 2024As research on in vitro cardiotoxicity assessment and cardiac disease modeling becomes more important, the demand for human pluripotent stem cell-derived cardiomyocytes...
As research on in vitro cardiotoxicity assessment and cardiac disease modeling becomes more important, the demand for human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) is increasing. However, it has been reported that differentiated hPSC-CMs are in a physiologically immature state compared to in vivo adult CMs. Since immaturity of hPSC-CMs can lead to poor drug response and loss of acquired heart disease modeling, various approaches have been attempted to promote maturation of CMs. Here, we confirm that peroxisome proliferator-activated receptor alpha (PPARα), one of the representative mechanisms of CM metabolism and cardioprotective effect also affects maturation of CMs. To upregulate PPARα expression, we treated hPSC-CMs with fenofibrate (Feno), a PPARα agonist used in clinical hyperlipidemia treatment, and demonstrated that the structure, mitochondria-mediated metabolism, and electrophysiology-based functions of hPSC-CMs were all mature. Furthermore, as a result of multi electrode array (MEA)-based cardiotoxicity evaluation between control and Feno groups according to treatment with arrhythmia-inducing drugs, drug response was similar in a dose-dependent manner. However, main parameters such as field potential duration, beat period, and spike amplitude were different between the 2 groups. Overall, these results emphasize that applying matured hPSC-CMs to the field of preclinical cardiotoxicity evaluation, which has become an essential procedure for new drug development, is necessary.
PubMed: 38946019
DOI: 10.1093/stcltm/szae029 -
The Ocular Surface Jun 2024Human donor corneas are an essential control tissue for corneal research. We utilized whole mount immunofluorescence (WM-IF) to evaluate how the storage affects the...
PURPOSE
Human donor corneas are an essential control tissue for corneal research. We utilized whole mount immunofluorescence (WM-IF) to evaluate how the storage affects the tissue integrity and putative limbal stem cells in human and porcine corneas. Moreover, we compare this information with the marker expression patterns observed in human pluripotent stem cell (hPSC)-derived LSCs.
METHODS
The expression of putative LSC markers was analyzed with WM-IF and the fluorescence intensity was quantified in human donor corneas stored for 1-30 days, and in porcine corneas processed 0-6 hours after euthanasia. The results were compared with the staining of human and porcine corneal cryosections and with both primary and hPSC-derived LSC cultures.
RESULTS
WM-IF analyses emerged as a more effective method when compared to tissue sections for visualizing the expression of LSC markers within human and porcine corneas. Storage duration was a significant factor influencing the expression of LSC markers, as human tissues stored longer exhibited notable epithelial degeneration and lack of LSC markers. Porcine corneas replicated the expression patterns observed in fresh human tissue. We validated the diverse expression patterns of PAX6 in the limbal-corneal region, which aligned with findings from hPSC-LSC differentiation experiments.
CONCLUSIONS
WM-IF coupled with quantification of fluorescence intensities proved to be a valuable tool for investigating LSC marker expression in both human and porcine tissues ex vivo. Prolonged storage significantly influences the expression of LSC markers, underscoring the importance of fresh human or substitute control tissue when studying limbal stem cell biology.
PubMed: 38945477
DOI: 10.1016/j.jtos.2024.06.004 -
Journal of Pharmacological and... Jun 2024This editorial prefaces the annual themed issue on safety pharmacology (SP) methods which has been published since 2004 in the Journal of Pharmacological and...
This editorial prefaces the annual themed issue on safety pharmacology (SP) methods which has been published since 2004 in the Journal of Pharmacological and Toxicological Methods (JPTM). Here we highlight content derived from the 2023 Safety Pharmacology Society (SPS) meeting held in Brussels, Belgium. The meeting generated 138 abstracts, reproduced in the current volume of JPTM. As in prior years, the manuscripts reflect various areas of innovation in SP including in silico modeling of stroke volume, cardiac output and systemic vascular resistance, computational approaches that compare drug-induced proarrhythmic sensitivity of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), an evaluation of the utility of the corrected J-Tpeak and Tpeak-to-Tend parameters from the ECG as potential proarrhythmia biomarkers, and the applicability of nonclinical concentration-QTc (C-QTc) modeling of data derived from the conduct of the in vivo QTc study as a component of the core battery of safety pharmacology studies.
PubMed: 38945308
DOI: 10.1016/j.vascn.2024.107533 -
Stem Cell Research Jun 2024Vascular Ehlers-Danlos Syndrome (vEDS) is an inherited connective tissue disorder caused by COL3A1 gene, mutations that encodes type III collagen, a crucial component of...
Vascular Ehlers-Danlos Syndrome (vEDS) is an inherited connective tissue disorder caused by COL3A1 gene, mutations that encodes type III collagen, a crucial component of blood vessels. vEDS can be life-threatening as these patients can have severe internal bleeding due to arterial rupture. Here, we generated induced pluripotent stem cell (iPSC) lines from two vEDS patients carrying a missense mutation in the COL3A1 (c.226A > G, p.Asn76Asp) gene. These lines exhibited typical iPSC characteristics including morphology, expression of pluripotency markers, and could differentiate to all three germ layer. These iPSC lines can serve as valuable tools for elucidating the pathophysiology underlying vEDS.
PubMed: 38944978
DOI: 10.1016/j.scr.2024.103485 -
Fluorescence-activated nuclear sorting (FANS) of nuclei from in vitro-generated syncytiotrophoblast.Placenta Jun 2024Large, multinucleated cells, like syncytiotrophoblasts (STB), are not readily analyzed by standard methods used for single cells, such as single-cell RNA-sequencing and...
Large, multinucleated cells, like syncytiotrophoblasts (STB), are not readily analyzed by standard methods used for single cells, such as single-cell RNA-sequencing and fluorescence-activated cellular sorting (FACS). Here we have demonstrated that fluorescence-activated nuclear sorting (FANS) is suitable to analyze nuclei from STB. Human pluripotent stem cells (PSCs) can be differentiated into a mixed trophoblast populations comprising approximately 20 % STB by treatment with BMP4 (Bone Morphogenetic Protein-4), plus A83-01 and PD173074, inhibitors of activin and FGF2 signaling, respectively (the BAP model) in about a week. Here we demonstrate that FANS can be used to separate two types of STB nuclei from the nine different clusters of trophoblast nuclei previously identified in the BAP model by single nucleus RNA sequencing (snRNAseq). Rather than using cell surface markers, as in FACS, transcription factors in various combinations were employed to target specific nuclear types. Nuclei were isolated at d 8 of BAP differentiation of H1 human embryonic stem cells and fixed in 4 % paraformaldehyde. After permeabilization in 0.1 % triton X-100, nuclei were incubated for 3 and 1 h at 4 °C with primary and secondary antibodies respectively and nuclear samples were then subjected to FANS. By using markers identified by snRNA and immunohistochemistry, nuclei were first sorted into a Topoisomerase-1, or TOP1, bright population and then into the two STB subpopulations by using antibodies to JUNB (Jun B Proto-Oncogene) and TFCP2L1 (Transcription Factor CP2 Like 1). The protocol established here is simple, straightforward, and efficient and can be used on a relatively large scale to sort individual subtypes of nuclei from mixed populations of trophoblasts for further analysis.
PubMed: 38944560
DOI: 10.1016/j.placenta.2024.06.007 -
Current Problems in Cardiology Jun 2024Congenital heart disease (CHD) affects approximately 1% of live births worldwide, making it the most common congenital anomaly in newborns. Recent advancements in... (Review)
Review
Congenital heart disease (CHD) affects approximately 1% of live births worldwide, making it the most common congenital anomaly in newborns. Recent advancements in genetics and genomics have significantly deepened our understanding of the genetics of CHDs. While the majority of CHD etiology remains unclear, evidence consistently indicates that genetics play a significant role in its development. CHD etiology holds promise for enhancing diagnosis and developing novel therapies to improve patient outcomes. In this review, we explore the contributions of both monogenic and polygenic factors of CHDs and highlight the transformative impact of emerging technologies on these fields. We also summarized the state-of-the-art techniques, including targeted next-generation sequencing (NGS), whole genome and whole exome sequencing (WGS, WES), single-cell RNA sequencing (scRNA-seq), human induced pluripotent stem cells (hiPSCs) and others, that have revolutionized our understanding of cardiovascular disease genetics both from diagnosis perspective and from disease mechanism perspective in children and young adults. These molecular diagnostic techniques have identified new genes and chromosomal regions involved in syndromic and non-syndromic CHD, enabling a more defined explanation of the underlying pathogenetic mechanisms. As our knowledge and technologies continue to evolve, they promise to enhance clinical outcomes and reduce the CHD burden worldwide.
PubMed: 38944223
DOI: 10.1016/j.cpcardiol.2024.102726 -
Stem Cell Research Jun 2024Recent studies reported that the mutation in the THAP11 gene (THAP11) could be responsible for the inborn vitamin deficiency known as cobalamin disorder, by affecting...
Recent studies reported that the mutation in the THAP11 gene (THAP11) could be responsible for the inborn vitamin deficiency known as cobalamin disorder, by affecting the expression of the enzyme MMACHC, key in the cobalamin metabolism. However, the specifics of the molecular mechanism are largely unknown. In here we generated genetically modified human pluripotent stem cell lines with THAP11 mutation, providing a new research tool for futher exploring the molecular mechanism. The established hPSC lines remain pluripotent, showing expression of OCT3/4, differentiation capacity to the three germ layers and displaying normal karyotype.
PubMed: 38943762
DOI: 10.1016/j.scr.2024.103483 -
Scientific Reports Jun 2024Eukaryotic cells can synthesize formyl-methionine (fMet)-containing proteins not only in mitochondria but also in the cytosol to some extent. Our previous study revealed...
Eukaryotic cells can synthesize formyl-methionine (fMet)-containing proteins not only in mitochondria but also in the cytosol to some extent. Our previous study revealed substantial upregulation of N-terminal (Nt)-fMet-containing proteins in the cytosol of SW480 colorectal cancer cells. However, the functional and pathophysiological implications remain unclear. Here, we demonstrated that removal of the Nt-formyl moiety of Nt-fMet-containing proteins (via expressing Escherichia coli PDF peptide deformylase) resulted in a dramatic increase in the proliferation of SW480 colorectal cancer cells. This proliferation coincided with the acquisition of cancer stem cell features, including reduced cell size, enhanced self-renewal capacity, and elevated levels of the cancer stem cell surface marker CD24 and pluripotent transcription factor SOX2. Furthermore, deformylation of Nt-fMet-containing proteins promoted the tumorigenicity of SW480 colorectal cancer cells in an in vivo xenograft mouse model. Taken together, these findings suggest that cytosolic deformylation has a tumor-enhancing effect, highlighting its therapeutic potential for cancer treatment.
Topics: Humans; Neoplastic Stem Cells; Animals; Cytosol; Mice; Cell Line, Tumor; Cell Proliferation; Amidohydrolases; Colorectal Neoplasms; CD24 Antigen; SOXB1 Transcription Factors; Disease Progression; Methionine
PubMed: 38942903
DOI: 10.1038/s41598-024-65701-1 -
Stem Cell Reports Jun 2024Genetic differences between pluripotent stem cell lines cause variable activity of extracellular signaling pathways, limiting reproducibility of directed differentiation...
Genetic differences between pluripotent stem cell lines cause variable activity of extracellular signaling pathways, limiting reproducibility of directed differentiation protocols. Here we used human embryonic stem cells (hESCs) to interrogate how exogenous factors modulate endogenous signaling events during specification of foregut endoderm lineages. We find that transforming growth factor β1 (TGF-β1) activates a putative human OTX2/LHX1 gene regulatory network which promotes anterior fate by antagonizing endogenous Wnt signaling. In contrast to Porcupine inhibition, TGF-β1 effects cannot be reversed by exogenous Wnt ligands, suggesting that induction of SHISA proteins and intracellular accumulation of Fzd receptors render TGF-β1-treated cells refractory to Wnt signaling. Subsequently, TGF-β1-mediated inhibition of BMP and Wnt signaling suppresses liver fate and promotes pancreas fate. Furthermore, combined TGF-β1 treatment and Wnt inhibition during pancreatic specification reproducibly and robustly enhance INSULIN cell yield across hESC lines. This modification of widely used differentiation protocols will enhance pancreatic β cell yield for cell-based therapeutic applications.
PubMed: 38942030
DOI: 10.1016/j.stemcr.2024.05.010 -
Stem Cell Reports Jun 2024A comprehensive understanding of the human pluripotent stem cell (hPSC) differentiation process stands as a prerequisite for the development of hPSC-based therapeutics....
A comprehensive understanding of the human pluripotent stem cell (hPSC) differentiation process stands as a prerequisite for the development of hPSC-based therapeutics. In this study, single-cell RNA sequencing (scRNA-seq) was performed to decipher the heterogeneity during differentiation of three hPSC lines toward corneal limbal stem cells (LSCs). The scRNA-seq data revealed nine clusters encompassing the entire differentiation process, among which five followed the anticipated differentiation path of LSCs. The remaining four clusters were previously undescribed cell states that were annotated as either mesodermal-like or undifferentiated subpopulations, and their prevalence was hPSC line dependent. Distinct cluster-specific marker genes identified in this study were confirmed by immunofluorescence analysis and employed to purify hPSC-derived LSCs, which effectively minimized the variation in the line-dependent differentiation efficiency. In summary, scRNA-seq offered molecular insights into the heterogeneity of hPSC-LSC differentiation, allowing a data-driven strategy for consistent and robust generation of LSCs, essential for future advancement toward clinical translation.
PubMed: 38942029
DOI: 10.1016/j.stemcr.2024.06.001