Manufacturing human pluripotent stem cell derived endothelial cells in scalable and cell-friendly microenvironments

2019 ◽  
Vol 7 (1) ◽  
pp. 373-388
Author(s):  
Haishuang Lin ◽  
Qian Du ◽  
Qiang Li ◽  
Ou Wang ◽  
Zhanqi Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Endothelial Cells ◽  
Stem Cell ◽  
Pluripotent Stem Cells ◽  
Pluripotent Stem Cell ◽  
Human Pluripotent Stem Cells ◽  
Human Pluripotent Stem Cell ◽  
Alginate Hydrogel

Alginate hydrogel tubes are designed for the scalable expansion of human pluripotent stem cells and efficient differentiation into endothelial cells.

scholarly journals Expression of miRNAs from the Imprinted DLK1/DIO3 Locus Signals the Osteogenic Potential of Human Pluripotent Stem Cells

Cells ◽  
2019 ◽  
Vol 8 (12) ◽  
pp. 1523 ◽  
Author(s):  
Laetitia Barrault ◽  
Jacqueline Gide ◽  
Tingting Qing ◽  
Lea Lesueur ◽  
Jorg Tost ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Osteogenic Differentiation ◽  
Cell Lines ◽  
Pluripotent Stem Cells ◽  
Pluripotent Stem Cell ◽  
Human Pluripotent Stem Cells ◽  
Osteogenic Potential ◽  
Human Pluripotent Stem Cell ◽  
Stem Cell Lines

Substantial variations in differentiation properties have been reported among human pluripotent cell lines (hPSC), which could affect their utility and clinical safety. We characterized the variable osteogenic capacity observed between different human pluripotent stem cell lines. By focusing on the miRNA expression profile, we demonstrated that the osteogenic differentiation propensity of human pluripotent stem cell lines could be associated with the methylation status and the expression of miRNAs from the imprinted DLK1/DIO3 locus. More specifically, quantitative analysis of the expression of six different miRNAs of that locus prospectively identified human embryonic stem cells and human-induced pluripotent stem cells with differential osteogenic differentiation capacities. At the molecular and functional levels, we showed that these miRNAs modulated the expression of the activin receptor type 2B and the downstream signal transduction, which impacted osteogenesis. In conclusion, miRNAs of the imprinted DLK1/DIO3 locus appear to have both a predictive value and a functional impact in determining the osteogenic fate of human pluripotent stem cells.

scholarly journals Human pluripotent stem cell-derived organoids and their potential to reduce the use of animal models in research

2021 ◽  
Vol 4 (s1) ◽  
Author(s):  
Salvatore Simmini ◽  
Allen C. Eaves ◽  
Sharon A. Louis ◽  
Wing Chang
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Animal Models ◽  
Pluripotent Stem Cells ◽  
Pluripotent Stem Cell ◽  
Human Pluripotent Stem Cells ◽  
Human Pluripotent Stem Cell ◽  
Tissue Specific ◽  
Organoid Cultures

Efficient and reproducible generation of tissue-specific organoids from Human Pluripotent Stem Cells (hPSCs) represents one of the key tools for reducing the use of animals in research. STEMCELL Technologies is committed to optimizing workflows that efficiently support the generation and maintenance of multiple types of organoid cultures derived from hPSCs.

scholarly journals Towards consistent generation of pancreatic lineage progenitors from human pluripotent stem cells

2015 ◽  
Vol 370 (1680) ◽  
pp. 20140365 ◽  
Author(s):  
Maria Rostovskaya ◽  
Nicholas Bredenkamp ◽  
Austin Smith
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Lines ◽  
Pluripotent Stem Cells ◽  
Pluripotent Stem Cell ◽  
Human Pluripotent Stem Cells ◽  
Type I ◽  
Pancreatic Progenitors ◽  
Stem Cell Lines

Human pluripotent stem cells can in principle be used as a source of any differentiated cell type for disease modelling, drug screening, toxicology testing or cell replacement therapy. Type I diabetes is considered a major target for stem cell applications due to the shortage of primary human beta cells. Several protocols have been reported for generating pancreatic progenitors by in vitro differentiation of human pluripotent stem cells. Here we first assessed one of these protocols on a panel of pluripotent stem cell lines for capacity to engender glucose sensitive insulin-producing cells after engraftment in immunocompromised mice. We observed variable outcomes with only one cell line showing a low level of glucose response. We, therefore, undertook a systematic comparison of different methods for inducing definitive endoderm and subsequently pancreatic differentiation. Of several protocols tested, we identified a combined approach that robustly generated pancreatic progenitors in vitro from both embryo-derived and induced pluripotent stem cells. These findings suggest that, although there are intrinsic differences in lineage specification propensity between pluripotent stem cell lines, optimal differentiation procedures may consistently direct a substantial fraction of cells into pancreatic specification.

Convergence of human pluripotent stem cell, organoid, and genome editing technologies

2021 ◽  
pp. 153537022098580
Author(s):  
Lin Wang ◽  
Zhaohui Ye ◽  
Yoon-Young Jang
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Genome Editing ◽  
Pluripotent Stem Cells ◽  
Pluripotent Stem Cell ◽  
Three Dimensional ◽  
Experimental Models ◽  
Human Pluripotent Stem Cell ◽  
Research Areas ◽  
Technological Advances

The last decade has seen many exciting technological breakthroughs that greatly expanded the toolboxes for biological and biomedical research, yet few have had more impact than induced pluripotent stem cells and modern-day genome editing. These technologies are providing unprecedented opportunities to improve physiological relevance of experimental models, further our understanding of developmental processes, and develop novel therapies. One of the research areas that benefit greatly from these technological advances is the three-dimensional human organoid culture systems that resemble human tissues morphologically and physiologically. Here we summarize the development of human pluripotent stem cells and their differentiation through organoid formation. We further discuss how genetic modifications, genome editing in particular, were applied to answer basic biological and biomedical questions using organoid cultures of both somatic and pluripotent stem cell origins. Finally, we discuss the potential challenges of applying human pluripotent stem cell and organoid technologies for safety and efficiency evaluation of emerging genome editing tools.

scholarly journals Remdesivir but not famotidine inhibits SARS-CoV-2 replication in human pluripotent stem cell-derived intestinal organoids

2020 ◽  
Author(s):  
Jana Krüger ◽  
Rüdiger Groß ◽  
Carina Conzelmann ◽  
Janis A. Müller ◽  
Lennart Koepke ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Pluripotent Stem Cells ◽  
Pluripotent Stem Cell ◽  
Gastrointestinal Symptoms ◽  
Cell Types ◽  
Human Pluripotent Stem Cell ◽  
Intestinal Organoids

Gastrointestinal symptoms in COVID-19 are associated with prolonged symptoms and increased severity. We employed human intestinal organoids derived from pluripotent stem cells (PSC-HIOs) to analyze SARS-CoV-2 pathogenesis and to validate efficacy of specific drugs in the gut. Certain, but not all cell types in PSC-HIOs express SARS-CoV-2 entry factors ACE2 and TMPRSS2, rendering them susceptible to SARS-CoV-2 infection. Remdesivir, a promising drug to treat COVID-19, effectively suppressed SARS-CoV-2 infection of PSC-HIOs. In contrast, the histamine-2-blocker famotidine showed no effect. Thus, PSC-HIOs provide an interesting platform to study SARS-CoV-2 infection and to identify or validate drugs.

Abstract 010: A Separable Upstream Promoter of the Slc8a1 (NCX1) Gene Efficiently Marks Cardiac Cell Populations Derived from Human Pluripotent Stem Cells

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Alejandro Hidalgo-Gonzalez ◽  
Dmitry A Ovchinnikov ◽  
James Hudson ◽  
Justin Cooper-White ◽  
Wolvetang Ernst
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Pluripotent Stem Cells ◽  
Pluripotent Stem Cell ◽  
Human Pluripotent Stem Cells ◽  
Cardiac Cells ◽  
Protein Quantification ◽  
Egfp Expression ◽  
Cardiac Cell

The sarcolemmal Na+/Ca2+ exchanger SLC8A1(NCX) regulates intracellular Ca+ in cardiomyocytes from early developmental stages. The upstream-most SLC8A1(NCX1) promoter is well conserved amongst the homoeothermic animals and contains putative binding sites for transcription factors of the NKX, GATA, STAT and CDX families. We hypothesized that functional cardiac cells with mature cardiac structural markers will express the sarcolemmal Na+/Ca2+ calcium antiporting channel, important for proper functional contractivity of in vitro differentiated human pluripotent stem cells. Pseudotyped lentiviral particles delivering NCX1cp-EGFP reporter cassette were used to confirm the efficiency and specificity of the reporter in rodent foetal cardiac cell isolates, and to establish stable human pluripotent stem cell lines. Cells were differentiated using a 2D induction protocol, and gene expression analysis and protein quantification carried at day 16. Initial NCX1cp-EGFP expression was observed from day 10-11 of cardiac differentiation. Beating foci were visualized 1-2 day after initial NCXCP-EGFP expression, reporter expression was confined to the grouped and individual beating cells, and highly correlated with the efficiency of spontaneously contractile cell production. At later stages, NCX1cp-EGFP expression correlated with clusters of formed spontaneously contractile units harbouring essentially all cardiomyocytes present in cultures, as evidenced by colocalization of high levels of cardiac troponin T (cTnT) and α-actinin proteins. The EGFP+ sorted fraction of differentiated cultures was found to be highly enriched in both early (ISL1, TBX5) and late (cTnT, MYH6) cardiomyocyte markers when compared to the EGFP- fraction. We conclude that a ~3 kb genomic fragment of the distal cardiac-specific promoter of the SLC8A1(NCX1) containing the upstream-most exon of the gene is sufficient to drive the expression of a lentiviral reporter in both rodent heart-derived primary and human (embryonic and induced) pluripotent stem cell-derived cardiac cells. Isolation of a homogenous and functional cardiomyogenic population represents one of the key objectives for cardiac tissue engineering, and in particular in vitro drug screening applications.

scholarly journals Autologous correction in patient induced pluripotent stem cell-endothelial cells to identify a novel pathogenic mutation of hereditary hemorrhagic telangiectasia

2020 ◽  
Vol 10 (4) ◽  
pp. 204589401988535
Author(s):  
Fang Zhou ◽  
Xiuli Zhao ◽  
Xiu Liu ◽  
Yanyan Liu ◽  
Feng Ma ◽  
...  
Keyword(s):  
Stem Cells ◽  
Endothelial Cells ◽  
Stem Cell ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Hereditary Hemorrhagic Telangiectasia ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Differentiation Potential ◽  
Induced Pluripotent

Hereditary hemorrhagic telangiectasia is a rare disease with autosomal dominant inheritance. More than 80% hereditary hemorrhagic telangiectasia patients carry heterozygous mutations of Endoglin or Activin receptor-like kinase-1 genes. Endoglin plays important roles in vasculogenesis and human vascular disease. In this report, we found a novel missense mutation (c.88T > C) of Endoglin gene in a hereditary hemorrhagic telangiectasia 1 patient. Induced pluripotent stem cells of the patient were generated and differentiated into endothelial cells. The hereditary hemorrhagic telangiectasia-induced pluripotent stem cells have reduced differentiation potential toward vascular endothelial cells and defective angiogenesis with impaired tube formation. Endoplasmic reticulum retention of the mutant Endoglin (Cys30Arg, C30R) causes less functional protein trafficking to cell surface, which contributes to the pathogenesis of hereditary hemorrhagic telangiectasia. Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 genetic correction of the c.88T > C mutation in induced pluripotent stem cells revealed that C30R mutation of Endoglin affects bone morphogenetic protein 9 downstream signaling. By establishing a human induced pluripotent stem cell from hereditary hemorrhagic telangiectasia patient peripheral blood mononuclear cells and autologous correction on mutant hereditary hemorrhagic telangiectasia-induced pluripotent stem cells, we were able to identify a new disease-causing mutation, which facilitates us to understand the roles of Endoglin in vascular development and pathogenesis of related vascular diseases.

Abstract 498: Combinatorial Extracellular Matrix Microarrays for Probing Endothelial Differentiation of Human Induced Pluripotent Stem Cells

2015 ◽  
Vol 35 (suppl_1) ◽  
Author(s):  
Luqia Hou ◽  
John Coller ◽  
Vanita Natu ◽  
Ngan Huang
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Stem Cell ◽  
Pluripotent Stem Cells ◽  
Pluripotent Stem Cell ◽  
Endothelial Differentiation ◽  
Induced Pluripotent

Human induced pluripotent stem cell (iPSC)-derived endothelial cells (iPSC-ECs) are a promising cell source for vascular regeneration in patients with peripheral arterial disease. However, a critical bottleneck to their clinical translation is the ability to differentiate the cells reproducibly at high yields. Since endothelial cells interact with the basement membrane extracellular matrix (ECM), we sought to examine the role of ECMs on endothelial differentiation using combinatorial ECM microenvironments. ECM microarrays were developed by covalent conjugation of ECMs (gelatin, fibronectin, laminin, heparin sulfate proteoglycans, collagen IV, matrigel) and the multi-component combinations thereof. The pluripotent stem cells attached to the ECMs and subsequently differentiated over the course of 5 days. Endothelial differentiation was semi-quantitatively scored based on the degree of CD31 staining. Our results demonstrated greater levels of CD31staining when cultured on gelatin + matrigel + laminin (G+M+L) or fibronectin + laminin + heparan sulfate (F+L+H), compared to other combinations across three human pluripotent stem cell lines (iPSC-Huf5, iPSC-CON1, and ESC-H1). This enhancement in endothelial differentiation on the microscale was confirmed at larger cell culture platforms in which a marked increase in CD31+ cells was observed in G+M+L modified-dishes (> 5 fold), and F+L+H combination (> 10 fold), compared to gelatin-modified dishes. RT-PCR further confirmed the transcriptional upregulation in endothelial markers for CD31 (> 2 fold) and VE-cadherin (> 4 fold) on G+M+L, compared to gelatin-modified dishes. To elucidate the role of cell-ECM interactions on endothelial differentiation, gene expression of integrin subunits were examined. Gene expression was markedly upregulated in integrins α1 (>10 fold); α4, α5, and αV (>5 fold); and β1, β3 (>50 fold), and β4, when comparing differentiated cells on day14 to undifferentiated cells. The upregulation of integrin subunits was concomitant with upregulation in endothelial genes. Together, this data suggested that combinatorial ECMs differentially promote endothelial differentiation, in part through integrin-mediated pathways.

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