A Rho-Associated Coiled-Coil Containing Kinase Inhibitor, Y-27632, Improves Viability of Dissociated Single Cells, Efficiency of Colony Formation, and Cryopreservation in Porcine Pluripotent Stem Cells

2019 ◽  
Vol 21 (1) ◽  
pp. 37-50 ◽  
Author(s):  
Sang-Ki Baek ◽  
Young-Soo Cho ◽  
Ik-Sung Kim ◽  
Soo-Been Jeon ◽  
Dae-Ky Moon ◽  
...  
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Colony Formation ◽  
Kinase Inhibitor ◽  
Single Cells ◽  
Coiled Coil

344 LOSS OF CONSTRAINTS IMPOSED BY TISSUE ENVIRONMENT ACTIVATES EARLY SIGNALS OF CELLULAR REPROGRAMMING

2015 ◽  
Vol 27 (1) ◽  
pp. 260
Author(s):  
D. A. Anzalone ◽  
D. Iuso ◽  
P. Toschi ◽  
F. Zacchini ◽  
G. E. Ptak ◽  
...  
Keyword(s):  
Stem Cells ◽  
Suspension Culture ◽  
Pluripotent Stem Cells ◽  
Single Cells ◽  
Adherent Cells ◽  
Pluripotency Factors ◽  
Differentiated Cells ◽  
Nanog Expression ◽  
Muse Cells

Pluripotency is the ability of one cell to generate every cell type of the 3 germ layers, a property typically owned by embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC), with some exceptions; multilineage-differentiating stress-enduring (Muse) cells are an example. Muse cells, described as pre-existing pluripotent stem cells in mesenchymal tissues (Kuroda et al. 2010) are able to form clusters from single cells in suspension culture, express pluripotency factors and differentiate into cell types of the 3 germ layers, like ESC and iPSC. In addition, Muse cells are proposed to be the only source of cells capable to generate iPSC by current methodologies (Wakao et al. 2011). However, it is unclear whether they are normally present in adult tissue, derive from precursors stem or differentiated cells, or are induced by the in vitro conditions. In our work, we tested the hypothesis that the transition from a committed (tissue) to an uncommitted (in vitro culture) environment triggers in the cells the activation of a default gene circuitry leading to pluripotency. Adult skin fibroblasts were obtained from sheep ear biopsy (n = 3) and expanded in vitro (A) or cultured in suspension in hanging drops (B) or in nonadherent dishes (C) in MEM with 10% FBS. In a subsequent experiment, clonal expansion was attempted by culturing single suspension cells in drops of medium (D). Pluripotency was assessed analysing Oct4 and Nanog expression, using real-time PCR (mRNA) and Western blotting (protein), in cultured fibroblasts compared to whole ear biopsy (30-day-old fetus was used as positive control, CTR). Furthermore, in adherent cells (A) and in clusters obtained from suspension culture (B, C, D), Oct4 and Nanog expression was compared by immunofluorescence. We found that while in the ear biopsy not one of these pluripotency markers was expressed, in in vitro-expanded fibroblasts both mRNA and protein expression was detected; mRNA expression value (mean ± s.e.m. relative to CTR) was 0.59 ± 0.18 for Nanog and 0.2 ± 0.07 for Oct4. Moreover, fibroblasts in suspension (B, C, D) were able to form clusters [obtained from 32% (16/50) of single cells, D] similar to those normally obtained with ESC, iPSC. and Muse cells. All the clusters (B, C, D) showed a more intensive signal of Oct4 and Nanog protein compared to adherent cells by immunofluorescence. In the present work we demonstrate that adult somatic cells (skin fibroblasts) express key pluripotency factors, such as OCT4 and Nanog, in both adherent and suspension culture, after removal from the tissue (ear). We can conclude that the simple in vitro culture switches on the expression of pluripotency markers in adult somatic cells. Removal from the context of the tissue probably leads the cells to lose their tissue-specific identity and acquire a new undifferentiated one, which in an optimal condition culture could result in pluripotency. Our interpretation is that reprogramming must be an automatic, default response when differentiated cells are removed from the constraints imposed by a multicellular environment.

scholarly journals Single cell transcriptome analysis reveals markers of naïve and lineage-primed hematopoietic progenitors derived from human pluripotent stem cells

2019 ◽  
Author(s):  
Antonella Fidanza ◽  
Nicola Romanò ◽  
Prakash Ramachandran ◽  
Sara Tamagno ◽  
Martha Lopez-Yrigoyen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Single Cells ◽  
Human Pluripotent Stem Cells ◽  
In Vitro Differentiation ◽  
Hematopoietic Progenitors ◽  
Lineage Priming ◽  
Cell Transcriptome ◽  
Single Cell Transcriptome

AbstractDuring embryogenesis the hematopoietic system develops through distinct waves that generate progenitors with increasing lineage potential, ultimately producing haematopoietic stem cells (HSCs). In vitro differentiation of human pluripotent stem cells (hPSCs) follows the early steps of haematopoietic development but the production of HSCs has proven more challenging. To study the dynamics and heterogeneity of hematopoietic progenitor cells generated in vitro from hPSCs, we performed RNA sequencing of over 10000 CD235a-CD43+single cells. We identified the transcriptome of naïve progenitors and those primed toward erythroid, megakaryocyte and leukocyte lineages, and revealed their markers by clustering, trajectory analyses and functional assays. CD44 marks naïve clonogenic progenitors that express the transcription factor, LMO4 and can be expanded upon BMP4 stimulation. Naïve progenitors give rise to primed CD326+erythroid, ICAM2+CD9+megakaryocyte, and monocyte, neutrophil and eosinophil progenitors. We have generated an online dataset of human hematopoietic progenitors and their transcriptional remodelling upon lineage priming.

scholarly journals Patient-specific hepatocyte-like cells derived from induced pluripotent stem cells model pazopanib-mediated hepatotoxicity

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Yukti Choudhury ◽  
Yi Chin Toh ◽  
Jiangwa Xing ◽  
Yinghua Qu ◽  
Jonathan Poh ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Drug Testing ◽  
Kinase Inhibitor ◽  
Patient Specific ◽  
Drug Induced ◽  
Clinical Phenotypes ◽  
Induced Pluripotent

Abstract Idiosyncratic drug-induced hepatotoxicity is a major cause of liver damage and drug pipeline failure, and is difficult to study as patient-specific features are not readily incorporated in traditional hepatotoxicity testing approaches using population pooled cell sources. Here we demonstrate the use of patient-specific hepatocyte-like cells (HLCs) derived from induced pluripotent stem cells for modeling idiosyncratic hepatotoxicity to pazopanib (PZ), a tyrosine kinase inhibitor drug associated with significant hepatotoxicity of unknown mechanistic basis. In vitro cytotoxicity assays confirmed that HLCs from patients with clinically identified hepatotoxicity were more sensitive to PZ-induced toxicity than other individuals, while a prototype hepatotoxin acetaminophen was similarly toxic to all HLCs studied. Transcriptional analyses showed that PZ induces oxidative stress (OS) in HLCs in general, but in HLCs from susceptible individuals, PZ causes relative disruption of iron metabolism and higher burden of OS. Our study establishes the first patient-specific HLC-based platform for idiosyncratic hepatotoxicity testing, incorporating multiple potential causative factors and permitting the correlation of transcriptomic and cellular responses to clinical phenotypes. Establishment of patient-specific HLCs with clinical phenotypes representing population variations will be valuable for pharmaceutical drug testing.

scholarly journals A Rainbow Reporter Tracks Single Cells and Reveals Heterogeneous Cellular Dynamics among Pluripotent Stem Cells and their Differentiated Derivatives

2020 ◽  
Author(s):  
Danny El-Nachef ◽  
Kevin Shi ◽  
Kevin M. Beussman ◽  
Refugio Martinez ◽  
Mary C. Regier ◽  
...  
Keyword(s):  
Stem Cells ◽  
Single Cell ◽  
Pluripotent Stem Cells ◽  
Cortical Neurons ◽  
Neural Differentiation ◽  
Single Cells ◽  
Directed Differentiation ◽  
Reporter Expression ◽  
Cell Lineages ◽  
Molecular Identity

SummaryRecent single cell analyses have found molecular heterogeneities within populations of pluripotent stem cells (PSCs). A tool that tracks single cell lineages and their phenotypes longitudinally would reveal whether heterogeneity extends beyond molecular identity. Hence, we generated a stable Cre-inducible rainbow reporter human PSC line that provides up to 18 unique membrane-targeted fluorescent barcodes. These barcodes enable repeated assessments of single cells as they clonally expand, change morphology, and migrate. Owing to the cellular resolution of this reporter, we identified subsets of PSCs with enhanced clonal expansion, synchronized cell divisions, and persistent localization to colony edges. Reporter expression was stably maintained throughout directed differentiation into cardiac myocytes, cortical neurons, and hepatoblasts. Repeated examination of neural differentiation revealed self-assembled cortical tissues derive from clonally dominant progenitors. Collectively, these findings demonstrate the broad utility and easy implementation of this reporter line for tracking single cell behavior.

scholarly journals Single-cell phenotyping of human induced pluripotent stem cells by high-throughput imaging

2015 ◽  
Author(s):  
Hans Christian Volz ◽  
Florian Heigwer ◽  
Tatjana Wuest ◽  
Marta Galach ◽  
Jochen Utikal ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Single Cell ◽  
High Throughput ◽  
Pluripotent Stem Cells ◽  
Single Cells ◽  
Phenotypic Variability ◽  
Screening Experiments ◽  
Induced Pluripotent ◽  
High Throughput Imaging

Single-cell phenotyping promises to yield insights into biological responses in heterogeneous cell populations. We developed a method based on single-cell analysis to phenotype human induced pluripotent stem cells (hIPSC) by high-throughput imaging. Our method uses markers for morphology and pluripotency as well as social features to characterize perturbations using a meta-phenotype based on mapping single cells to distinct phenotypic classes. Analysis of perturbations on a single cell level enhances the applicability of human induced pluripotent stem cells (hIPSC) for screening experiments taking the inherently increased phenotypic variability of these cells into account. We adapted miniaturized culture conditions to allow for the utilization of hIPSC in RNA interference (RNAi) high-throughput screens and single cell phenotyping by image analysis. We identified key regulators of pluripotency in hIPSC masked in a population-averaged analysis and we confirmed several candidate genes (SMG1, TAF1) and assessed their effect on pluripotency.

scholarly journals Improving Generation of Cardiac Organoids from Human Pluripotent Stem Cells Using the Aurora Kinase Inhibitor ZM447439

Biomedicines ◽  
2021 ◽  
Vol 9 (12) ◽  
pp. 1952
Author(s):  
Su-Jin Lee ◽  
Hyeon-A Kim ◽  
Sung-Joon Kim ◽  
Hyang-Ae Lee
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Drug Testing ◽  
Kinase Inhibitor ◽  
Aurora Kinase ◽  
Gene Profiling ◽  
Success Rates ◽  
Aurora Kinase Inhibitor ◽  
Drug Induced

Drug-induced cardiotoxicity reduces the success rates of drug development. Thus, the limitations of current evaluation methods must be addressed. Human cardiac organoids (hCOs) derived from induced pluripotent stem cells (hiPSCs) are useful as an advanced drug-testing model; they demonstrate similar electrophysiological functionality and drug reactivity as the heart. How-ever, similar to other organoid models, they have immature characteristics compared to adult hearts, and exhibit batch-to-batch variation. As the cell cycle is important for the mesodermal differentiation of stem cells, we examined the effect of ZM447439, an aurora kinase inhibitor that regulates the cell cycle, on cardiogenic differentiation. We determined the optimal concentration and timing of ZM447439 for the differentiation of hCOs from hiPSCs and developed a novel protocol for efficiently and reproducibly generating beating hCOs with improved electrophysiological functionality, contractility, and yield. We validated their maturity through electro-physiological- and image-based functional assays and gene profiling with next-generation sequencing, and then applied these cells to multi-electrode array platforms to monitor the cardio-toxicity of drugs related to cardiac arrhythmia; the results confirmed the drug reactivity of hCOs. These findings may enable determination of the regulatory mechanism of cell cycles underlying the generation of iPSC-derived hCOs, providing a valuable drug testing platform.

Hematopoiesis of Human Induced Pluripotent Stem Cells Derived From Patients with Down Syndrome

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 881-881
Author(s):  
Natsumi Nishihama ◽  
Yasuhiro Ebihara ◽  
Feng Ma ◽  
Wenyu Yang ◽  
Daisuke Tomizawa ◽  
...  
Keyword(s):  
Stem Cells ◽  
Down Syndrome ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Colony Formation ◽  
Trisomy 21 ◽  
Conflicts Of Interest ◽  
P Value ◽  
Increased Risk ◽  
Induced Pluripotent

Abstract Abstract 881 Trisomy 21, genetic hallmark of Down syndrome, is the most frequent human chromosomal abnormality. Infants and children with Down Syndrome (DS) are known to have some hematological disorders with an increased risk of developing leukemia. Ten to 20% of newborn with DS are diagnosed as neonatal preleukemic status, Transient Myeloproliferative Disorder (TMD), and approximately 30% of TMD patients are predisposed to acute megakaryoblastic leukemia (AMKL). Recently, acquired mutations in the N-terminal activation domain of the GATA1 gene, leading to expression of a shorter GATA1 isoform (GATA1s), have been reported in AMKL and TMD (Wechsler et al., 2002; Mundschau et al., 2003), but neither patients nor mice with germline mutations leading to expression of GATA1s developed AMKL and TMD in the absent of trisomy 21. These findings suggested that trisomy 21 itself directly contributes to the development of AMKL and TMD. However, the role of trisomy 21 in hematopoiesis, particularly in the human fetus remains poorly understood. To better understand the effects of trisomy 21 on hematopoiesis in embryonic stage and leukemogenesis, we employed human induced pluripotent stem cells (hiPSCs) derived from patients with DS (DS-hiPSCs). Six DS-hiPS and 5 hiPS cell lines (control) from healthy donors, which we used here, were all created from skin fibroblasts and reprogrammed by the defined 3 or 4 reprogramming factors (OCT3/4, KLF4, and SOX2, or c-MYC in addition to the 3 factors, respectively). We generated blood cells from DS-hiPSCs and controls with coculture system using murine aorta-gonad-mesonephros (AGM)-derived stromal cell line (Ma et al., 2009). The cells from hiPSCs were harvested at D11 or D12 of coculture and analyzed the presence of hematopoietic markers and the potentials of hematopoietic colony formation. In the experiments using hiPSCs reprogrammed by 3 factors, human CD34 expression in harvested cells from DS-hiPSCs or controls were detected 10.06 ± 4.35% and 3.04%, respectively. CD45 expression of CD34+ cells was small proportion in both DS-hiPSCs and controls. We next examined the hematopoietic colony formation. Both myeloid and erythroid colonies were detected. Number of colonies formed from DS-hiPSCs was 43.7±11.1 to 74.3±11.2 per an iPSC colony. It's approximately 2 to 3.5 folds numbers of control (p-value<0.05). Similar results were obtained in the experiments using hiPSCs reprogrammed by 4 factors. These results indicated that hiPSCs derived from patients with Down syndrome could differentiate into multiple hematopoietic cell lineages and the differentiation into hematopoietic lineage was promoted in DS patients. Further researches are under investigation to identify the responsible genes in trisomy 21 for acceleration of hematopoiesis with microarray analysis. Our study may contribute to understanding of the effects of trisomy 21 on hematopoiesis and effective use of patients derived hiPSCs in research and clinical application. Disclosures: No relevant conflicts of interest to declare.

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