scholarly journals 4241 Identification of small molecules that facilitate the efficient differentiation of stem cell derived β-cells

2020 ◽  
Vol 4 (s1) ◽  
pp. 8-8
Author(s):  
Yuhao Min ◽  
Chris Clifford ◽  
Quinn P. Peterson
Keyword(s):  
Stem Cells ◽  
Small Molecules ◽  
High Throughput ◽  
Single Cells ◽  
Embryonic Stem ◽  
Immunocytochemical Staining ◽  
Β Cells ◽  
Cell Stage ◽  
Chemical Screening ◽  
Screening Platform

OBJECTIVES/GOALS: In this study, we established a high-throughput chemical screening platform to identify small molecules that facilitates efficient differentiation of stem cells derived β (SC-β) cells. Using this platform, we identified several compounds that potentially increase the differentiation efficiency. METHODS/STUDY POPULATION: Differentiation of human embryonic stem cells (HUES8) into SC-β was carried out using previously published protocols in a 3D cell suspension. Single cells were replated in Matrigel-coated well plates at the start of different stages depending on experiments. Differentiation medium supplemented with small molecules at a final concentration of 2 M and 0.2 M was used throughout the stage. All the cells were then fixed and permeabilized. Immunocytochemical staining was performed. Images of each well were taken and analyzed. Numbers of the total cell, insulin-positive cell, NKX6.1-positive cell, and co-positive cell were recorded. Candidate compounds were validated using flow cytometry or ICC. RESULTS/ANTICIPATED RESULTS: We identified several hit compounds that significantly increase the NKX6.1 positive cell percentage compared to the DMSO-treated controls when treated at the PP1 cell stage. Follow up assays demonstrated that at least one of these putative hits reproducibly increased NKX6.1 expression. In addition, we identified other compounds that significantly increase the insulin and NKX6.1 copositive SC-β cell population when treated at the later PP2 cell stage during the differentiation. We expect a dosage-dependent response when the candidate hits are validated using more accurate assays. DISCUSSION/SIGNIFICANCE OF IMPACT: We established a high-throughput screening platform to identify small molecules that increase the efficiency of SC-β direct differentiation. Successful generation of SC-β allows cell replacement therapy in diabetes patients, and a better understanding of pancreatic biology and development.

scholarly journals Induction of Stem Cell Like Cells from Mouse Embryonic Fibroblast by Short-Term Shear Stress and Vitamin C

2021 ◽  
Vol 11 (4) ◽  
pp. 1941
Author(s):  
Seungmin Yeom ◽  
Myung Chul Lee ◽  
Shambhavi Pandey ◽  
Jaewoon Lim ◽  
Sangbae Park ◽  
...  
Keyword(s):  
Stem Cells ◽  
Shear Stress ◽  
Stem Cell ◽  
Vitamin C ◽  
Suspension Culture ◽  
Small Molecules ◽  
Tumor Development ◽  
Embryonic Stem ◽  
Short Term ◽  
External Stimuli

Induced pluripotent stem cells (iPSCs) are a good medicine source because of their potential to differentiate into various tissues or cells. However, traditionally, iPSCs made by specific transgenes and virus vectors are not appropriate for clinical use because of safety concerns and risk of tumor development. The goal of this research was to develop an alternative method for reprogramming, using small molecules and external stimuli. Two groups were established: short-term shear stress (STSS) under suspension culture and a combination of short-term shear stress and vitamin C (SSVC) under suspension culture. For STSS, the pipetting was carried out for cells twice per day for 2 min for 14 days in the embryonic stem cell (ES) medium. In the case of SSVC, the procedure was the same as for STSS however, its ES medium included 10 µM of vitamin C. After 14 days, all spheroids were picked and checked for pluripotency by ALP (alkaline phosphatase) assay and immunocytochemistry. Both groups partially showed the characteristics of stem cells but data demonstrated that the spheroids under shear stress and vitamin C had improved stem cell-like properties. This research showed the possibility of external stimuli and small molecules to reprogram the somatic cells without the use of transgenes.

scholarly journals Non-invasive and high-throughput interrogation of exon-specific isoform expression

2021 ◽  
Author(s):  
Dong-Jiunn Jeffery Truong ◽  
Teeradon Phlairaharn ◽  
Bianca Eßwein ◽  
Christoph Gruber ◽  
Deniz Tümen ◽  
...  
Keyword(s):  
Stem Cells ◽  
High Throughput ◽  
High Throughput Screening ◽  
Embryonic Stem ◽  
High Sensitivity ◽  
Therapeutic Interventions ◽  
Dominant Factor ◽  
Reporter System ◽  
Isoform Expression ◽  
Induced Pluripotent

AbstractExpression of exon-specific isoforms from alternatively spliced mRNA is a fundamental mechanism that substantially expands the proteome of a cell. However, conventional methods to assess alternative splicing are either consumptive and work-intensive or do not quantify isoform expression longitudinally at the protein level. Here, we therefore developed an exon-specific isoform expression reporter system (EXSISERS), which non-invasively reports the translation of exon-containing isoforms of endogenous genes by scarlessly excising reporter proteins from the nascent polypeptide chain through highly efficient, intein-mediated protein splicing. We applied EXSISERS to quantify the inclusion of the disease-associated exon 10 in microtubule-associated protein tau (MAPT) in patient-derived induced pluripotent stem cells and screened Cas13-based RNA-targeting effectors for isoform specificity. We also coupled cell survival to the inclusion of exon 18b of FOXP1, which is involved in maintaining pluripotency of embryonic stem cells, and confirmed that MBNL1 is a dominant factor for exon 18b exclusion. EXSISERS enables non-disruptive and multimodal monitoring of exon-specific isoform expression with high sensitivity and cellular resolution, and empowers high-throughput screening of exon-specific therapeutic interventions.

scholarly journals A Novel High-Throughput Screening Platform Identifies Itaconate Derivatives from Marine Penicillium antarcticum as Inhibitors of Mesenchymal Stem Cell Differentiation

Marine Drugs ◽  
2020 ◽  
Vol 18 (4) ◽  
pp. 192
Author(s):  
Pietro Marchese ◽  
Nipun Mahajan ◽  
Enda O’Connell ◽  
Howard Fearnhead ◽  
Maria Tuohy ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cell Differentiation ◽  
Small Molecules ◽  
High Throughput ◽  
High Throughput Screening ◽  
Chondrogenic Differentiation ◽  
Stem Cell Differentiation ◽  
Ectopic Calcification ◽  
Tissue Calcification ◽  
Screening Platform

Worldwide diffused diseases such as osteoarthritis, atherosclerosis or chronic kidney disease are associated with a tissue calcification process which may involve unexpected local stem cell differentiation. Current pharmacological treatments for such musculoskeletal conditions are weakly effective, sometimes extremely expensive and often absent. The potential to develop new therapies is represented by the discovery of small molecules modulating resident progenitor cell differentiation to prevent aberrant tissue calcification. The marine environment is a rich reserve of compounds with pharmaceutical potential and many novel molecules are isolated from macro and microorganisms annually. The potential of small molecules synthetized by marine filamentous fungi to influence the osteogenic and chondrogenic differentiation of human mesenchymal stem/stromal cells (hMSCs) was investigated using a novel, high-throughput automated screening platform. Metabolites synthetized by the marine-derived fungus Penicillium antarcticum were evaluated on the platform. Itaconic acid derivatives were identified as inhibitors of calcium elaboration into the matrix of osteogenically differentiated hMSCs and also inhibited hMSC chondrogenic differentiation, highlighting their capacity to impair ectopic calcification. Bioactive small molecule discovery is critical to address ectopic tissue calcification and the use of biologically relevant assays to identify naturally occurring metabolites from marine sources represents a strategy that can contribute to this effort.

scholarly journals High-Throughput Screening Assay for the Identification of Compounds Regulating Self-Renewal and Differentiation in Human Embryonic Stem Cells

2008 ◽  
Vol 2 (6) ◽  
pp. 602-612 ◽  
Author(s):  
Sabrina C. Desbordes ◽  
Dimitris G. Placantonakis ◽  
Anthony Ciro ◽  
Nicholas D. Socci ◽  
Gabsang Lee ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
High Throughput ◽  
Human Embryonic Stem Cells ◽  
High Throughput Screening ◽  
Embryonic Stem ◽  
Screening Assay ◽  
Self Renewal ◽  
High Throughput Screening Assay

47 DIFFERENTIAL DEVELOPMENTAL ABILITY OF EMBRYOS CLONED FROM TISSUE-SPECIFIC STEM CELLS

2007 ◽  
Vol 19 (1) ◽  
pp. 142
Author(s):  
K. Inoue ◽  
N. Ogonuki ◽  
H. Miki ◽  
S. Noda ◽  
S. Inoue ◽  
...  
Keyword(s):  
Stem Cells ◽  
Nuclear Transfer ◽  
Embryonic Stem ◽  
Donor Cell ◽  
Fibroblast Cell ◽  
High Rate ◽  
Full Potential ◽  
Cell Nuclei ◽  
Cell Stage

Although cloning animals by somatic cell nuclear transfer is generally an inefficient process, use of appropriate donor cell types may improve the cloning outcome significantly. Among the donor cells tested so far, mouse embryonic stem cells have given the best efficiency in terms of the development of reconstructed embryos into offspring. In this study, we examined whether 2 in vitro-produced pluripotent stem cells—neural stem cells (NSCs) and mesenchymal stem cells (MSCs)—could be better nuclear donors than other differentiated cells. Embryos were reconstructed by transfer of nuclei from NSCs or MSCs with full potential for differentiation in vitro. Most (76%) of the 2-cell NCS embryos developed to the 4-cell stage; 43% implanted and 1.6% developed to term after transfer to pseudopregnant recipients. These rates were very similar to those of embryos cloned from fibroblast cell nuclei. Interestingly, in the patterns of zygotic gene expression, NSC embryos were more similar to in vitro-fertilized embryos than fibroblast cloned embryos. By contrast, embryos reconstructed using MSC nuclei showed lower developmental ability and no implantation was obtained after embryo transfer. Chromosomal analysis of the donor MSCs revealed very high frequencies of monosomy and trisomy, which might have caused the very poor post-implantation development of embryos following nuclear transfer. Thus, in vitro-produced pluripotent cells can serve as donors of nuclei for cloning mice, but may be prone to chromosomal aberrations leading to a high rate of cloned embryo death.

scholarly journals Expression of Genes Related to Germ Cell Lineage and Pluripotency in Single Cells and Colonies of Human Adult Germ Stem Cells

2016 ◽  
Vol 2016 ◽  
pp. 1-17 ◽  
Author(s):  
Sabine Conrad ◽  
Hossein Azizi ◽  
Maryam Hatami ◽  
Mikael Kubista ◽  
Michael Bonin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Gene Expression Profile ◽  
Expression Profile ◽  
Adult Stem Cells ◽  
Single Cells ◽  
Human Fibroblasts ◽  
Embryonic Stem ◽  
Human Adult

The aim of this study was to elucidate the molecular status of single human adult germ stem cells (haGSCs) and haGSC colonies, which spontaneously developed from the CD49f MACS and matrix- (collagen−/laminin+ binding-) selected fraction of enriched spermatogonia. Single-cell transcriptional profiling by Fluidigm BioMark system of a long-term cultured haGSCs cluster in comparison to human embryonic stem cells (hESCs) and human fibroblasts (hFibs) revealed that haGSCs showed a characteristic germ- and pluripotency-associated gene expression profile with some similarities to hESCs and with a significant distinction from somatic hFibs. Genome-wide comparisons with microarray analysis confirmed that different haGSC colonies exhibited gene expression heterogeneity with more or less pluripotency. The results of this study confirm that haGSCs are adult stem cells with a specific molecular gene expression profilein vitro, related but not identical to true pluripotent stem cells. Under ES-cell conditions haGSC colonies could be selected and maintained in a partial pluripotent state at the molecular level, which may be related to their cell plasticity and potential to differentiate into cells of all germ layers.

Efficient propagation of single cells accutase-dissociated human embryonic stem cells

2008 ◽  
Vol 75 (5) ◽  
pp. 818-827 ◽  
Author(s):  
Ruchi Bajpai ◽  
Jacqueline Lesperance ◽  
Min Kim ◽  
Alexey V. Terskikh
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Single Cells ◽  
Embryonic Stem

Niche-Based Screening Reveals Leukemia Stem Cell Specific Therapeutics

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 760-760
Author(s):  
Kimberly A. Hartwell ◽  
Peter G. Miller ◽  
Alison L. Stewart ◽  
Alissa R. Kahn ◽  
David J. Logan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Drug Discovery ◽  
Small Molecules ◽  
Small Molecule ◽  
High Throughput ◽  
Hematopoietic Cells ◽  
Leukemia Stem Cells

Abstract Abstract 760 Recent insights into the molecular and cellular processes that drive leukemia have called attention to the limitations intrinsic to traditional drug discovery approaches. To date, the majority of cell-based functional screens have relied on probing cell lines in vitro in isolation to identify compounds that decrease cellular viability. The development of novel therapeutics with greater efficacy and decreased toxicity will require the identification of small molecules that selectively target leukemia stem cells (LSCs) within the context of their microenvironment, while sparing normal cells. We hypothesized that it would be possible to systematically identify LSC susceptibilities by modeling key elements of bone marrow niche interactions in high throughput format. We tested this hypothesis by creating and optimizing an assay in which primary murine stem cell-enriched leukemia cells are plated on bone marrow stromal cells in 384-well format, and examined by a high content image-based readout of cobblestoning, an in vitro morphological surrogate of cell health and self-renewal. AML cells cultured in this way maintained their ability to reinitiate disease in mice with as few as 100 cells. 14,720 small molecule probes across diverse chemical space were screened at 5uM in our assay. Retest screening was performed in the presence of two different bone marrow stromal types in parallel, OP9s and primary mesenchymal stem cells (MSCs). Greater than 60% of primary screen hits positively retested (dose response with IC50 at or below 5 μM) on both types of stroma. Compounds that inhibited leukemic cobblestoning merely by killing the stroma were identified by CellTiter-Glo viability analysis and excluded. Compounds that killed normal primary hematopoietic stem and progenitor cell inputs, as assessed by a related co-culture screen, were also excluded. Selectivity for leukemia over normal hematopoietic cells was additionally examined in vitro by comingling these cells on stroma within the same wells. Primary human CD34+ AML leukemia and normal CD34+ cord blood cells were also tested, by way of the 5 week cobblestone area forming cell (CAFC) assay. Additionally, preliminary studies of human AML cells pulse-treated with small molecules ex vivo, followed by in vivo transplantation, provided further evidence of potent leukemia kill across genotypes. A biologically complex functional approach to drug discovery, such as the novel method described here, has previously been thought impossible, due to presumed incompatibility with high throughput scale. We show that it is possible, and that it bears fruit in a first pilot screen. By these means, we discover small molecule perturbants that act selectively in the context of the microenvironment to kill LSCs while sparing stroma and normal hematopoietic cells. Some hits act cell autonomously, and some do not, as evidenced by observed leukemia kill when only the stromal support cells are treated prior to the plating of leukemia. Some hits are known, such as parthenolide and celastrol, and some are previously underappreciated, such as HMG-CoA reductase inhibition. Others are entirely new, and would not have been revealed by conventional approaches to therapeutic discovery. We therefore present a powerful new approach, and identify drug candidates with the potential to selectively target leukemia stem cells in clinical patients. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Controlling Embryonic Stem Cell Growth and Differentiation by Automation

2012 ◽  
Vol 17 (9) ◽  
pp. 1171-1179 ◽  
Author(s):  
Michael P. Kowalski ◽  
Amy Yoder ◽  
Li Liu ◽  
Laura Pajak
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Drug Discovery ◽  
Embryonic Stem Cells ◽  
Embryonic Stem Cell ◽  
High Throughput ◽  
Embryoid Body ◽  
Embryonic Stem ◽  
Basic Research ◽  
Complex Biological Process

Despite significant use in basic research, embryonic stem cells have just begun to be used in the drug discovery process. Barriers to the adoption of embryonic stem cells in drug discovery include the difficulty in growing cells and inconsistent differentiation to the desired cellular phenotype. Embryonic stem cell cultures require consistent and frequent handling to maintain the cells in a pluripotent state. In addition, the preferred hanging drop method of embryoid body (EB) differentiation is not amenable to high-throughput methods, and suspension cultures of EBs show a high degree of variability. Murine embryonic stem cells passaged on an automated platform maintained ≥90% viability and pluripotency. We also developed a method of EB formation using 384-well microplates that form a single EB per well, with excellent uniformity across EBs. This format facilitated high-throughput differentiation and enabled screens to optimize directed differentiation into a desired cell type. Using this approach, we identified conditions that enhanced cardiomyocyte differentiation sevenfold. This optimized differentiation method showed excellent consistency for such a complex biological process. This automated approach to embryonic stem cell handling and differentiation can provide the high and consistent yields of differentiated cell types required for basic research, compound screens, and toxicity studies.

Definitive endoderm: a key step in coaxing human embryonic stem cells into transplantable β-cells

2008 ◽  
Vol 36 (3) ◽  
pp. 272-275 ◽  
Author(s):  
Henrik Semb
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Insulin Pump ◽  
Embryonic Stem ◽  
Definitive Endoderm ◽  
Attractive Alternative ◽  
Β Cells ◽  
Number Of Patients

Using the Edmonton protocol, a number of patients with Type 1 diabetes mellitus have remained insulin-independent for prolonged periods of time. In spite of this success, transplantation of islets from cadaver donors will remain a therapy for very few patients owing to a lack of donors. Thus, if cell therapy should be widely available, it will require an unlimited source of cells to serve as a ‘biological’ insulin pump. At this time, the development of β-cells from hESCs (human embryonic stem cells) has emerged as the most attractive alternative. It is envisioned that ultimate success of an in vitro approach to programme hESCs into β-cells will depend on the ability, at least to a certain degree, to sequentially reproduce the individual steps that characterizes normal β-cell ontogenesis during fetal pancreatic development, including definitive endoderm from which all gastrointestinal organs, including the pancreas, originate. In the present article, differentiation of hESCs into putative definitive endodermal cell types is reviewed.

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