High-Throughput Screening-Compatible Single-Step Protocol to Differentiate Embryonic Stem Cells in Neurons

2008 ◽  
Vol 17 (3) ◽  
pp. 573-584 ◽  
Author(s):  
Annalisa Fico ◽  
Genesia Manganelli ◽  
Marino Simeone ◽  
Stefano Guido ◽  
Gabriella Minchiotti ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
High Throughput ◽  
High Throughput Screening ◽  
Embryonic Stem ◽  
Single Step

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

Array‐Based High‐Throughput Screening in Mouse Embryonic Stem Cells with sh RNAs

2013 ◽  
Vol 26 (1) ◽  
Author(s):  
Chia‐Hui Wang ◽  
Nianhan Ma ◽  
Yu‐Tsen Lin ◽  
Cheng‐Chung Wu ◽  
Hong‐Jin Wu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
High Throughput ◽  
High Throughput Screening ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells

scholarly journals Human IL-12 p40 as a reporter gene for high-throughput screening of engineered mouse embryonic stem cells

2008 ◽  
Vol 8 (1) ◽  
pp. 52 ◽  
Author(s):  
Leonardo D'Aiuto ◽  
Clinton S Robison ◽  
Margherita Gigante ◽  
Edward Nwanegbo ◽  
Benjamin Shaffer ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Reporter Gene ◽  
High Throughput ◽  
High Throughput Screening ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells

Image Analysis and Classification for High-Throughput Screening of Embryonic Stem Cells

2015 ◽  
pp. 17-31
Author(s):  
Laura Casalino ◽  
Pasqua D’Ambra ◽  
Mario R. Guarracino ◽  
Antonio Irpino ◽  
Lucia Maddalena ◽  
...  
Keyword(s):  
Stem Cells ◽  
Image Analysis ◽  
Embryonic Stem Cells ◽  
High Throughput ◽  
High Throughput Screening ◽  
Embryonic Stem

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.

A Novel, Three Dimensional (3D) Culture System for the Efficient, Single-Step Erythropoietic Differentiation of Mouse Embryonic Stem Cells (mESCs)

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2044-2044
Author(s):  
Iliana Fauzi ◽  
Nicki Panoskaltsis ◽  
Athanasios Mantalaris
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Conditioned Medium ◽  
Hematopoietic Cells ◽  
Embryonic Stem ◽  
3D Culture ◽  
Single Step ◽  
Rotating Wall ◽  
Rotating Wall Vessel ◽  
Mesoderm Formation

Abstract Abstract 2044 Current established protocols for the culture and differentiation of embryonic stem cells (ESCs) utilise two-dimensional (2D) tissue culture flasks/dishes. These culture methods are cumbersome and inefficient involving three stages: a) maintenance/expansion of undifferentiated ESCs, b) spontaneous differentiation through formation of embryoid bodies (EBs), and c) dissociation of EBs and replating leading to the terminal differentiation to the desired lineages. One of the major challenges in the use of ESCs for the production of red blood cells is controlling their differentiation pathway(s). Optimal culture conditions and requirements as well as precise differentiation mechanisms and cellular interactions within EBs are still not well characterised, resulting in sub-optimal control of homogenous differentiation especially due to the formation of all three germ layers. Furthermore, cavitation within EBs results in loss of available cell numbers, which reduces the yield and quality of the cellular product outcomes. To date, the most efficient protocols for the generation of oxygen-transporting, enucleated red blood cells from ESCs require co-culture with feeder cells and a multi-step process that lasts for approximately one month rendering such protocols difficult to scale-up. We have developed an integrated, single-step bioprocess that: a) uses conditioned medium (CM) derived from HepG2, a human hepatocarcinoma cell line, that stimulates mesoderm formation, b) facilitates 3D culture through encapsulation of undifferentiated mESCs in hydrogels, c) bypasses EB formation, and d) involves culture in a rotating wall vessel bioreactor that does not require passaging of the cells and is scalable and automatable. Previously, we have shown that in traditional 2D culture systems use of HepG2-CM facilitated early differentiation of mESCs into hematopoietic cells, as shown by expression of C-Myb, C-kit, Gata-2, SCL, and beta-globin genes, in comparison with that of control cultures. A significantly higher number (p≤0.001) of hematopoietic colonies was also achieved in conditioned medium-treated murine embryonic stem cells (CM)-mESC, at day 7 and 14, with a two-fold enhancement of all myeloid-erythroid progenitor colonies. Nucleated eythrocytes and macrophages were identified in the CM-mESC group as early as day 7 of culture. However, attempting to bypass the EB formation step in the 2D culture system did not produce any hematopoietic cells even by the conditioned medium-treated embryonic stem cells. Here, we now demonstrate, that single-step 3D cultures of encapsulated mESCs can produce hematopoietic cells bypassing EB formation. Specifically, undifferentiated mESCs were encapsulated (20,000 cells per hydrogel bead) and placed inside the rotating wall vessel bioreactor. The experimental group was exposed to conditioned medium supplemented with LIF for 3 days to stimulate mesoderm formation bypassing EB formation and terminal hematopoietic differentiation was accomplished by simply changing the culture medium and replacing it with 3U/ml hEPO and 40ng/ml mSCF. A significant increase in the number (p≤ 0.05) of hematopoietic colonies was observed from CM-mESCs at day 14, with a total five-fold expansion as well as enhancement of erythroid progenitors, BFU-E and CFU-E formation. A higher expression of hematopoietic genes, C-Myb, C-kit, Gata-2, SCL, as well as erythroid genes, EKLF and beta-major globin was also reported at 3 weeks of culture in low concentrations of cytokines in the bioreactor. Immunophenotypic analysis of the highly viable cells collected from the CM-mESCs group confirmed the positive expression of the proerythroblast markers (TER-119 and CD71). In conclusion, we have devised a scalable, automatable, single-step process for the derivation of mature erythrocytes from mESCs which may be used for further study of erythroid development and applications in the human system. Disclosures: No relevant conflicts of interest to declare.

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