Precise Patterning of Mouse Embryonic Stem Cells on Glass Cover Slips for High-Throughput Analysis Using Laser Direct-Write

2011 ◽  
Author(s):  
Andrew D. Dias ◽  
Nathan R. Schiele ◽  
Brendan M. Carr ◽  
Nurazhani Abdul Raof ◽  
Yubing Xie ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Embryonic Stem Cells ◽  
High Throughput ◽  
Embryonic Stem ◽  
Direct Write ◽  
Glass Cover ◽  
Computer Aided ◽  
Petri Dishes ◽  
Laser Direct Write

Engineering a microenvironment where the growth substrate and distance between cells are controlled is highly desirable to understand how cellular interactions affect stem cell differentiation. Laser direct-write (LDW) allows rapid and precise placement of living cells via computer-aided design/computer-aided manufacturing (CAD/CAM) control. Application of this technique to study the effects of various stem cell microenvironments on differentiation requires a high-throughput experimental setup [1]. Recently, our lab has developed a gelatin-based LDW method for the precise patterning of sensitive cell types, such as mouse embryonic stem cells (mESCs), at a resolution of about 5 μm [2]. Although viable mESCs were successfully printed with maintained pluripotency, this technique required cells to be patterned onto polystyrene Petri dishes [2,3], which may limit high-throughput efficiency. Moreover, the use of polystyrene Petri dishes requires large quantities of culture medium and is not convenient for biological analysis of mESC differentiation. Therefore, the objective of this study was to adapt the LDW method, without altering its prior success, to transfer patterns of viable mESCs to glass cover slips. However, this adaptation to cover slips could not be achieved through simple downscaling due to the unique challenges of providing sufficient moisture for viable cell transfer while maintaining pattern registry on a cover slip. Once cells have been laser patterned, cover slips can then be moved to a 24-well plate so that separate sets of laser patterned cells can be analyzed in parallel for higher experimental throughput utilizing fewer resources to maintain the cells.

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.

Laser Direct-Write of Embryonic Stem Cells and Cells Encapsulated in Alginate Beads for Engineered Biological Constructs

2012 ◽  
Vol 1418 ◽  
Author(s):  
T.B. Phamduy ◽  
A.D. Dias ◽  
N. Abdul Raof ◽  
N.R. Schiele ◽  
D.T. Corr ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Alginate Beads ◽  
Stem Cell Marker ◽  
Direct Write ◽  
Pluripotency Marker ◽  
Single Bead ◽  
Substrate Surfaces ◽  
Laser Direct Write

AbstractThe ability to control the deposition of mouse embryonic stem cells (mESCs), and mESCs encapsulated in 200-μm diameter alginate microbeads, into customized patterns has recently been achieved using laser direct-write (LDW). Gelatin-based LDW was utilized to target and reproducibly deposit groups of cells directly onto receiving substrate surfaces. Live/dead staining for cell viability and immunocytochemistry for the pluripotency marker, Oct-4, indicated that transferred mESCs were viable following transfer, and maintained an important embryonic stem cell marker, respectively. LDW was further used to print mESCs encapsulated in hydrogel microbeads into customized patterns on a single-bead basis. Recent efforts have also achieved patterns of discrete co-cultures of mESCs and breast cancer cells in separate hydrogel microbeads. Altogether, we demonstrated the feasibility of LDW to print patterns of mESCs and mESC-microbeads for the biomimetic assembly of engineered cellular constructs and tissue models.

High-content screening of small compounds on human embryonic stem cells

2010 ◽  
Vol 38 (4) ◽  
pp. 1046-1050 ◽  
Author(s):  
Ivana Barbaric ◽  
Paul J. Gokhale ◽  
Peter W. Andrews
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Embryonic Stem Cells ◽  
High Throughput ◽  
Cell Biology ◽  
Es Cells ◽  
Embryonic Stem ◽  
High Content Screening ◽  
Cellular Models ◽  
Compound Libraries

Human ES (embryonic stem) cells and iPS (induced pluripotent stem) cells have been heralded as a source of differentiated cells that could be used in the treatment of degenerative diseases, such as Parkinson's disease or diabetes. Despite the great potential for their use in regenerative therapy, the challenge remains to understand the basic biology of these remarkable cells, in order to differentiate them into any functional cell type. Given the scale of the task, high-throughput screening of agents and culture conditions offers one way to accelerate these studies. The screening of small-compound libraries is particularly amenable to such high-throughput methods. Coupled with high-content screening technology that enables simultaneous assessment of multiple cellular features in an automated and quantitative way, this approach is proving powerful in identifying both small molecules as tools for manipulating stem cell fates and novel mechanisms of differentiation not previously associated with stem cell biology. Such screens performed on human ES cells also demonstrate the usefulness of human ES/iPS cells as cellular models for pharmacological testing of drug efficacy and toxicity, possibly a more imminent use of these cells than in regenerative medicine.

scholarly journals The maintenance of pluripotency following laser direct-write of mouse embryonic stem cells

Biomaterials ◽  
2011 ◽  
Vol 32 (7) ◽  
pp. 1802-1808 ◽  
Author(s):  
Nurazhani Abdul Raof ◽  
Nathan R. Schiele ◽  
Yubing Xie ◽  
Douglas B. Chrisey ◽  
David T. Corr
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells ◽  
Direct Write ◽  
Laser Direct Write

scholarly journals Strategy to Establish Embryo-Derived Pluripotent Stem Cells in Cattle

2021 ◽  
Vol 22 (9) ◽  
pp. 5011
Author(s):  
Daehwan Kim ◽  
Sangho Roh
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Embryonic Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Stem Cell Research ◽  
Pluripotent Stem Cells ◽  
Embryonic Stem ◽  
Culture Conditions ◽  
Human Diseases ◽  
Induced Pluripotent

Stem cell research is essential not only for the research and treatment of human diseases, but also for the genetic preservation and improvement of animals. Since embryonic stem cells (ESCs) were established in mice, substantial efforts have been made to establish true ESCs in many species. Although various culture conditions were used to establish ESCs in cattle, the capturing of true bovine ESCs (bESCs) has not been achieved. In this review, the difficulty of establishing bESCs with various culture conditions is described, and the characteristics of proprietary induced pluripotent stem cells and extended pluripotent stem cells are introduced. We conclude with a suggestion of a strategy for establishing true bESCs.

Stress resistant human embryonic stem cells as a potential source for the identification of novel cancer stem cell markers

2010 ◽  
Vol 289 (2) ◽  
pp. 208-216 ◽  
Author(s):  
Shaker A. Mousa ◽  
Thangirala Sudha ◽  
Evgeny Dyskin ◽  
Usawadee Dier ◽  
Christine Gallati ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Embryonic Stem Cells ◽  
Cancer Stem Cell ◽  
Human Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Stem Cell Markers ◽  
Cell Markers ◽  
Cancer Stem Cell Markers ◽  
Potential Source

Developmentally regulated use of alternative promoters creates a novel platelet-derived growth factor receptor transcript in mouse teratocarcinoma and embryonic stem cells

1989 ◽  
Vol 9 (10) ◽  
pp. 4563-4567
Author(s):  
T H Vu ◽  
G R Martin ◽  
P Lee ◽  
D Mark ◽  
A Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Embryonic Stem Cells ◽  
Growth Factor ◽  
Short Form ◽  
Embryonic Stem ◽  
Growth Factor Receptor ◽  
Extracellular Domain ◽  
Platelet Derived Growth Factor ◽  
Alternative Promoters

Embryonal carcinoma and embryonic stem cells expressed a novel form of platelet-derived growth factor receptor mRNA which was approximately 1,100 base pairs shorter than the 5.3-kilobase (kb) transcript expressed in fibroblasts and other cell types. The 4.2-kb stem cell transcript was initiated within the genomic region immediately upstream of exon 6 of the 5.3-kb transcript and therefore lacked the first five exons, which encode much of the extracellular domain of the receptor expressed in fibroblasts. In stem cells, the short form was predominant, although both forms were present at low levels. Following differentiation in vitro, expression levels of the long form increased dramatically. These findings suggest that during early embryogenesis, a stem cell-specific promoter is used in a stage- and cell type-specific manner to express a form of the platelet-derived growth factor receptor that lacks much of the extracellular domain and may function independently of ligand.

scholarly journals First person – Federico Pecori

2020 ◽  
Vol 133 (20) ◽  
pp. jcs255166
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Embryonic Stem Cells ◽  
Cell Biology ◽  
Embryonic Stem ◽  
Early Career ◽  
First Person ◽  
Receptor Endocytosis ◽  
Early Career Researchers ◽  
Selection Of

ABSTRACTFirst Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Federico Pecori is first author on ‘Mucin-type O-glycosylation controls pluripotency in mouse embryonic stem cells via Wnt receptor endocytosis’, published in JCS. Federico is a PhD student in the lab of Shoko Nishihara at the Laboratory of Cell Biology, Department of Bioinformatics, Soka University, Tokyo, Japan, where he is interested in the mechanisms regulating stem cell identity.

Types of Stem Cells Used in US-Based Clinical Trials between 1999 and 2014

2021 ◽  
Vol 26 ◽  
pp. 169-191
Author(s):  
Emma E. Redfield ◽  
Erin K. Luciano ◽  
Monica J. Sewell ◽  
Lucas A. Mitzel ◽  
Isaac J. Sanford ◽  
...  
Keyword(s):  
Stem Cells ◽  
Clinical Trials ◽  
Stem Cell ◽  
Embryonic Stem Cells ◽  
Adult Stem Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
The Us ◽  
Health Database ◽  
Induced Pluripotent

This study looks at the number of clinical trials involving specific stem cell types. To our knowledge, this has never been done before. Stem cell clinical trials that were conducted at locations in the US and registered on the National Institutes of Health database at ‘clinicaltrials.gov’ were categorized according to the type of stem cell used (adult, cancer, embryonic, perinatal, or induced pluripotent) and the year that the trial was registered. From 1999 to 2014, there were 2,357 US stem cell clinical trials registered on ‘clinicaltrials.gov,’ and 89 percent were from adult stem cells and only 0.12 percent were from embryonic stem cells. This study concludes that embryonic stem cells should no longer be used for clinical study because of their irrelevance, moral questions, and induced pluripotent stem cells.

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