A technology of a different sort: microraft arrays

Cortés‐Llanos, Belén; Wang, Yuli; Sims, Christopher E.; Allbritton, Nancy L.

doi:10.1039/d1lc00506e

Cited by 10 publications

(9 citation statements)

References 79 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The three transgenes displayed varying degrees of integration efficiencies, which improved using ribonucleoprotein (RNP)-mediated Cas9 targeting as opposed to plasmid-based targeting ( Figure S1 ). Microraft arrays 46,47 , an established technology utilizing magnetic cell culture elements for single-cell sorting applications, were used after gene targeting to identify, isolate, and expand individual stem cells into monoclonal colonies ( Figure 1D ). A key feature of the microraft arrays is that the adhered and spatially separated cells can exchange media between adjacent cells, creating a colony-like environment ( Experimental Procedures ).…”

Section: Resultsmentioning

confidence: 99%

“…Here, we used microraft arrays to generate clonal CRISPR/Cas9-edited reporter cell lines for real-time monitoring of the pluripotent transcription factors OCT4, SOX2, and NANOG, in hESCs. We are the initial implementors in using microraft array technology for developing reporters for pluripotency transcription factors in human stem cells 46,47 . In contrast to methods based on flow sorting or single-cell well plate seeding, our approach provides a gentle process for clonal selection.…”

Section: Discussionmentioning

confidence: 99%

“…Such reagents would allow the field to properly evaluate current methods for cellular differentiation to increase efficacy, improve our understanding of pluripotency, and elucidate real-time expression dynamics during these developmental processes. Here, we address the gap in tools to elucidate pluripotent transcription factors by providing an efficient and gentle method to create stable monoclonal CRISPR/Cas9 gene-edited hESC lines with OCT4, SOX2, and/or NANOG fluorescently tagged utilizing microraft array technology 46,47 . Our work establishes a robust method for generating live-cell reporters in human pluripotent stem cells lines that will be useful to the human stem cell field.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Single-cell dynamics of core pluripotency factors in human pluripotent stem cells

Mihailovic

Wolff

Kedziora

et al. 2022

Preprint

Self Cite

View full text Add to dashboard Cite

The human transcription factors OCT4, SOX2, and NANOG form a core signaling network critical for maintaining stem cell pluripotency and self-renewal potential. The spatiotemporal expression dynamics of these pluripotency factors throughout differentiation is unclear, limiting our understanding of stem cell fate decisions. Here, we combined CRISPR/Cas9-mediated gene editing with microraft array technology to generate human embryonic stem cell lines with endogenously tagged fluorophores for OCT4, SOX2, and NANOG. Fluorescence time-lapse imaging revealed that pluripotent stem cells show gastrulation-like patterning without direct chemical induction. Directed differentiation to the three primary germ layers - endoderm, mesoderm, and ectoderm - revealed distinct spatiotemporal patterns of SOX2 and NANOG expression in single cells. Finally, we captured dynamic changes in cell morphology during ectoderm differentiation corresponding to the formation of neural rosettes. This study provides a robust method for generating live-cell reporters in human stem cells and describes the single-cell dynamics of human pluripotency factors during differentiation.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Single-cell dynamics of core pluripotency factors in human pluripotent stem cells

Mihailovic

Wolff

Kedziora

et al. 2022

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…Time-lapse images were performed every 2 h for 24 h. The microarray was maintained in focus using a customized software autofocus (MATLAB) employing a modified Laplacian focus algorithm. 21,24 During time-lapse imaging, a custom-made incubator maintained the cells on the arrays at 37 C, 60% humidity, and 5% CO 2 .…”

Section: Automated Imagingmentioning

confidence: 99%

“…Microraft arrays were developed for imaging of single cells over time followed by isolation of the single cells for downstream assays including gene expression. 21 In this study, microraft arrays were adapted to track and sort individual lymphocytes with and without applying LRAs. The microraft arrays were optimized to track the latency and kinetics of reactivation in large numbers of single cells, followed by identification of reactivated cells with unique properties or reactivation behaviors.…”

mentioning

confidence: 99%

Automated microarray platform for single‐cell sorting and collection of lymphocytes following HIV reactivation

Cortés‐Llanos

Jain

Volkheimer

et al. 2023

Bioengineering & Transla Med

Self Cite

View full text Add to dashboard Cite

A promising strategy to cure HIV‐infected individuals is to use latency reversing agents (LRAs) to reactivate latent viruses, followed by host clearance of infected reservoir cells. However, reactivation of latent proviruses within infected cells is heterogeneous and often incomplete. This fact limits strategies to cure HIV which may require complete elimination of viable virus from all cellular reservoirs. For this reason, understanding the mechanism(s) of reactivation of HIV within cellular reservoirs is critical to achieve therapeutic success. Methodologies enabling temporal tracking of single cells as they reactivate followed by sorting and molecular analysis of those cells are urgently needed. To this end, microraft arrays were adapted to image T‐lymphocytes expressing mCherry under the control of the HIV long terminal repeat (LTR) promoter, in response to the application of LRAs (prostratin, iBET151, and SAHA). In response to prostratin, iBET151, and SAHA, 30.5%, 11.2%, and 12.1% percentage of cells, respectively. The arrays enabled large numbers of single cells (>25,000) to be imaged over time. mCherry fluorescence quantification identified cell subpopulations with differing reactivation kinetics. Significant heterogeneity was observed at the single‐cell level between different LRAs in terms of time to reactivation, rate of mCherry fluorescence increase upon reactivation, and peak fluorescence attained. In response to prostratin, subpopulations of T lymphocytes with slow and fast reactivation kinetics were identified. Single T‐lymphocytes that were either fast or slow reactivators were sorted, and single‐cell RNA‐sequencing was performed. Different genes associated with inflammation, immune activation, and cellular and viral transcription factors were found.

show abstract

A microwell platform for high-throughput longitudinal phenotyping and selective retrieval of organoids

Sockell,

Wong,

Longwell

et al. 2023

Cell Systems

View full text Add to dashboard Cite

A technology of a different sort: microraft arrays

Abstract: A common procedure performed throughout biomedical research is the selection and isolation of biological entities such as organelles, cells and organoids from a mixed population. In this review, we describe...

Cited by 10 publications

References 79 publications

Single-cell dynamics of core pluripotency factors in human pluripotent stem cells

Single-cell dynamics of core pluripotency factors in human pluripotent stem cells

Automated microarray platform for single‐cell sorting and collection of lymphocytes following HIV reactivation

A microwell platform for high-throughput longitudinal phenotyping and selective retrieval of organoids

Contact Info

Product

Resources

About