CellTagging: combinatorial indexing to simultaneously map lineage and identity at single-cell resolution

Kong, Wenjun; Biddy, Brent A.; Kamimoto, Kenji; Amrute, Junedh M.; Butka, Emily G.; Morris, Samantha A.

doi:10.1038/s41596-019-0247-2

Cited by 58 publications

(46 citation statements)

References 70 publications

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“…Other advances can be applied for a different problem of characterizing molecular cell state, and include those utilizing transcriptomics (mRNA) as one of its modalities, in addition to either DNA, 50 protein expression, 10,51 chromatin accessibility 52,53 or DNA methylation 54–57 . Lineage tracing of immune cells that do not carry a natural bar code, such as TCR or B‐cell receptor, can also benefit from recent technologies using synthetic barcodes, such as CellTagging which uses a lentiviral approach 58 . This approach offers advantages over alternate approaches where gene editing is challenging 58 …”

Section: Discussionmentioning

confidence: 99%

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Recent advances in single‐cell multimodal analysis to study immune cells

Louie

Luciani

2021

Immunol. Cell Biol.

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Recent advances in single‐cell technologies have enabled the profiling of the genome, epigenome, transcriptome and proteome, along with temporal and spatial information of individual cells. These technologies have provided unique opportunities to understand mechanisms underpinning the immune system, such as characterizations of the molecular cell state, how the cell state evolves along its lineage and the impact of spatial location on cell state. In this review, we discuss how these mechanisms have been studied through recent advances in single‐cell multimodal technologies.

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Section: Discussionmentioning

confidence: 99%

“…Lineage tracing of immune cells that do not carry a natural bar code, such as TCR or B‐cell receptor, can also benefit from recent technologies using synthetic barcodes, such as CellTagging which uses a lentiviral approach 58 . This approach offers advantages over alternate approaches where gene editing is challenging 58 …”

Section: Discussionmentioning

confidence: 99%

Recent advances in single‐cell multimodal analysis to study immune cells

Louie

Luciani

2021

Immunol. Cell Biol.

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“…For example, iTracer ( He et al, 2020b ) is a novel technique that takes advantage of previously developed complex barcode libraries ( Weinreb et al, 2020 ) and an inducible Cas9 scarring system similar to the one used in the lineage tracing by nuclease-activated editing of ubiquitous sequences (LINNAEUS) method( Spanjaard et al, 2018 ) to reconstruct the lineage trajectories and fate decisions of induced pluripotent stem cells (iPSCs). CellTagging is another recently developed method that allows for the parallel capture of lineage information and cell identity through a combinatorial cell indexing approach with lentiviral barcoding ( Kong et al, 2020 ). This protocol consists of generating complex plasmid and lentivirus CellTag libraries to label cells, followed by sequential CellTagging throughout a given biological process.…”

Section: Lineage Tracing Coupled With Single-cell Rna Sequencingmentioning

confidence: 99%

Single-Cell Toolkits Opening a New Era for Cell Engineering

Lee

Kim

Park

2021

Molecules and Cells

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Since the introduction of RNA sequencing (RNA-seq) as a high-throughput mRNA expression analysis tool, this procedure has been increasingly implemented to identify cell-level transcriptome changes in a myriad of model systems. However, early methods processed cell samples in bulk, and therefore the unique transcriptomic patterns of individual cells would be lost due to data averaging. Nonetheless, the recent and continuous development of new single-cell RNA sequencing (scRNA-seq) toolkits has enabled researchers to compare transcriptomes at a single-cell resolution, thus facilitating the analysis of individual cellular features and a deeper understanding of cellular functions. Nonetheless, the rapid evolution of high throughput single-cell “omics” tools has created the need for effective hypothesis verification strategies. Particularly, this issue could be addressed by coupling cell engineering techniques with single-cell sequencing. This approach has been successfully employed to gain further insights into disease pathogenesis and the dynamics of differentiation trajectories. Therefore, this review will discuss the current status of cell engineering toolkits and their contributions to single-cell and genome-wide data collection and analyses.

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“…Aspirate medium and wash once with glioblastoma dissection medium by letting GBOs sink to the bottom of the conical tube before carefully aspirating the supernatant. 22. Transfer GBOs to a glass dissection dish containing 10 mL glioblastoma dissection medium within a laminar flow biosafety cabinet with a suitable dissection microscope.…”

Section: Passaging Glioblastoma Organoids (Timing 1-2 H)mentioning

confidence: 99%

Generation and biobanking of patient-derived glioblastoma organoids and their application in CAR-T cell testing

Jacob

Ming

Song

2020

Preprint

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Short Summary: Detailed procedures for generating and biobanking glioblastoma organoids from resected patient tumor tissue and testing CAR-T cell efficacy by co-culture. Additional procedures for tissue processing, immunohistology, and detecting hypoxia gradients and actively proliferating cells. AbstractGlioblastoma tumors exhibit extensive inter-and intra-tumoral heterogeneity, which has contributed to the poor outcomes of numerous clinical trials and continues to complicate the development of effective therapeutic strategies. Current in vitro models do not preserve the cellular and mutational diversity of parent tumors and often require a lengthy generation time with variable efficiency. Here, we describe detailed procedures for generating glioblastoma organoids (GBOs) from surgically resected patient tumor tissue using a chemically defined medium without cell dissociation. By preserving cell-cell interactions and minimizing clonal selection, GBOs maintain the cellular heterogeneity of parent tumors. We include methods for passaging and cryopreserving GBOs for continued use, biobanking, and long-term recovery. We further describe procedures for investigating patient-specific responses to immunotherapies by co-culturing GBOs with chimeric antigen receptor (CAR) T cells. This protocol takes approximately 2-4 weeks to generate GBOs and 5-7 days to perform CAR-T cell co-culture. Competence with human cell culture, tissue processing, and immunohistology is required for optimal results.An ideal in vitro model for glioblastoma would maintain cellular diversity, preserve native cell-cell interactions, recapitulate defining histological characteristics, and maintain the transcriptomes and mutation profiles of parent tumors. To be useful for personalized medicine approaches, the model would need a quick generation time with high fidelity for success and be scalable to test potential therapeutics. This would require maintaining patient-derived glioblastoma tissue in culture with minimal perturbation. Towards this goal, we opted to directly culture micro-dissected tumor pieces without cell dissociation to preserve cell-cell interactions and minimize clonal selection. We start from resected patient tumor tissue that can be sampled from different tumor foci or subdivided into regional samples to analyze intra-tumoral heterogeneity. Through testing of many medium formulations, we learned that tumor pieces did not require basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), serum, or added extracellular matrix such as Matrigel to survive, form organoids, and proliferate at rates similar to parent tumors 11 . We optimized a chemically defined medium that contains basic nutrients and components needed to support glial cell health with few exogenous factors to minimize clonal selection induced by growth factors and decrease potential treatment confounders. This resulted in an optimized version of our medium used to maintain human brain organoid cultures 12,13 . We culture tumor pieces on an orbital shaker to inc...

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CellTagging: combinatorial indexing to simultaneously map lineage and identity at single-cell resolution

Cited by 58 publications

References 70 publications

Recent advances in single‐cell multimodal analysis to study immune cells

Recent advances in single‐cell multimodal analysis to study immune cells

Single-Cell Toolkits Opening a New Era for Cell Engineering

Generation and biobanking of patient-derived glioblastoma organoids and their application in CAR-T cell testing

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