Single-Cell RNA Sequencing to Disentangle the Blood System

Acosta, Jean; Ssozi, Daniel; Galen, Peter van

doi:10.1161/atvbaha.120.314654

Cited by 10 publications

(11 citation statements)

References 80 publications

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“…In order to explore the design space more broadly in a detailed way, more sophisticated high resolution and single cell based analytic techniques, such as single cell RNA‐Seq, and proteomic and metabolomic analysis of cells and culture components should be more routinely used by the CGT field. [ 22–24 ] Using a broad range of analytics for characterization enables the discovery of the most relevant CQAs that can then be routinely monitored and serve as the basis of process development. In addition to a diverse set of analytics, a robust testing panel should also be multi‐dimensional in nature, leveraging multiple methods to assess the same CQAs.…”

Section: Broad and High‐resolution Analytics Are The Cornerstone To Enable Iterative Developmentmentioning

confidence: 99%

Process evolution in cell and gene therapy from discovery to commercialization

Csaszar¹,

Mills²,

Zandstra

2021

Can J Chem Eng

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The field of cell and gene therapy (CGT) is maturing at a rapid rate. Manufacturing remains a major challenge and manufacturing failures and limitations are a notable source of CGT products not meeting specification or being held back from regulatory approvals. To ensure that products remain compliant and competitive throughout their lifecycle, CGT process development requires both ongoing advancement of fit‐for‐purpose technologies to allow for and support well‐designed development studies, and a methodology that enables these studies to occur effectively during focused product development campaigns. We describe a framework for ongoing innovation in CGT process development, manufacturing, and clinical testing, based on the quality by design (QbD) approach. We propose use of the concepts of process discovery, process characterization, and process development in an iterative way to refine the design space as a product matures. This strategy is enabled by the early implementation of broad and robust analytics, and a modular approach to accelerate optimization. With these strategies, CGT developers can prospectively plan for comparability studies and develop in a stage‐appropriate manner, to allow for ongoing innovation and improvements from discovery through to commercialization.

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Section: Broad and High‐resolution Analytics Are The Cornerstone To Enable Iterative Developmentmentioning

confidence: 99%

Process evolution in cell and gene therapy from discovery to commercialization

Csaszar¹,

Mills²,

Zandstra

2021

Can J Chem Eng

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“…The copyright holder for this preprint this version posted October 14, 2021. ; https://doi.org/10.1101/2021.10. 13.464251 doi: bioRxiv preprint Each NBC population is then infected with a unique, heritable molecular barcode (Fig. 1A); in this case, we used the PRISM library of lentiviral vectors that each contains a unique, heritable 24-base-pair (bp) DNA barcode sequence designed to avoid sequence homology with the genome 28,35 .…”

Section: Suncatcher Clonal Barcoding Approachmentioning

confidence: 99%

“…Although differences in the mutational profile of tumor cell subpopulations may be the best-documented parameter of tumor heterogeneity, it is ultimately the functional heterogeneity, a result of both genetic and non-genetic sources of heterogeneity, that impacts the course of disease progression [7][8][9][10] . Large scale sequencing efforts and technologies such as single cell RNA sequencing and single nucleus sequencing have revealed the diverse mutational and transcriptional landscape of human cancers [11][12][13][14][15] . Nevertheless, we still have much to learn about the effects of functional heterogeneity on disease progression.…”

Section: Introductionmentioning

confidence: 99%

“…The copyright holder for this preprint this version posted October 14, 2021. ; https://doi.org/10.1101/2021.10. 13.464251 doi: bioRxiv preprint in order to generate appropriately matched control and edited cells 16 . Moreover, clonal dynamics-driven by clone frequency, spatial proximity, heterotypic interactions, and/or cell plasticity-can profoundly affect disease progression 4,10,17,18 .…”

Section: Introductionmentioning

confidence: 99%

“…The copyright holder for this preprint this version posted October 14, 2021. ; https://doi.org/10.1101/2021.10. 13.464251 doi: bioRxiv preprint phenotypic, and functional basis for heterogeneity and clonal fitness during experimentation. Importantly, our approach even enables one to study clones that are negatively selected during any process so that their specific vulnerabilities can be uncovered.…”

Section: Introductionmentioning

confidence: 99%

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SunCatcher: Clonal Barcoding with qPCR-Based Detection Enables Functional Analysis of Live Cells and Generation of Custom Combinations of Cells for Research and Discovery

Guo

Spasic

Maynard

et al. 2021

Preprint

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Over recent decades, cell lineage tracing, clonal analyses, molecular barcoding, and single cell-omic analysis methods have proven to be valuable tools for research and discovery. Here, we report a clonal molecular barcoding method, which we term SunCatcher, that enables longitudinal tracking and retrieval of live barcoded cells for further analysis. Briefly, single cell-derived clonal populations are generated from any complex cell population and each is infected with a unique, heritable molecular barcode. One can combine the barcoded clones to recreate the original parental cell population or generate custom pools of select clones, while also retaining stocks of each individual barcoded clone. We developed two different barcode deconvolution methods: a Next-Generation Sequencing method and a highly sensitive, accurate, rapid, and inexpensive quantitative PCR-based method for identifying and quantifying barcoded cells in vitro and in vivo. Because stocks of each individual clone are retained, one can analyze not only the positively selected clones but also the negatively selected clones result from any given experiment. We used SunCatcher to barcode individual clones from mouse and human breast cancer cell lines. Heterogeneous pools of barcoded cells reliably reproduced the original proliferation rates, tumor-forming capacity, and disease progression as the original parental cell lines. The SunCatcher PCR-based approach also proved highly effective for detecting and quantifying early spontaneous metastases from orthotopic sites that otherwise would not have been detected by conventional methods. We envision that SunCatcher can be applied to any cell-based studies and hope it proves a useful tool for the research community.

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Clonal barcoding with qPCR detection enables live cell functional analyses for cancer research

et al. 2022

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Single-cell analysis methods are valuable tools; however, current approaches do not easily enable live cell retrieval. That is a particular issue when further study of cells that were eliminated during experimentation could provide critical information. We report a clonal molecular barcoding method, called SunCatcher, that enables longitudinal tracking and live cell functional analysis. From complex cell populations, we generate single cell-derived clonal populations, infect each with a unique molecular barcode, and retain stocks of individual barcoded clones (BCs). We develop quantitative PCR-based and next-generation sequencing methods that we employ to identify and quantify BCs in vitro and in vivo. We apply SunCatcher to various breast cancer cell lines and combine respective BCs to create versions of the original cell lines. While the heterogeneous BC pools reproduce their original parental cell line proliferation and tumor progression rates, individual BCs are phenotypically and functionally diverse. Early spontaneous metastases can also be identified and quantified. SunCatcher thus provides a rapid and sensitive approach for studying live single-cell clones and clonal evolution, and performing functional analyses.

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Single-Cell RNA Sequencing to Disentangle the Blood System

Cited by 10 publications

References 80 publications

Process evolution in cell and gene therapy from discovery to commercialization

Process evolution in cell and gene therapy from discovery to commercialization

SunCatcher: Clonal Barcoding with qPCR-Based Detection Enables Functional Analysis of Live Cells and Generation of Custom Combinations of Cells for Research and Discovery

Clonal barcoding with qPCR detection enables live cell functional analyses for cancer research

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