Stress relief: emerging methods to mitigate dissociation-induced artefacts

Machado, Léo; Relaix, Frédéric; Mourikis, Philippos

doi:10.1016/j.tcb.2021.05.004

Cited by 28 publications

(22 citation statements)

References 77 publications

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“…Therefore, it is currently difficult to analyze a small amount of starting cells such as those in human clinical samples. The dissociation process may cause a stress‐induced bias in gene expression of the tissue (Machado et al , 2021; Miyawaki‐Kuwakado et al , 2021). For this reason, epigenomic analysis using tissue sections has been widely used.…”

Section: Resultsmentioning

confidence: 99%

Modeling population size independent tissue epigenomes by ChIL‐seq with single thin sections

Maehara

Tomimatsu

Harada

et al. 2021

Molecular Systems Biology

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Recent advances in genome-wide technologies have enabled analyses using small cell numbers of even single cells. However, obtaining tissue epigenomes with cell-type resolution from large organs and tissues still remains challenging, especially when the available material is limited. Here, we present a ChIL-based approach for analyzing the diverse cellular dynamics at the tissue level using high-depth epigenomic data. "ChIL for tissues" allows the analysis of a single tissue section and can reproducibly generate epigenomic profiles from several tissue types, based on the distribution of target epigenomic states, tissue morphology, and number of cells. The proposed method enabled the independent evaluation of changes in cell populations and gene activation in cells from regenerating skeletal muscle tissues, using a statistical model of RNA polymerase II distribution on gene loci. Thus, the integrative analyses performed using ChIL can elucidate in vivo cell-type dynamics of tissues.

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Section: Resultsmentioning

confidence: 99%

Modeling population size independent tissue epigenomes by ChIL‐seq with single thin sections

Maehara

Tomimatsu

Harada

et al. 2021

Molecular Systems Biology

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“…Previous methods for adult stem cell isolation required extensive tissue dissection and digestions, which were recently shown to elicit stress responses in stem cells. As a result, the transcriptome profiles of freshly isolated stem cells were skewed by disassociationinduced artefacts and may have failed to represent the genuine quiescent state [173][174][175]. Thus, previous transcriptome profiles derived from freshly isolated MuSCs should be revisited.…”

Section: Discussionmentioning

confidence: 99%

“…Thus, previous transcriptome profiles derived from freshly isolated MuSCs should be revisited. New methods aiming to preserve the true quiescent state of stem cells by employing in situ fixation or nascent mRNA tagging have already advanced our understanding of the true quiescence signatures [38,119,161,174]. For example, IEGs such as Jun and Fos are highly expressed in freshly isolated MuSCs, but their mRNA levels are actually much lower in fixed MuSCs [38,175].…”

Section: Discussionmentioning

confidence: 99%

A Long Journey before Cycling: Regulation of Quiescence Exit in Adult Muscle Satellite Cells

Zhou

Han

2022

IJMS

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Skeletal muscle harbors a pool of stem cells called muscle satellite cells (MuSCs) that are mainly responsible for its robust regenerative capacities. Adult satellite cells are mitotically quiescent in uninjured muscles under homeostasis, but they exit quiescence upon injury to re-enter the cell cycle to proliferate. While most of the expanded satellites cells differentiate and fuse to form new myofibers, some undergo self-renewal to replenish the stem cell pool. Specifically, quiescence exit describes the initial transition of MuSCs from quiescence to the first cell cycle, which takes much longer than the time required for subsequent cell cycles and involves drastic changes in cell size, epigenetic and transcriptomic profiles, and metabolic status. It is, therefore, an essential period indispensable for the success of muscle regeneration. Diverse mechanisms exist in MuSCs to regulate quiescence exit. In this review, we summarize key events that occur during quiescence exit in MuSCs and discuss the molecular regulation of this process with an emphasis on multiple levels of intrinsic regulatory mechanisms. A comprehensive understanding of how quiescence exit is regulated will facilitate satellite cell-based muscle regenerative therapies and advance their applications in various disease and aging conditions.

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“…Further examination using makers such as CD206, Siglec-H and CD38 that are expressed on BAMs and not microglia ( 51 ), as well as using histological techniques to define the locations of the chemotherapy-affected cells will be important to more accurately define the impacts of chemotherapy on the brain immune microenvironment especially given that the vasculature in the border regions would have different barrier properties compared to the blood-brain barrier of the parenchyma. Future work aimed at better understanding the immune cell dynamics within medulloblastomas may also consider first enriching for CD45 + cells prior to bulk RNA sequencing or employing single cell sequencing or single cell proteomics technologies to detect immune signatures in medulloblastoma tissue, though these methods are accompanied with the caveat of tissue processing induced artefacts [reviewed in ( 54 )]. Our sequencing data was limited by small sample sizes in this exploratory study and, in the case of CSI-treated tumors, by the choice to sample later time points when tumors were larger, but possibly too late after treatment cessation to detect gene expression differences.…”

Section: Discussionmentioning

confidence: 99%

Conventional Therapies Deplete Brain-Infiltrating Adaptive Immune Cells in a Mouse Model of Group 3 Medulloblastoma Implicating Myeloid Cells as Favorable Immunotherapy Targets

et al. 2022

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Medulloblastoma is the most common childhood brain cancer. Mainstay treatments of radiation and chemotherapy have not changed in decades and new treatment approaches are crucial for the improvement of clinical outcomes. To date, immunotherapies for medulloblastoma have been unsuccessful, and studies investigating the immune microenvironment of the disease and the impact of current therapies are limited. Preclinical models that recapitulate both the disease and immune environment are essential for understanding immune-tumor interactions and to aid the identification of new and effective immunotherapies. Using an immune-competent mouse model of aggressive Myc-driven medulloblastoma, we characterized the brain immune microenvironment and changes induced in response to craniospinal irradiation, or the medulloblastoma chemotherapies cyclophosphamide or gemcitabine. The role of adaptive immunity in disease progression and treatment response was delineated by comparing survival outcomes in wildtype C57Bl/6J and in mice deficient in Rag1 that lack mature T and B cells. We found medulloblastomas in wildtype and Rag1-deficient mice grew equally fast, and that craniospinal irradiation and chemotherapies extended survival equally in wildtype and Rag1-deficient mice, suggesting that tumor growth and treatment response is independent of T and B cells. Medulloblastomas were myeloid dominant, and in wildtype mice, craniospinal irradiation and cyclophosphamide depleted T and B cells in the brain. Gemcitabine treatment was found to minimally alter the immune populations in the brain, resulting only in a depletion of neutrophils. Intratumorally, we observed an abundance of Iba1+ macrophages, and we show that CD45high cells comprise the majority of immune cells within these medulloblastomas but found that existing markers are insufficient to clearly delineate resident microglia from infiltrating macrophages. Ultimately, brain resident and peripheral macrophages dominate the brain and tumor microenvironment and are not depleted by standard-of-care medulloblastoma therapies. These populations therefore present a favorable target for immunotherapy in combination with front-line treatments.

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Stress relief: emerging methods to mitigate dissociation-induced artefacts

Cited by 28 publications

References 77 publications

Modeling population size independent tissue epigenomes by ChIL‐seq with single thin sections

Modeling population size independent tissue epigenomes by ChIL‐seq with single thin sections

A Long Journey before Cycling: Regulation of Quiescence Exit in Adult Muscle Satellite Cells

Conventional Therapies Deplete Brain-Infiltrating Adaptive Immune Cells in a Mouse Model of Group 3 Medulloblastoma Implicating Myeloid Cells as Favorable Immunotherapy Targets

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