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Impacts of cryopreservation on cell type composition and gene expression in bone marrow biopsy from multiple myeloma patients

ABSTRACT: As the development of the single cell sequencing technology, the workflow of preserving and processing cells needs to be more flexible and manageable to fit the longitudinal or complex single cell study. Consequently, considering the upstream cell cryopreservation protocol instead of using fresh cells become more common in single cell sequencing experiment. There have been several studies reported that cryopreservation protocol could largely preserve the transcriptomic profiles but it brought slight perturbations to the cells. In this case, the effect of cryopreservation on transcriptomic changes needs to be studied to understand whether there will be any bias in the downstream analysis. In this paper, we compared fresh and cryopreserved CD138+ and CD138- cells from multiple myeloma patients’ bone marrow aspirates to evaluate the impact of cryopreservation on cell population frequency and transcriptome. We found the cryopreservation protocol maintained highly consistent gene expression profiles for myeloma cells (R ≥ 0.96) and their microenvironment (R ≥ 0.9), while it slightly impacted the proportion of certain immune cell subtypes in the CD138- samples. In general, our cryopreservation protocol could well preserve bone marrow aspirate samples from multiple patients.

ORGANISM(S): Homo sapiens

PROVIDER: GSE161722 | GEO | 2020/11/19

REPOSITORIES: GEO

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Project description:in order to document molllecular signature of iMycCα B-cell lymphomas, RNA sequecing of CD138+ and CD138-B-cell lymphomas was performed and normalized expression data was obtained for all protein-coding genes Methods: B220+IgM+IgD+CD138+ and B220+IgM+IgD+CD138- B-cell lymphomas were investigated in the iMycCα mice. Total RNA from lymphoma cells (purified with B220-coupled beads from Miltenyi Biotech) was extracted and analysed by microarray (Génome et Transcriptome, GenoToul, Toulouse, France; get.genotoul.fr). RNA-seq paired-end libraries were prepared according to the Illumina protocol with some adjustments, using the TruSeq Stranded Total RNA Gold library prep Kit (Illumina, San Diego, USA). Libraries were quantified by qPCR using the KAPA Library Quantification Kit (Roche, Basel, Switzerland). Libraries quality was assessed by the HS NGS kit on the Fragment Analyzer (Agilent Technologies, Santa Clara, USA). Libraries were equimolarly pooled and RNA sequencing was then performed on one S4 lane of the Illumina NovaSeq 6000 instrument (Illumina, San Diego, USA), using the NovaSeq 6000 S4 v1.5 Reagent Kit (300 cycles), and a paired-end 2 x 150 pb strategy Results: Bioinformatic analysis defined CD138+ and CD138- B-cell lymphomas as two groups with a different transcriptome signature ( 2522 genes differentially expressed) driving various arrays of the immune responses and expressing different signaling/metabolic pathways. Enrichment of previously established human Burkitt lymphoma (BL) and multiple myeloma signatures was tested in our CD138+ B-cell lymphomas. In contrast to BL, myeloma up and down signatures were significantly enriched, emphasizing the molecular similarity of mouse CD138+ B-cell lymphomas to human multiple myeloma cells and the relevance of our mice as pertinent model of plasma B-cell lymphomas. Conclusions: Our mouse model carrying a homozygous insertion of c-myc into the IgH locus develops B-cell lymphoma with a large part of CD138+ plasma cell neoplasms. Its interesting and large transcriptome similarities with human myelomas make this mouse model an accurate, reliable experimental myeloma model.

Project description:Cerebrospinal fluid (CSF) matrix biomarkers have become increasingly valuable surrogate markers of neuropsychiatric diseases in research and clinical practice. In contrast, CSF cells have been rarely investigated due to their relative scarcity and fragility, and lack of common collection and cryopreservation protocols, with limited exceptions for neurooncology and primary immune-based diseases like multiple sclerosis. The advent of a microfluidics-based multi-omics approaches to studying individual cells has allowed for the study of cellular phenotyping, intracellular dynamics, and intercellular relationships that provide multidimensionality unable to be obtained through acellular fluid-phase analyses. challenges to cell-based research include site to- site differences in handling, storage, and thawing methods, which can lead to inaccuracy and inter-assay variability. In the present study, we performed single-cell RNA sequencing (10x Genomics) on fresh (Samples labeled as FRE) or previously cryopreserved human CSF samples from three alternative cryopreservation methods: Fetal Bovine Serum with Dimethyl sulfoxide (FBS/DMSO, Samples labeled as FBS), FBS/DMSO after a DNase step (a step often included in epigenetic studies, samples labeled as DNA), and cryopreservation using commercially available Recovery©media (Samples labeled as REC). In comparing relative differences between fresh and cryopreserved samples, we found little effect of the cryopreservation method on being able to resolve donor-linked cell type proportions, markers of cellular stress, and overall gene expression at the single-cell level, whereas donor-specific differences were readily discernable. We further, demonstrate the compatibility of fresh and cryopreserved CSF immune cell sequencing using biologically relevant sexually dimorphic gene expression differences by donor. Our findings support the utility and interchangeability of FBS/DMSO and Recovery cryopreservation with fresh sample analysis, providing a methodological grounding that will enable researchers to further expand our understanding of the CSF immune cell contributions to neurological and psychiatric disease.

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Impacts of cryopreservation on cell type composition and gene expression in bone marrow biopsy from multiple myeloma patients

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