Combined single-cell gene and isoform expression analysis in haematopoietic stem and progenitor cells

AbstractSingle-cell RNA sequencing (scRNA-seq) enables gene expression profiling and characterization of novel cell types within heterogeneous cell populations. However, most approaches cannot detect alternatively spliced transcripts, which can profoundly shape cell phenotype by generating functionally distinct proteins from the same gene. Here, we integrate short- and long-read scRNA-seq of hematopoietic stem and progenitor cells to characterize changes in cell type abundance, gene and isoform expression during differentiation and ageing.

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Comprehensive single-cell gene and isoform expression analysis reveals signatures of ageing in haematopoietic stem and progenitor cells

10.21203/rs.3.rs-151546/v1 ◽

2021 ◽

Author(s):

Laura Mincarelli ◽

Vladimir Uzun ◽

David Wright ◽

Stuart Rushworth ◽

Wilfried Haerty ◽

...

Keyword(s):

Stem Cells ◽

Single Cell ◽

Progenitor Cells ◽

Cell Type ◽

Aberrant Expression ◽

Hematopoietic Stem ◽

Long Read ◽

Stem And Progenitor Cells ◽

Cell Type Specific ◽

Isoform Expression

Abstract Single-cell approaches have revealed that the haematopoietic hierarchy is a continuum of differentiation, from stem cell to committed progenitor, marked by changes in gene expression. However, many of these approaches neglect isoform level information, and thus do not capture the extent and effect of alternative splicing within the system. Here, we present the first integrated short- and long-read single-cell RNA-seq of haematopoietic stem and progenitor cells. We demonstrate that over half of genes detected in standard short-read single-cell analyses are expressed as multiple, often functionally distinct, isoforms. This includes many transcription factors and key cytokine receptors, and in particular the Thrombopoietin receptor Mpl, which displays complex isoform expression patterns between individual hematopoietic stem cells. The dataset further reveals novel signatures of hematopoietic ageing, including a global increase in lncRNA expression. Strikingly, the long-read sequencing enables us to observe aberrant expression of full-length VJ-rearranged immunoglobulin kappa transcripts in aged haematopoietic stem cells, prior to lymphoid commitment. Integrating single cell and cell-type specific isoform landscape in normal and aged hematopoiesis provides a new reference for accurate molecular profiling of heterogeneous tissues, as well as novel insights into transcriptional complexity, cell-type specific splicing events and effects of ageing.

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A Meta-Analytic Single-Cell Atlas of Mouse Bone Marrow Hematopoietic Development

10.1101/2021.08.12.456098 ◽

2021 ◽

Author(s):

Benjamin D Harris ◽

John Lee ◽

Jesse Gillis

Keyword(s):

Bone Marrow ◽

Single Cell ◽

Progenitor Cells ◽

Cell Types ◽

Marker Genes ◽

Mouse Bone Marrow ◽

Clear Understanding ◽

Hematopoietic Stem ◽

Hematopoietic Development ◽

Stem And Progenitor Cells

The clinical importance of the hematopoietic system makes it one of the most heavily studied lineages in all of biology. A clear understanding of the cell types and functional programs during hematopoietic development is central to research in aging, cancer, and infectious diseases. Known cell types are traditionally identified by the expression of proteins on the surface of the cells. Stem and progenitor cells defined based on these markers are assigned functions based on their lineage potential. The rapid growth of single cell RNA sequencing technologies (scRNAseq) provides a new modality for evaluating the cellular and functional landscape of hematopoietic stem and progenitor cells. The popularity of this technology among hematopoiesis researchers enables us to conduct a robust meta-analysis of mouse bone marrow scRNAseq data. Using over 300,000 cells across 12 datasets, we evaluate the classification and function of cell types based on discrete clustering, in silico FACS sorting, and a continuous trajectory. We identify replicable signatures that define cell types based on genes and known cellular functions. Additionally, we evaluate the conservation of signatures associated with erythroid and monocyte lineage development across species using co-expression networks. The co-expression networks predict the effectiveness of the signature at identifying erythroid and monocyte cells in zebrafish and human scRNAseq data. Together, this analysis provides a robust reference, particularly marker genes and functional annotations, for future experiments in hematopoietic development.

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Characterization of hematopoietic stem and progenitor cells

Immunologic Research ◽

10.1007/bf02935524 ◽

1987 ◽

Vol 6 (4) ◽

pp. 294-304 ◽

Cited By ~ 9

Author(s):

Douglas E. Williams ◽

Li Lu ◽

Hal E. Broxmeyer

Keyword(s):

Progenitor Cells ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells

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Single-Cell RNA Sequencing of Hematopoietic Stem and Progenitor Cells Treated with Gemcitabine and Carboplatin

Genes ◽

10.3390/genes11050549 ◽

2020 ◽

Vol 11 (5) ◽

pp. 549

Author(s):

Niclas Björn ◽

Ingrid Jakobsen ◽

Kourosh Lotfi ◽

Henrik Gréen

Keyword(s):

Cell Cycle ◽

Cell Proliferation ◽

Single Cell ◽

Progenitor Cells ◽

Myeloid Cell ◽

Enrichment Analysis ◽

Myelogenous Leukemia ◽

Transcriptional Level ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells

Treatments that include gemcitabine and carboplatin induce dose-limiting myelosuppression. The understanding of how human bone marrow is affected on a transcriptional level leading to the development of myelosuppression is required for the implementation of personalized treatments in the future. In this study, we treated human hematopoietic stem and progenitor cells (HSPCs) harvested from a patient with chronic myelogenous leukemia (CML) with gemcitabine/carboplatin. Thereafter, scRNA-seq was performed to distinguish transcriptional effects induced by gemcitabine/carboplatin. Gene expression was calculated and evaluated among cells within and between samples compared to untreated cells. Cell cycle analysis showed that the treatments effectively decrease cell proliferation, indicated by the proportion of cells in the G2M-phase dropping from 35% in untreated cells to 14.3% in treated cells. Clustering and t-SNE showed that cells within samples and between treated and untreated samples were affected differently. Enrichment analysis of differentially expressed genes showed that the treatments influence KEGG pathways and Gene Ontologies related to myeloid cell proliferation/differentiation, immune response, cancer, and the cell cycle. The present study shows the feasibility of using scRNA-seq and chemotherapy-treated HSPCs to find genes, pathways, and biological processes affected among and between treated and untreated cells. This indicates the possible gains of using single-cell toxicity studies for personalized medicine.

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Single-cell RNA-seq reveals a distinct transcriptome signature of aneuploid hematopoietic cells

Blood ◽

10.1182/blood-2017-08-803353 ◽

2017 ◽

Vol 130 (25) ◽

pp. 2762-2773 ◽

Cited By ~ 26

Author(s):

Xin Zhao ◽

Shouguo Gao ◽

Zhijie Wu ◽

Sachiko Kajigaya ◽

Xingmin Feng ◽

...

Keyword(s):

Immune Response ◽

Single Cell ◽

Progenitor Cells ◽

Rna Seq ◽

Hematopoietic Stem ◽

Monosomy 7 ◽

Key Points ◽

Stem And Progenitor Cells ◽

Diploid Cells ◽

Transcriptome Signature

Key Points We distinguished aneuploid cells from diploid cells within the hematopoietic stem and progenitor cells using scRNA-seq. Monosomy 7 cells showed downregulated pathways involved in immune response and maintenance of DNA stability.

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Characterization of transcriptional networks in blood stem and progenitor cells using high-throughput single-cell gene expression analysis

Nature Cell Biology ◽

10.1038/ncb2709 ◽

2013 ◽

Vol 15 (4) ◽

pp. 363-372 ◽

Cited By ~ 197

Author(s):

Victoria Moignard ◽

Iain C. Macaulay ◽

Gemma Swiers ◽

Florian Buettner ◽

Judith Schütte ◽

...

Keyword(s):

Gene Expression ◽

Single Cell ◽

Progenitor Cells ◽

High Throughput ◽

Gene Expression Analysis ◽

Transcriptional Networks ◽

Stem And Progenitor Cells ◽

Cell Gene Expression ◽

Cell Gene

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Simultaneous tracking of division and differentiation from individual hematopoietic stem and progenitor cells reveals within-family homogeneity despite population heterogeneity

10.1101/586354 ◽

2019 ◽

Cited By ~ 1

Author(s):

Tamar Tak ◽

Giulio Prevedello ◽

Gaël Simon ◽

Noémie Paillon ◽

Ken R. Duffy ◽

...

Keyword(s):

Progenitor Cells ◽

High Throughput ◽

Common Ancestor ◽

Single Cells ◽

Population Level ◽

Cell Types ◽

Culture Conditions ◽

Population Heterogeneity ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells

AbstractThe advent of high throughput single cell methods such as scRNA-seq has uncovered substantial heterogeneity in the pool of hematopoietic stem and progenitor cells (HSPCs). A significant issue is how to reconcile those findings with the standard model of hematopoietic development, and a fundamental question is how much instruction is inherited by offspring from their ancestors. To address this, we further developed a high-throughput method that enables simultaneously determination of common ancestor, generation, and differentiation status of a large collection of single cells. Data from it revealed that while there is substantial population-level heterogeneity, cells that derived from a common ancestor were highly concordant in their division progression and share similar differentiation outcomes, revealing significant familial effects on both division and differentiation. Although each family diversifies to some extent, the overall collection of cell types observed in a population is largely composed of homogeneous families from heterogeneous ancestors. Heterogeneity between families could be explained, in part, by differences in ancestral expression of cell-surface markers that are used for phenotypic HSPC identification: CD48, SCA-1, c-kit and Flt3. These data call for a revision of the fundamental model of haematopoiesis from a single tree to an ensemble of trees from distinct ancestors where common ancestor effect must be considered. As HSPCs are cultured in the clinic before bone marrow transplantation, our results suggest that the broad range of engraftment and proliferation capacities of HSPCs could be consequences of the heterogeneity in their engrafted families, and altered culture conditions might reduce heterogeneity between families, possibly improving transplantation outcomes.

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S. Aureus Cas9 Leads to Higher Sickle Mutation Correction Compared to S. Pyogenes Cas9 in Patient Hematopoietic Stem and Progenitor Cells

Blood ◽

10.1182/blood-2021-150830 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1861-1861

Author(s):

Byoungyong Yoo ◽

So Hyun Julie Park Park ◽

Yankai Zhang ◽

Vivien A. Sheehan ◽

Gang Bao

Keyword(s):

Progenitor Cells ◽

Research Funding ◽

Gene Editing ◽

Cell Phenotype ◽

High Morbidity ◽

Gene Correction ◽

Hematopoietic Stem ◽

Nucleotide Mutation ◽

Stem And Progenitor Cells ◽

Donor Template

Abstract Introduction: Sickle cell disease (SCD) is a red blood cell disorder caused by a single nucleotide mutation in the β-globin gene (HBB). Allogeneic hematopoietic stem cell transplantation (HSCT) is the only available cure, but is available to only a minority of patients and can be associated with high morbidity and mortality. CRISPR/Cas9 mediated genome editing may provide a permanent cure for SCD patients by correcting the sickle mutation in HBB in hematopoietic stem and progenitor cells (HSPCs). Previously, we achieved ~39% sickle mutation correction in SCD HSPCs by delivering S. pyogenes (Spy) Cas9/R-66S gRNA as ribonucleoprotein (RNP) and single-stranded oligodeoxynucleotides (ssODN) corrective donor template. S. aureus (Sau) Cas9 has potentially advantageous properties to improve therapeutic gene editing efficiency and safety, including smaller size allowing for efficient in vivo delivery and longer Protospacer Adjacent Motif (PAM) sequence for higher specificity. However, although in general, the cutting efficiency of SauCas9 is lower than SpyCas9, the differences in gene correction and other gene-editing outcomes between SpyCas9 and SauCas9 have not been well studied. Methods: With our R-66S gRNA sequence targeting the sickle mutation, the PAM sequence of SauCas9 (NGGRRT) is mutually permissive with that of SpyCas9 (NGG), allowing the same sequence to be targeted by both Cas9 nucleases. We delivered R-66S gRNA with SpyCas9 and SauCas9 respectively as RNP, along with corrective ssODN donor template into SCD HSPCs. We analyzed sickle mutation correction rate and small insertions and deletions (INDELs) profile by Next Generation Sequencing (NGS). Results/discussions: We found that although the INDEL rate of SpyCas9 is higher than SauCas9 at the same molar concentration of RNP, SauCas9 gave 43% sickle mutation correction, slightly higher than SpyCas9 (39%), demonstrating efficient homology-directed repair (HDR) mediated gene correction by SauCas9. To further investigate the potential for clinical translation, we will perform in-depth efficiency and safety characterization comparing SauCas9 and SpyCas9 mediated sickle mutation correction therapy in SCD HSPCs. Conclusion: In this work, we showed that, compared with the highly-optimized and widely-used SpyCas9, SauCas9 leads to a higher sickle mutation correction in SCD HSPCs, demonstrating the therapeutic potential of SauCas9 for treating SCD. We will further investigate the efficiency and safety of gene-edited therapy mediated by these two Cas9 orthologs, including in-depth characterization of off-target effects, chromosomal rearrangement and aberrations, and large genomic modifications. We will differentiate gene-corrected SCD HSPCs to study erythropoiesis and red cell phenotype, including normal hemoglobin production and reduced sickling under hypoxic conditions. Lastly, we will evaluate the engraftment efficiency of gene-edited cells in Nonirradiated NOD,B6.SCID Il2rγ -/- Kit (W41/W41) (NBSGW) mice that support the engraftment of human hematopoietic stem cells. Disclosures Sheehan: Forma Therapeutics: Research Funding; Beam Therapeutics: Research Funding; Novartis: Research Funding.

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A Tm4sf1-Marked Subpopulation of Endothelial Stem/Progenitor Cells Identified by Lung Single-Cell Omics of Pulmonary Arterial Hypertension

10.1101/2022.01.09.475566 ◽

2022 ◽

Author(s):

Jason Hong ◽

Brenda Wong ◽

Caroline Huynh ◽

Brian Tang ◽

Soban Umar ◽

...

Keyword(s):

Endothelial Cells ◽

Pulmonary Arterial Hypertension ◽

Arterial Hypertension ◽

Single Cell ◽

Progenitor Cells ◽

Cell Communication ◽

Cell Phenotype ◽

Hematopoietic Stem ◽

Pulmonary Arterial ◽

Lung Endothelial Cells

Rationale: The identification and role of endothelial progenitor cells (EPCs) in pulmonary arterial hypertension (PAH) remains controversial. Single-cell omics analysis can shed light on EPCs and their potential contribution to PAH pathobiology. Objectives: We aim to identify EPCs in rat lungs and assess their relevance to preclinical and human PAH. Methods: Differential expression, gene set enrichment, cell-cell communication, and trajectory reconstruction analyses were performed on lung endothelial cells from single-cell RNA-seq of Sugen-hypoxia, monocrotaline, and control rats. Relevance to human PAH was assessed in multiple independent blood and lung transcriptomic datasets. Measurements and Main Results: A subpopulation of endothelial cells (EA2) marked by Tm4sf1, a gene strongly implicated in cancer, harbored a distinct transcriptomic signature including Bmpr2 downregulation that was enriched for pathways such as inflammation and angiogenesis. Cell-to-cell communication networks specific to EA2 were activated in PAH such as CXCL12 signaling. Trajectory analysis demonstrated EA2 has a stem/progenitor cell phenotype. Analysis of independent datasets revealed Tm4sf1 is a marker for hematopoietic stem cells and is upregulated in PAH peripheral blood, particularly in patients with worse WHO functional class. EA2 signature genes including Procr and Sulf1 were found to be differentially regulated in the lungs of PAH patients and in PAH models in vitro, such as BMPR2 knockdown. Conclusions: Our study uncovered a novel Tm4sf1-marked stem/progenitor subpopulation of rat lung endothelial cells and demonstrated its relevance to preclinical and human PAH. Future experimental studies are warranted to further elucidate the pathogenic role and therapeutic potential of targeting EA2 and Tm4sf1 in PAH.

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The Bone’s Role in Myeloid Neoplasia

International Journal of Molecular Sciences ◽

10.3390/ijms21134712 ◽

2020 ◽

Vol 21 (13) ◽

pp. 4712

Author(s):

Lukas Kazianka ◽

Philipp B Staber

Keyword(s):

Bone Marrow ◽

Single Cell ◽

Significant Progress ◽

Hematopoietic Stem ◽

Myeloid Neoplasms ◽

Stem And Progenitor Cells ◽

Myeloid Neoplasia ◽

Novel Therapeutic Strategies ◽

Hematopoietic Niche

The interaction of hematopoietic stem and progenitor cells with their direct neighboring cells in the bone marrow (the so called hematopoietic niche) evolves as a key principle for understanding physiological and malignant hematopoiesis. Significant progress in this matter has recently been achieved making use of emerging high-throughput techniques that allow characterization of the bone marrow microenvironment at single cell resolution. This review aims to discuss these single cell findings in the light of other conventional niche studies that together define the current notion of the niche’s implication in (i) normal hematopoiesis, (ii) myeloid neoplasms and (iii) disease-driving pathways that can be exploited to establish novel therapeutic strategies in the future.

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