Genetic Evidence of a Novel Blood Differentiation Pathway from Lympho-Myeloid Hematopoietic Stem/Progenitor Cells, Independent of Common Myeloid Progenitors.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1696-1696
Author(s):  
Anne Hultquist ◽  
Robert Mansson ◽  
Mikael Sigvardsson ◽  
Jorgen Adolfsson ◽  
David Bryder ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cell ◽  
Single Cell ◽  
Progenitor Cells ◽  
Genetic Evidence ◽  
Differentiation Potential ◽  
Hematopoietic Stem ◽  
Genetic Profiling ◽  
Road Map ◽  
Single Cell Pcr

Abstract We have recently identified three novel subsets of multipotent hematopoietic stem/progenitor cells (HSCs) in the Lin−Sca-1+c-Kithi (LSK) compartment of adult murine bone marrow based on differential expression of CD34 and the cytokine tyrosine kinase receptor Flt3. Long-term HSCs (LT-HSCs) lack CD34 and Flt3 expression (LSKCD34-flt3-), whereas short-term HSCs (ST-HSCs) are LSKCD34+flt3−. A third LSK population is characterized by co-expression of CD34 and Flt3 (LSKCD34+flt3+) and possess a combined myeloid (granulocyte and monocyte) and lymphoid (B and T cell) differentiation potential, but surprisingly lack megakaryocytic (Mk) and erythroid (E) potential in vitro and in vivo. These findings implicate an alternative road map for blood lineage development distinct from the classical model in which the first lineage commitment step of HSCs is thought to result in a strict separation into myelopoiesis and lymphopoiesis. In the current study we sought genetic evidence in further support of this new model through genetic profiling of these three HSC subpopulations, using affymetrix chips, quantitative (Q)-PCR and single cell PCR. In contrast to the pluripotent LSKCD34−Flt3− LT-HSCs and LSKCD34+Flt3− ST-HSCs, LSKCD34+Flt3+ cells downregulated or turned of genes critically involved in promoting Mk and E lineage development, such as the Epo and Tpo receptors as well as the transcription factor Gata-1. In contrast, the gene for Il-7rα, critically involved in early B and T cell development was upregulated in LSKFlt3+ cells, but absent in LT-HSCs and ST-HSCs. However, in agreement with their sustained ability to produce granulocytes and monocytes, G-CSFR and Pu.1 expression was sustained from LT-HSC through the LSKCD34+flt3+ stage Particularly noteworthy, single cell PCR demonstrated that a fraction of single LSKCD34+Flt3+ cells upregulating Il-7rα gene expression, sustained and co-expressed G-CSFR expression. Thus, genetic profiling at the single cell level provide further and compelling evidence for a novel road map for blood lineage development, independent of the common myeloid progenitor (CMP). Our biological and gene expression data rather supports the existence of a pathway in which HSCs lose their lineage potentials one by one, starting with the Mk and E lineages.

Evidence for a Novel Blood Lineage Commitment Pathway from Lympho-Myeloid Hematopoietic Stem Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 802-802 ◽  
Author(s):  
Sten Eirik W. Jacobsen ◽  
Robert Mansson ◽  
Anne Hultquist ◽  
Mikael Sigvardsson ◽  
Natalija Buza-Vidas ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cell ◽  
Hematopoietic Stem Cells ◽  
Single Cell ◽  
Lineage Commitment ◽  
Mouse Bone Marrow ◽  
Differentiation Potential ◽  
Hematopoietic Stem ◽  
Stage Of Development ◽  
Number Of Genes

Abstract We recently identified a novel Lin−Sca-1+c-kithiCD34+Flt3hi (LSKCD34+Flt3hi) lymphoid-primed multipotent progenitor (LMPP) in adult mouse bone marrow which, although possessing a combined lymphoid (B and T cell) and myeloid (granulocyte-monocyte; GM) differentiation potential, have little or no ability to adopt erythroid (E) and megakaryocyte (MK) lineage fates (Adolfsson et al, Cell121:295, 2005). The identification of this lineage restricted lymphomyeloid progenitor implicates the existence of alternative roadmaps for lineage commitment of pluripotent hematopoietic stem cells (HSCs), distinct from the classical model suggesting that the first HSC commitment step results in a strict separation into common lymphoid and myeloid progenitors. Herein we provide further, genetic evidence for such a model. Affymetrix global gene profiling, quantitative PCR, and multiplex single cell PCR analysis of LSKCD34−Flt3− long-term (LT)-HSCs, LSKCD34+Flt3− short-term (ST)-HSCs and LSKCD34+Flt3hi LMPPs, demonstrate that LMPPs in contrast to LT-HSCs and ST-HSCs down-regulate or turn off a number of genes critically involved in MkE lineage development, including GATA-1 and the receptors for erythropoietin and thrombopoietin. In contrast, a number of genes specific for early lymphoid development, including Rag-1, sterile Ig and IL-7 receptor are upregulated in LMPPs but absent in LT-HSCs and ST-HSCs. Importantly, within the LMPP, these lymphoid genes are typically co-expressed with a number of GM associated genes such as G-CSF receptor and MPO, but virtually never co-expressed with MkE associated genes. Investigating fetal liver day 14.5 we also provide evidence for existence of the LSKCD34+Flt3hi LMPPs at this early stage of development, and using a single cell clonal assay promoting combined B, T and myeloid lineage development, we demonstrate that a large fraction of fetal LMPPs lacking MkE potential possess a combined GM, B and T cell potential. Thus, evaluation at the single cell level of combined lineage potentials and multilineage gene expression provide compelling evidence for lymphoid-priming within the HSC compartment being preceeded by a loss of MkE potential, but occurring prior to loss of GM potential.

scholarly journals Dissecting Ex Vivo Expansion of Mobilized Peripheral Blood Hematopoietic Stem and Progenitor Cells By Single Cell RNA Sequencing

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3343-3343
Author(s):  
Matteo Maria Naldini ◽  
Gabriele Casirati ◽  
Erika Zonari ◽  
Giacomo Desantis ◽  
Andrea Cammarata ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Gene Therapy ◽  
Single Cell ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Single Cell Rna Sequencing ◽  
Mobilized Peripheral Blood

Abstract Hematopoietic stem and progenitor cell (HSPC) expansion remains an important unmet goal for ex vivo gene therapy based on gene addition and editing to compensate for the negative impact of the gene transfer procedure enabling faster engraftment and less complications. Additionally, ex vivo expansion of corrected cells may improve efficacy at more sustainable manufacturing costs by downscaling transduction. To date, our knowledge of precise mechanisms of action of expansion compounds is limited, and it remains unclear whether cord blood expansion protocols also maintain stemness of mobilized peripheral blood CD34+ cells (mPB), the preferred HSPC source for gene therapy. We performed serial (day 0,4,8) droplet-based single cell RNA sequencing (scRNAseq) on lentivirally transduced mPB expanded with UM171 to dissect cellular heterogeneity, monitor population dynamics over time and identify a transcriptional profile of primitive cells in culture. By associating published HSPC gene expression profiles to our scRNAseq dataset from uncultured mPB, we found that 45% of cells harbored a myelo-lymphoid signature. Smaller cell clusters expressed a shared erythroid (ERY) and megakaryocytic (MK) signature (20%), or a more primitive multipotent HSC-like signature (15%) characterized by enhanced JAK/STAT signaling and expression of HSC associated genes (AVP, HOPX, ID3). Unsupervised ordering of cells within pseudotime separated emerging MK/ERYpoiesis (FCER1A, HBD) from lympho-myelopoiesis (CD52, JUN), with intermediate states of more primitive progenitors located in between. After 4 days in culture, we noted a general increase in nuclear and mitochondrial gene transcription with activation of oxidative metabolism, paralleled by cell cycle activation, as expected from cytokine stimulation. By d8 of culture these changes leveled off but remained higher than uncultured cells. Of note, cells at d8 revealed an activation of cellular stress response pathways (e.g. TNFa, IFNg) hinting towards a compromised culture that may eventually exhaust HSC. Unsupervised clustering of cultured mPB highlighted a dramatic expansion (70-80%) of MK/ERY progenitor cells with high cycling activity with only 20-30% cells showing myelo-lymphoid transcriptional features. In line, pseudotime analysis highlighted a main ERY and MK trajectory separated from that of cells characterized by the expression of HSPC genes (HOPX, SPINK2) and of an emerging myeloid trajectory (MPO). To profile HSC in culture, we sorted and sequenced CD34+90+201+ cells from d4 expansion culture (3% of total cells), which we show to contain >70% of SCID repopulating potential. ScRNAseq revealed transcriptional similarity with the myelo-lymphoid progenitor cluster identified in the unsorted d4 culture. Unsupervised clustering of the CD34+90+201+ population revealed cell cycle dependent heterogeneity, identifying a highly quiescent cluster with expression of HSC-like signatures. This cluster was also characterized by relatively low gene expression, possibly reflecting a non-activated cell state consistent with primitive HSPC. Pseudotime analysis produced a four-branched minimum spanning tree, which retained a clear cell cycle and metabolic effect. Top variable genes included cell cycle, glycolytic, mitochondrial and ribosomal genes, identifying different metabolic modules along the branched trajectory. These results highlight that cell heterogeneity within a purified, HSC-enriched population is driven mainly by metabolic activation and cell cycle status. As a complementary approach, we purified LT-HSC from uncultured mPB (CD34+38-90+45RA-49f+), marked them with CFSE and expanded them in UM171 culture. LT-HSCs expanded on average 3.5 fold in 7 days, with the following distribution: 0 divisions: 3%; 1: 26%; 2: 47%; 3: 21%; 4: 3%. We performed scRNAseq on LT-HSC pre culture and after 7d separating a highly proliferative (≥2 divisions) and quiescent (0 - 1 division) fraction, allowing us to obtain unprecedented insight into the response of engrafting cells to ex vivo culture and set a framework to dissect self-renewal (HSC expansion), HSC maintenance and loss through differentiation as potential culture outcomes. Our combined functional/transcriptomic approach will define new HSC markers in culture and greatly facilitate side-by-side comparison of different expansion protocols towards rapid clinical translation. Disclosures No relevant conflicts of interest to declare.

scholarly journals Single-Cell RNA Sequencing of Healthy Human Marrow Hematopoietic Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4997-4997
Author(s):  
Xin Zhao ◽  
Shouguo Gao ◽  
Sachiko Kajigaya ◽  
Qingguo Liu ◽  
Zhijie Wu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Blood Cell ◽  
Single Cell ◽  
Progenitor Cells ◽  
Rna Sequencing ◽  
Hematopoietic Stem ◽  
Cell Numbers ◽  
Lineage Differentiation ◽  
Cd34 Cells ◽  
Single Cell Rna Sequencing

Hematopoiesis, especially the early events of blood cell formation, has been mainly studied in bulk populations of cells and using progenitor colony formation assays; the familiar hierarchy of cell lineage differentiation and maturation, and associated regulatory factors have been inferred from these methods. However, these techniques often require extensive manipulation of cells, the exposure of cells to unphysiological conditions, aggregation of heterogeneous populations, and prior assumptions concerning cell function and gene expression. New single cell methodology avoids many of these potential experimental deficiencies. Here we have applied single-cell RNA-sequencing(scRNA-seq)to fresh human bone marrow CD34+cells: we profiled 391 single hematopoietic stem/progenitor cells (HSPCs) from four healthy donors by deep sequencing of individual cell transcriptomes. An average of 4560 protein-coding genes were detected per cell. Cells clustered into six distinct groups, which could be assigned to known HSPC subpopulations (Fig 1A), based on expression of lineage-specific genes. Lin-CD34+CD38+cells emerged as locally clustered cell populations (Clusters 2-6, including MEP, GMP, ETP and ProB), while Lin-CD34+CD38-cells formed a single cluster (HSC/MLP). Reconstruction of differentiation trajectories by transcription in single cells revealed four committed lineages derived from stem cell compartment. The earliest fate split separates MEPs from MLPs, which partition further into lymphoid, and granulocyte-monocyte progenitors (Fig 1B). The overall pattern differs from the classical hematopoietic model describing a single binary split between myeloid and lymphoid differentiation immediately downstream of multipotent cells. However, our data align well to recently published scRNA-seq data showing sequential commitment of stem cells to the lymphoid, erythroid/megakaryocytic, and finally myeloid lineages (Setty M, Nat Biotechnol2019; Pellin D, Nat Commun2019). We further examined trends in gene expression in each of the branches and found dynamic expression changes underlying cell fate during early lineage differentiation (Fig 1C). As confirmation, PCA plot of published single-cell assay for transposase-accessible chromatin (scATAC-seq) shows similar differentiation pattern. After projecting scATAC-seq data to our transcriptomic clusters' specific genes, MEP-dependent and myeloid/lymphoid-dependent genes were located on opposing sides of the PC1 with same direction (Fig 1D), indicating transcriptome and epigenome work on differentiation in concerted effort. scRNA-seq provides opportunities for discovery and characterization at the molecular levels of early HSC differentiation and developmental intermediates, retrospectively, without the need to isolate purified populations. However, information inferred from scRNA-seq may be obscured due to missing reads and limited cell numbers. More cells would provide greater detail and higher resolution mapping.Given the low frequency of megakaryocyte progenitors within the CD34+cells as well as the neglected Lin-CD34-BM compartment, we could not fully resolve the separation and maturation of all lineages. Nonetheless, we found good coverage of cell types and a similar HSPC Atlas as other published studies (Velten L, Nat Cell Biol2017; Pellin D, Nat Commun2019)despite our limited numbers of starting cells. Our data accurately reflect the pattern of normal hematopoiesis, which may help to revise and refine characterization of hematopoiesis and provide a general reference framework to investigate the complexities of blood cell production at single-cell resolution - especially when cell numbers are limited, as from patient samples and in marrow failure syndromes. Fig. 1scRNA-seq of human hematopoietic stem and progenitor cells. (A) Unsupervised hierarchical clustering of gene expression data for all cells. C1, HSC/MLP; C2, MEP; C3, GMP; C4, ProB; C5-C6, ETP. (B)Visualization of the HSPC continuum. Each ball represents one cell.(C) Large-scale shifts in gene expression during development of hematopoietic cells.Bars on top indicate locations of individual cells, colored by stages of development, along this developmental trajectory. (D) Projections of five transcriptomic gene modules onto PCA of scATAC-seq data (Buenrostro JD,Cell 2018). Each dot represents a transcriptional factor. Figure 1 Disclosures No relevant conflicts of interest to declare.

Genome-Wide Characterization of Human Hematopoietic Progenitor Cell Heterogeneity by Expression Profiling of Single Cells: A Pilot Study

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1231-1231
Author(s):  
Chih Long Liu ◽  
Bo Dai ◽  
Aaron M. Newman ◽  
Ravi Majeti ◽  
Ash A Alizadeh
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Progenitor Cells ◽  
Expression Profiles ◽  
Single Cells ◽  
Gene Expression Profiles ◽  
Rna Seq ◽  
Hematopoietic Stem ◽  
Single Donor ◽  
Normal Human

Abstract Abstract 1231 Background: Current methods for defining and isolating human hematopoietic stem and progenitor cells using surface markers enrich for unique functional properties of these populations. However, significant functional heterogeneity in these compartments remains with important implications for understanding normal and altered hematopoiesis. Using flow sorting to enrich >10,000 cells as progenitor subpopulations, we previously characterized the gene expression signature of normal human HSC (Majetiet al 2009 PNAS 106(9):3396–3401). We hypothesized that interrogation of the transcriptomes of single cells from this compartment could resolve remaining heterogeneity and help identify and better define features of progenitor cells and hematopoietic stem cells (HSCs). Methods: Using normal human bone marrow aspirates and a FACS Aria II instrument equipped with a specialized single-cell sorting apparatus, we sorted cells enriched for HSCs based on expression of Lin-CD34+CD38-CD90+CD45RA− into 1-cell, 10-cell, 100-cell, and 40000-cell (bulk) representations. We used at least 5 replicates per group and verified single cell deposition by direct visualization. We amplified cDNA from these corresponding inputs using an exponential whole transcriptome amplification (WTA) scheme (Miltenyi SuperAmp), and evaluated gene expression profiles by two microarray platforms (Agilent/GE Healthcare 60K, and Affymetrix U133 plus 2.0), and by RNA-Seq (Illumina). We used gene expression correlation between replicates within and between microarrays as means of assessing methodological reproducibility and estimating population heterogeneity. Results: Whole transcriptome amplification yielded cDNA ranging from 0.2–1 kb for 10 and 100 cells, with significantly lower size distribution of amplified cDNA observed for single cells. Gene expression profiles had significantly better replicate reproducibility and array coverage with the Agilent microarray platform when compared with the Affymetrix U133 Plus 2.0 platform (gene coverage of 84 % for 100 cells, 73 % for 10 cells and 50% for 1 cell for Agilent vs 24 % for 100 cells, 11 % for 10 cells and 5.7% for 1 cell for Affymetrix). RNA-Seq profiling of the same populations is ongoing with major technical optimizations focused on reducing amplification of non-human templates while maintaining library complexity and representation. Using biological replicates for each input size, we observed high inter-replicate correlation levels for expression profiles obtained for bulk sorted HSCs from 8 healthy donors (∼40000-cells, average r=0.97) and for 100-cell and 10-cell inputs from a single donor (r=0.96–0.99, respectively). While intra-array concordance of replicate measurements (n=14642) was high (r>0.91) within each of 5 single cells from a single donor, comparison of 5-single cells from the same donor identified significant heterogeneity, when compared to the 10-cell and 100-cell sub-clusters (Figure 1). Individual genes characteristically expressed by these heterogeneous single cell populations are currently being investigated by FACS and Fluidigm arrays. A larger experiment characterizing 192 single progenitor cells, employing Agilent microarrays and RNA-Seq is currently in progress. Conclusions: Single cell transcriptome profiling is feasible, with best performance on 60-mer microarrays. Single cell transcriptomes exhibit lower, but reasonable levels of reproducibility (r>0.7) and precision as compared with higher cell numbers. Gene expression profiles of single cells capture gene expression heterogeneity in HSCs. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The order and logic of CD4 versus CD8 lineage choice and differentiation in mouse thymus

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Mohammad M. Karimi ◽  
Ya Guo ◽  
Xiaokai Cui ◽  
Husayn A. Pallikonda ◽  
Veronika Horková ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cell ◽  
Single Cell ◽  
Signal Strength ◽  
Gene Activity ◽  
Cytotoxic T Cell ◽  
Individual Selection ◽  
Cell Lineages ◽  
Single Cell Rna Sequencing ◽  
Tcr Signals

AbstractCD4 and CD8 mark helper and cytotoxic T cell lineages, respectively, and serve as coreceptors for MHC-restricted TCR recognition. How coreceptor expression is matched with TCR specificity is central to understanding CD4/CD8 lineage choice, but visualising coreceptor gene activity in individual selection intermediates has been technically challenging. It therefore remains unclear whether the sequence of coreceptor gene expression in selection intermediates follows a stereotypic pattern, or is responsive to signaling. Here we use single cell RNA sequencing (scRNA-seq) to classify mouse thymocyte selection intermediates by coreceptor gene expression. In the unperturbed thymus, Cd4+Cd8a- selection intermediates appear before Cd4-Cd8a+ selection intermediates, but the timing of these subsets is flexible according to the strength of TCR signals. Our data show that selection intermediates discriminate MHC class prior to the loss of coreceptor expression and suggest a model where signal strength informs the timing of coreceptor gene activity and ultimately CD4/CD8 lineage choice.

scholarly journals Delineating spatiotemporal and hierarchical development of human fetal innate lymphoid cells

Cell Research ◽  
2021 ◽  
Author(s):  
Chen Liu ◽  
Yandong Gong ◽  
Han Zhang ◽  
Hua Yang ◽  
Yang Zeng ◽  
...  
Keyword(s):  
Single Cell ◽  
Progenitor Cells ◽  
Fetal Liver ◽  
Innate Lymphoid Cells ◽  
Lymphoid Cells ◽  
Lymphoid Tissues ◽  
Hematopoietic Stem ◽  
Molecular Features ◽  
Lymphoid Progenitors ◽  
B Lineage

AbstractWhereas the critical roles of innate lymphoid cells (ILCs) in adult are increasingly appreciated, their developmental hierarchy in early human fetus remains largely elusive. In this study, we sorted human hematopoietic stem/progenitor cells, lymphoid progenitors, putative ILC progenitor/precursors and mature ILCs in the fetal hematopoietic, lymphoid and non-lymphoid tissues, from 8 to 12 post-conception weeks, for single-cell RNA-sequencing, followed by computational analysis and functional validation at bulk and single-cell levels. We delineated the early phase of ILC lineage commitment from hematopoietic stem/progenitor cells, which mainly occurred in fetal liver and intestine. We further unveiled interleukin-3 receptor as a surface marker for the lymphoid progenitors in fetal liver with T, B, ILC and myeloid potentials, while IL-3RA– lymphoid progenitors were predominantly B-lineage committed. Notably, we determined the heterogeneity and tissue distribution of each ILC subpopulation, revealing the proliferating characteristics shared by the precursors of each ILC subtype. Additionally, a novel unconventional ILC2 subpopulation (CRTH2– CCR9+ ILC2) was identified in fetal thymus. Taken together, our study illuminates the precise cellular and molecular features underlying the stepwise formation of human fetal ILC hierarchy with remarkable spatiotemporal heterogeneity.

scholarly journals Identification of leukemic and pre-leukemic stem cells by clonal tracking from single-cell transcriptomics

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Lars Velten ◽  
Benjamin A. Story ◽  
Pablo Hernández-Malmierca ◽  
Simon Raffel ◽  
Daniel R. Leonce ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Cancer Stem Cells ◽  
Single Cell ◽  
Lineage Tracing ◽  
Specific Gene ◽  
Leukemic Stem Cells ◽  
Hematopoietic Stem ◽  
Mutational Status

AbstractCancer stem cells drive disease progression and relapse in many types of cancer. Despite this, a thorough characterization of these cells remains elusive and with it the ability to eradicate cancer at its source. In acute myeloid leukemia (AML), leukemic stem cells (LSCs) underlie mortality but are difficult to isolate due to their low abundance and high similarity to healthy hematopoietic stem cells (HSCs). Here, we demonstrate that LSCs, HSCs, and pre-leukemic stem cells can be identified and molecularly profiled by combining single-cell transcriptomics with lineage tracing using both nuclear and mitochondrial somatic variants. While mutational status discriminates between healthy and cancerous cells, gene expression distinguishes stem cells and progenitor cell populations. Our approach enables the identification of LSC-specific gene expression programs and the characterization of differentiation blocks induced by leukemic mutations. Taken together, we demonstrate the power of single-cell multi-omic approaches in characterizing cancer stem cells.

IMMU-50. GLIOBLASTOMA MANIPULATES HEMATOPOIETIC STEM CELL DIFFERENTIATION OUTCOMES AND DRIVES ALTERED IMMUNE RECONSTITUTION

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi104-vi104
Author(s):  
Bayli DiVita Dean ◽  
Tyler Wildes ◽  
Joseph Dean ◽  
David Shin ◽  
Connor Francis ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
Progenitor Cells ◽  
Rna Sequencing ◽  
Cell Fate ◽  
Immune Reconstitution ◽  
Stem Cell Differentiation ◽  
Cell Fate Determination ◽  
Hematopoietic Stem ◽  
Fate Determination

Abstract INTRODUCTION Bone marrow-derived hematopoietic stem and progenitor cells (HSPCs) give rise to the cellular components of the immune system. Unfortunately, immune reconstitution from HSPCs are negatively impacted by solid cancers, including high-grade gliomas. For example, an expansion of myeloid progenitor cells has been previously described across several cancers that originate outside the CNS. A similar expansion of MDSCs coupled with diminished T cell function has also been described in the peripheral blood of patients with newly-diagnosed GBM. Alterations in both lymphoid and myeloid compartments due to CNS malignancy led us to determine how intracranial gliomas impact HSPCs in both their capacity to reconstitute the immune compartment and in their cell fate determination. This is important to better understand the impact of gliomas on immunity and how we can leverage these findings to better develop cellular immunotherapeutics. METHODS HSPCs were isolated from bone marrow of C57BL/6 mice with orthotopic KR158B glioma, or age-matched naïve mice. Experiments were conducted to compare relative changes in: gene expression (RNA-sequencing), precursor frequencies, cell fate determination, and cellular function of cells derived from HSPCs of glioma-bearing mice. RESULTS RNA-sequencing revealed 700+ genes whose expression was significantly up- or downregulated in HSPCs from glioma-bearing mice, particularly those involved with stemness and metabolic activity. Importantly, HSPCs from glioma-bearing mice expressed upregulation of genes involved in myelopoiesis relative to naïve mice. This was coupled with an expansion of granulocyte macrophage precursors (GMPs), the progenitors to gMDSCs. Next, differentiation assays revealed that HSPCs from glioma-bearing mice had higher propensity of differentiating into MDSC under homeostatic conditions relative to controls both in vitro and in vivo. Furthermore, mice bearing intracranial gliomas possess an expansion of MDSCs which are more suppressive on T cell proliferation and hinders T cell-mediated tumor cell killing relative to MDSCs derived from naïve control mice.

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