MLL-AF9 Initiates Leukemia from a Single Committed Hematopoietic Progenitor.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2534-2534
Author(s):  
Andrei V. Krivtsov ◽  
Matthew C. Stubbs ◽  
Renee Wright ◽  
Zhaohui Feng ◽  
Andrew L. Kung ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Single Cell ◽  
Prognostic Significance ◽  
Cell Types ◽  
Stem Cell Population ◽  
Leukemia Stem Cells ◽  
Cell Of Origin ◽  
Median Latency ◽  
Hematopoietic Stem

Abstract Recent experiments have demonstrated that MLL-translocation associated fusion proteins can transform either hematopoietic stem cells (HSC) or granulocyte macrophage progenitors (GMP) into leukemia stem cells. However, it may be that leukemogenic process differs when HSC are the cell of origin as compared to myeloid progenitors. We transduced either HSC or GMP with a retrovirus expressing MLL-AF9 and GFP followed by single cell sorting of transduced cells. Approximately 80% of singly sorted MLL-AF9 transduced GMP (MLL-AF9-GMP) and about 25% of MLL-AF9 transduced HSC (MLL-AF9-HSC) could be serially re-plated over 9 passages. Upon transplantation into syngeneic mice, 83% (n=12) of MLL-AF9-HSC single cell derived clones induced AML with a median latency 70 days. Approximately 30% (n=20) of MLL-AF9-GMP single cell derived clones induced AML, with median latency 112 days. When MLL-AF9-GMP single cell derived clones were co-infected with an empty retrovirus (to provide additional oncogenic events as a result of retroviral integration) before transplantation into recipient mice, 93% of the transplanted mice (n=15) developed AML with mean latency 65 days, similar to leukemia initiated from HSC. This suggests that either single GMP or HSC can be transformed into leukemia initiating cells. However, extra mutations appear to be required to induce leukemia from committed progenitors. Consistent with this hypothesis, southern blot analysis performed on leukemias initiated from 5,000 and 15,000 MLL-AF9 transduced HSC or GMP demonstrated polyclonal AML arising from HSC compared to oligoclonal AML arising from GMP. Next, we used bioluminescent imaging to follow disease kinetics. When 15,000 MLL-AF9 transduced HSC were injected into recipient mice, the disease accumulated in a linear fashion over 42 days. However, when 15,000 MLL-AF9 transduced GMP were injected the disease developed more slowly over 75 days. Immunophenotypic analysis of the resultant leukemias demonstrated that the HSC-derived and GMP-derived leukemias were quite similar, with a GMP-like population containing LSC in both cases. Globally, the two cell types were also very similar with their gene expression profile being more similar to GMP than any other progenitor or stem cell population. However, we found that in addition to the previously reported 363-gene “self-renewal associated signature” LSC derived from HSC also possessed high-level expression of genes such as Flt3, Mcl1, and Notch-1. Preliminary analysis also suggests that gene expression differences between HSC and GMP-derived leukemia stem cells may have prognostic significance in human AML. These data suggest that AML derived from different cells of origin, while globally quite similar, require a different number of genetic events, and have gene expression differences that may influence drug response.

Cell of Origin Influences Leukemia Stem Cell Phenotype.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3459-3459
Author(s):  
Andrei V. Krivtsov ◽  
Amit U. Sinha ◽  
Matthew C. Stubbs ◽  
Andrew Kung ◽  
Scott Armstrong
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Single Cell ◽  
Conflicts Of Interest ◽  
Genetic Program ◽  
Cell Phenotype ◽  
Data Sets ◽  
Cell Of Origin ◽  
Median Latency ◽  
Hematopoietic Stem

Abstract Abstract 3459 Poster Board III-347 MLL-fusion proteins can transform either hematopoietic stem cells (HSC) or granulocyte macrophage progenitors (GMP) into leukemia stem cells (LSC). However, the leukemogenic process in HSC may differ from that in GMP. We transduced HSC and GMP with MLL-AF9 or control retroviruses. Single-cell sorted MLL-AF9 expressing HSC or GMP could be serially replated for over 9 passages. Upon transplantation into syngeneic mice, 86.3% (n=22) of HSC:MLL-AF9 single cell derived clones (SCC) induced AML with a median latency of 61 days, while 33.3% of GMP:MLL-AF9 SCC induced AMLs with median latency of 100 days. Immunophenotype analysis of the resultant leukemias demonstrated that long-term repopulating HSC (LT-HSC) and GMP-derived leukemias were quite similar, with a GMP-like (LGMP) population enriched in LSC in both cases. Gene expression analysis demonstrated that globally the LGMP isolated from HSC derived AMLs (AML:HSC) and GMP derived AMLs (AML:GMP) were similar to each other but possessed specific genetic programs reminiscent of the cell of origin (HSC or GMP). For example Evi1, Jun, and Fos oncogenes were highly expressed in HSC and AML:HSC, but expressed at low level in GMP or AML:GMP. The genetic program that distinguished LGMP:HSC from LGMP:GMP was found to be enriched in hematopoietic stem cells compared to more differentiated myeloid progenitors and correlate with genetic programs in and human MLL-rearranged AML associated with a poor clinical outcome in two independent MLL-rearranged AML data sets. In order to directly assess differences in treatment response for leukemias derived from different cells of origin, we treated leukemic mice with a chemotherapeutic agent often used to treatment human AMLs. Treatment of leukemic mice with Etoposide reduced the spleen weights in mice transplanted with AML:HSC to a lesser extent (28%) than in mice transplanted with AML:GMP (88%). Altogether, these data indicate that cell of origin of AML can influence the genetic program of fully developed leukemia, and thus could account for some of the heterogeneity in human leukemias and perhaps outcome. Disclosures No relevant conflicts of interest to declare.

scholarly journals Single Cell Transcriptomic Analysis of the Histone H3 K27M Mutation in Pre-Leukemic Hematopoietic Stem Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3773-3773
Author(s):  
Meaghan Boileau ◽  
Selin Jessa ◽  
Samantha Worme ◽  
Damien Faury ◽  
Nada Jabado ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Single Cell ◽  
Histone H3 ◽  
Cell Types ◽  
Clonal Expansion ◽  
Transcriptomic Analysis ◽  
Hematopoietic Stem ◽  
K27m Mutation

Acute myeloid leukemia (AML) develops in a step-wise manner from pre-leukemic clonal expansion to full-blown disease driven by aberrant epigenetic changes. Indeed, regulators of the epigenome such as DNMT3A, TET2, IDH1/2, EZH2 and ASXL1 are often mutated in pre-leukemia and myeloid malignancies. We and others identified K27M/I mutations in histone H3 in AML (Boileau et al. Nat Commun, 2019; Lehnertz et al. Blood, 2017). We demonstrated that K27 mutations are found in pre-leukemic hematopoietic stem cells (HSCs), are enriched in secondary AML, expand the functional human HSC pool and increase leukemic aggressiveness. Transcriptomic and epigenomic analysis determined that K27 mutations alter gene expression through a global decrease in promoter H3K27 tri-methylation and a gene-specific increase in H3K27 acetylation in leukemic cells (Boileau et al. Nat Commun, 2019). Here, we have analyzed the effects of the K27M mutation on HSCs at the single-cell level to understand its role in pre-leukemic clonal expansion. Healthy CD34+CD38- human cord blood cells were transduced with HIST1H3H WT or K27M and injected intrafemorally into sub-lethally irradiated NSG mice. After 14 weeks, bone marrow cells from the femur were collected and sorted for CD34+ transduced (GFP+) cells. Single-cell transcriptomics were performed by generating gene expression libraries from ~8,000 CD34+ cells using the 10X Genomics technology and sequenced using HiSeq4000. We have performed initial clustering and dimensionality reduction (t-SNE and UMAP) and identified 10 and 11 distinct clusters in the WT and K27M samples, respectively. Gene sets distinguishing the individual clusters have been determined. Using published gene lists for primitive hematopoietic cell types, the clusters have been assigned to specific cell types such as HSC, granulocyte-monocyte progenitors (GMP), common myeloid progenitors (CMP), multi-lymphoid progenitors (MLP) and megakaryocyte-erythroid progenitors (MEP) (Laurenti et al. Nat Immunol, 2013). Preliminary joint clustering analysis indicates the presence of two distinct clusters for the WT and K27M samples that were both assigned as "HSCs" in individual clustering. Further analysis to identify the differences in the clusters and cell populations between WT and K27M samples is being performed and will be presented at this meeting. Overall, this single-cell transcriptomic analysis will aid in determining the mechanism of action of the K27M mutant histone in pre-leukemic HSC clonal expansion. In addition, we will be performing similar single-cell analysis on HSCs expressing mutant ASXL1 as a comparison. Further understanding of the role of mutations in epigenetic regulators, such as histone H3 and ASXL1, in pre-leukemic clonal hematopoiesis will provide valuable insight on how to better prevent and treat AML and other myeloid malignancies. Disclosures No relevant conflicts of interest to declare.

scholarly journals Epigenetic Signature of Leukemia Stem Cells Defines Subgroups Associated with Clinical Outcome and Cell of Origin in AML

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2147-2147
Author(s):  
Namyoung Jung ◽  
Bo Dai ◽  
Andrew J. Gentles ◽  
Peter Murakami ◽  
Ravindra Majeti ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Stem Cells ◽  
Conflicts Of Interest ◽  
Cell Model ◽  
Leukemia Stem Cells ◽  
Functional Difference ◽  
Cell Of Origin ◽  
Hematopoietic Stem ◽  
Blast Cells

Abstract Acute myeloid leukemia (AML) is a hematologic malignancy initiated by leukemia-initiating or leukemia stem cells (LSC) which can differentiate into clonally related leukemic blast cells. This leukemia stem cell model proposes that functional properties of LSC and their blast progeny must be derived by epigenetic differences. Here, we examined genome wide DNA methylation of LSC-enriched populations and blast cells from 15 AML patients, along with 6 well-defined hematopoietic stem and progenitor cell (HSPC) populations from 5 normal controls using Illumina Infinium Human Methylation 450 BeadChip array. Strikingly, LSC-enriched populations exhibited global hypomethylation compared to non-engrafting blast cells, demonstrating that epigenetic change could drive the functional difference of LSC and their blast progeny. We defined an LSC epigenetic signature by integrating DNA methylation and gene expression analysis. The signature independently predicted overall survival of patients in both DNA methylation and gene expression data sets. Finally, we identified that LSC-enriched populations formed two major clusters when compared to normal HSPC: a granulocyte-macrophage progenitor (GMP)-like and a lymphoid-primed multipotential progenitor (L-MPP)-like subgroup that may reflect the cell of origin for these cases. These subgroups showed strong association with cytogenetic abnormalities and molecular mutations associated with the cell of origin. These results provide the first evidence for epigenetic variation between LSC and their blast progeny that are prognostic, and for epigenetically defined cell of origin of AML LSC. Disclosures No relevant conflicts of interest to declare.

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.

scholarly journals Molecular hallmarks of heterochronic parabiosis at single cell resolution

2020 ◽  
Author(s):  
Róbert Pálovics ◽  
Andreas Keller ◽  
Nicholas Schaum ◽  
Weilun Tan ◽  
Tobias Fehlmann ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Disease Risk ◽  
Global Gene Expression ◽  
Cell Types ◽  
The Body ◽  
Hematopoietic Stem ◽  
Gene Sets ◽  
Genes Encoding ◽  
Systemic Understanding

Slowing or reversing biological ageing would have major implications for mitigating disease risk and maintaining vitality. While an increasing number of interventions show promise for rejuvenation, the effectiveness on disparate cell types across the body and the molecular pathways susceptible to rejuvenation remain largely unexplored. We performed single-cell RNA-sequencing on 13 organs to reveal cell type specific responses to young or aged blood in heterochronic parabiosis. Adipose mesenchymal stromal cells, hematopoietic stem cells, hepatocytes, and endothelial cells from multiple tissues appear especially responsive. On the pathway level, young blood invokes novel gene sets in addition to reversing established ageing patterns, with the global rescue of genes encoding electron transport chain subunits pinpointing a prominent role of mitochondrial function in parabiosis-mediated rejuvenation. Intriguingly, we observed an almost universal loss of gene expression with age that is largely mimicked by parabiosis: aged blood reduces global gene expression, and young blood restores it. Altogether, these data lay the groundwork for a systemic understanding of the interplay between blood-borne factors and cellular integrity.

scholarly journals CD9 Defines Acute Myeloid Leukemia Stem Cells and is Regulated by Alpha-2-Macroglobulin

2020 ◽  
Author(s):  
Yongliang Liu ◽  
Guiqin Wang ◽  
Jiasi Zhang ◽  
Xue Chen ◽  
Huailong Xu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Drug Resistance ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Leukemia Stem Cells ◽  
Hematopoietic Stem ◽  
Therapy Strategy ◽  
And Migration ◽  
Acute Myeloid

Abstract Background: Leukemia stem cells (LSCs) are responsible for the initiation, progress and relapse of acute myeloid leukemia (AML). Therefore, the therapy strategy of targeting LSCs is hopeful to eradicate AML. In this study, we aim to identify LSC-specific surface markers and uncover the underlying mechanism of AML LSCs.Methods: Microarray gene expression data were used to investigate the candidate AML-LSC specific markers. CD9 expression was evaluated by flow cytometry in AML cell lines, patients with AML and normal donors. The biological characteristics of CD9-positive (CD9+) cells were analyzed by in vitro proliferation, chemotherapeutic drug resistance, migration and in vivo xenotransplantation assays. The molecular mechanism involved in CD9+ cell function was investigated by gene expression profiling. Effect of alpha-2-macroglobulin (A2M) on CD9+ cells was analyzed by proliferation, drug resistance and migration assays.Results: CD9 as a cell surface protein was specifically expressed on AML LSCs, but almost not expressed on normal hematopoietic stem cells (HSCs). CD9+ cells exhibited more resistance to chemotherapy drugs and higher migration potential than CD9-negative (CD9-) cells. More importantly, CD9+ cells possess the ability to reconstitute human AML in immunocompromised mice and promote tumor growth, suggesting CD9+ cells define the LSC population. Furthermore, we identified A2M plays a crucial role in CD9+ LSCs stemness maintenance. Down-regulation of A2M impairs drug-resistance and migration of CD9+ cells.Conclusion: Our findings suggest that CD9 is a new biomarker of AML LSCs and may serve as a promising therapeutic target.

scholarly journals A Unique Crosstalk between Tumor Cells and Hematopoietic Stem Cells Reveals a Myeloid Differentiation Pattern Signature Contributing to Metastasis

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2465-2465
Author(s):  
Ksenia Magidey ◽  
Ksenya Kveler ◽  
Rachelly Normand ◽  
Tongwu Zhang ◽  
Michael Timaner ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Single Cell ◽  
Tumor Cells ◽  
Expression Patterns ◽  
Cell Types ◽  
Myeloid Differentiation ◽  
Hematopoietic Stem ◽  
Tumor Bearing ◽  
Differentiation Pattern

Metastasis is the major cause of death in cancer patients. Recent studies have demonstrated that the crosstalk between different host and tumor cells in the tumor microenvironment regulates tumor progression and metastasis. Specifically, immune cell myeloid skewing is a prominent promoter of metastasis. While previous studies have demonstrated that the recruitment of myeloid cells to tumors is a critical step in dictating tumor fate, the reservoir of these cells in the bone marrow (BM) compartment and their differentiation pattern has not been explored. Here we utilized a unique model system consisting of tumor cell clones with low and high metastatic potential (met-low and met-high, respectively) derived from melanoma and breast carcinoma cell lines. Hematopoietic stem cells (HSCs) and their early progenitor subset were defined as Lin-/Sca1+/CD117+, representing LSK cells. BM transplantation experiments using GFP-positive LSK cells derived from met-low and met-high tumor bearing mice were carried out to study lineage differentiation. The genetic signatures of LSK cells were analyzed by single cell RNA-sequencing (scRNA-seq). This analysis included unbiased automated annotation of individual cell types by correlating single-cell gene expression with reference transcriptomic data sets (SingleR algorithm) in order to evaluate the proportions of cell types in BM and reveal cell type-specific differentially expressed genes. Expression patterns of proteins originated from tumor cells were analyzed using a range of multi-omics techniques including nanostring, protein array, and mass spectrometry analysis. Tumor proteomic data was integrated with differential receptor expression patterns in BM cell types to reveal novel crosstalk between tumor cells and HSCs in the BM compartment. Mice bearing met-high tumors exhibited a significant increase in the percentage of LSK cells in the BM in comparison to tumor-free mice or mice bearing met-low tumors. These results were confirmed by functional CFU assays of peripheral blood of met-high compared to met-low tumor bearing mice. In addition, mice that underwent BM transplantation with GFP-positive LSK cells obtained from met-high inoculated donors exhibited an increased percentage of circulating GFP-positive myeloid cells in comparison to counterpart mice transplanted with LSK cells from met-low inoculated donors. Moreover, scRNA-seq analysis of LSK cells obtained from the BM of met-low and met-high tumor bearing mice revealed that met-high tumors induce the enrichment of monocyte-dendritic progenitor population (MDP), confirmed also by flow cytometry. To uncover the possible factors involved in myeloid programming of LSK cells, we performed a proteomic screen of tumor conditioned medium and integrated the results with the scRNA-seq data analysis. This analysis revealed that the IL-6-IL-6R axis is highly active in LSK-derived MDP cells from mice bearing met-high tumors. An adoptive transfer experiment using MDP-GFP+ cells obtained from BM of met-high tumor bearing mice demonstrated that met-high tumors directly dictate HSC fate decision towards myeloid bias, resulting in increased metastasis. Evidently, blocking IL-6 in mice bearing met-high tumors reduced the number of MDP cells, and consequently decreased metastasis. Our study reveals a unique crosstalk between tumor cells and HSCs. It provides new insight into the mechanism by which tumors contribute to the presence of supporting stroma. Specifically, tumors secreting IL-6 dictate a specific genetic signature in HSCs that programs them towards myeloid differentiation, thereby inducing a metastatic switch. Disclosures No relevant conflicts of interest to declare.

scholarly journals Single Cell Transcriptome Analysis of GATA2 Deficiency in Hematopoiesis Modeled with Induced Pluripotent Stem Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5087-5087
Author(s):  
Francis Guitart ◽  
Moonjung Jung ◽  
Stefan Cordes ◽  
Shiqin Yu ◽  
Jizhong Zou ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Single Cell ◽  
Differential Gene Expression ◽  
Hematopoietic Stem ◽  
Manifold Alignment ◽  
Gata2 Deficiency ◽  
Differential Gene ◽  
Cell Transcriptome ◽  
Single Cell Transcriptome

Abstract GATA2 deficiency is a rare, inherited or sporadic genetic disorder characterized by variable onset of a pleomorphic constellation of immune, hematologic and lymphatic abnormalities linked to heterozygous mutations in the Gata2 gene. Patients develop monocyte, B cell, NK cell and dendritic cell deficiencies resulting in vulnerabilities to unusual infections. Patients with GATA2 deficiency also frequently progress to bone marrow failure, myelodysplastic syndrome and/or acute myelogenous leukemia. GATA proteins are transcription factors with central roles in early embryonic development and lineage specification. GATA2 is a master regulator of hematopoiesis, implicated in the initial generation and maintenance of hematopoietic stem cells (HSC). Murine models recapitulate the human phenotype incompletely: GATA2 heterozygous knockout mice do not manifest loss of monocyte, B cells or NK cells; however, serial repopulation assays show decreased engraftment potential. Direct studies of primary HSC from patients with GATA2 deficiency are challenging due to the generally hypocellular marrow. We hypothesized that human pluripotent stem cells, particularly patient-specific iPSC, could be used to study potential developmental defects in GATA2 deficiency, overcoming a lack of primary HSC. In order to gain insights into the impact of human GATA2 deficiency on hematopoietic differentiation, we compared the single cell transcriptomes of HSPC differentiated from (i) iPSCs from a patient with GATA2 deficiency due to a mutation p.R337X (c.1009C>T) (ii) isogenic iPSCs created via homology-directed repair of Gata2 p.R337X, (iii) iPSCs from a healthy control and (iv) isogenic Gata2 heterozygous mutant iPSCs with monoallelic frameshift mutations in the second zinc finger domain. Mesodermal and hematopoietic differentiation was performed under feeder-free, defined media conditions. At day 0, iPSCs were plated in mesodermal induction media containing VEGF, SCF, Activin A and Y27632 in STEMdiff APEL media. Mesodermal induction was continued until day 4, when embryoid bodies were cultured in hematopoietic specification media with SCF, FLT3L, IL3, IL6, G-CSF and BMP4 until day 16, when CD34+CD45+ iPSC-derived hematopoietic stem and progenitor cells (iHSC) were enumerated and sorted by fluorescence-activated cell sorting. Single cell RNA-seq was performed using the 10XGenomics Chromium platform and primary analysis via CellRanger. Scater was used to filter outlier cells. Seurat as used to compute multiple manifold alignment and differential gene expression. Cell classification, pseudotemporal ordering and branch point analysis were performed with monocle. URD was used to calculate confirmatory diffusion maps and pseudotemporal ordering. We analyzed 7,855 iHSPC (2952 from GATA2-deficient patient, 241 isogenic iHSPCs after repair of Gata2 mutation, 2,605 from a healthy volunteer and 2,057 from isogenic heterozygous Gata2 knockout iHSPCs) after filtering of outliers. We computed multiple manifold alignment to mitigate batch effects. Differential gene expression across Gata2 mutation status found that 42 out of 102 (42%) target genes of GATA2 (c.f. TRANSFAC database of curated transcription factor targets) were differentially expressed with adjusted p-values less than 0.05. Semi-supervised classification of cell-types and pseudotemporal ordering via monocle revealed two branch points, consistent with developmental branchings at the level of CLP and CMP multipotent progenitors. The numbers of cells along each branch was found to be statistically different (χ2=30.07, p-value = 3e-7) with the biggest differences noted in the lymphoid branch (state 4). Differential gene expression in this branch revealed a differential up-regulation of Notch1, CD69 and FKBPs and differential down-regulation of CD14. In conclusion, iPSC/iHSPC differentiation models combined with single cell transcriptome analysis may be a valuable tool to identify pathways responsible for impaired hematopoietic/lymphatic development in GATA2 deficiency. Figure. Figure. Disclosures Dunbar: National Institute of Health: Research Funding. Winkler:National Institute of Health: Research Funding.

Molecular and Functional Characterization of Early Lineage Commitment of Human Hematopoietic Stem Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 907-907
Author(s):  
Elisa Laurenti ◽  
Sergei Doulatov ◽  
Sasan Zandi ◽  
Jing Chen ◽  
Craig April ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Transcription Factor ◽  
Hematopoietic Stem Cells ◽  
B Cell ◽  
Cell Types ◽  
Cell Maturation ◽  
Hematopoietic Stem ◽  
B Cell Maturation ◽  
Early Human

Abstract Abstract 907 The hematopoietic system is a highly regulated cellular hierarchy, responsible for the day-to-day production of mature blood cells which can be divided in two major lineages, myeloid and lymphoid. Hematopoietic stem cells (HSCs) have the unique ability to give rise to all hematopoietic cell types, by first generating lineage-commited progenitors which in turn will produce terminally differentiated cells. HSCs are characterized by their extensive self-renewal and differentiation capacities. While in mice the mechanisms underlying early HSC differentiation and lineage determination are well understood at the molecular level, very few transcription factors regulating lineage decisions have been identified in human hematopoiesis. Our group has recently established a novel cell sorting strategy for human HSCs and early lineage committed progenitors (Doulatov et al., Nature Immunology, 2010; Notta et al., Science, 2011) which uncovered the existence of a novel human multilymphoid progenitor (MLP). MLPs give rise to all lymphoid cell types, as well as dendritic cells and monocytic cells. Here we report a comprehensive analysis of gene expression at each developmental stage of the early human hematopoietic hierarchy, ranging from the long-term repopulating stem cells to lineage-restricted progenitors through multipotent progenitors such as MLP, CMP (common myeloid progenitor), GMP (granulocyte-monocyte progenitor) and MEP (megakaryocyte-erythroid progenitor). We show that hematopoietic specification is defined by a small number of global gene expression clusters that correspond to major biological lineages and that lineage programs in committed progenitors are paired with HSC-shared priming programs. HSCs display most extensive priming along the lympho-myeloid branch (MLP). In contrast early progenitors of the megakaryocytic/erythrocytic lineage form a distinct cluster, highly enriched for cell cycle genes. To identify regulators of each major developmental transition, we computationally extracted population-specific gene-sets (“signatures”). We then integrated transcription factor expression data and enrichment of transcription factors binding sites in the promoters of each “signature” to obtain a map of transcriptional regulators in the context of the developmental hierarchy. Based on this model, we selected more than 15 candidate genes for functional validation. We chose genes predicted to act either on lymphoid (MLP), myeloid (MLP, CMP) or erythroid (MEP) commitment. Among these, we investigated the function of BCL11a, a C2H2 zinc finger transcriptional repressor, which expression is primed in HSCs then peaks in the newly discovered MLP population, indicating a putative role in lymphocyte specification. Consistent with this hypothesis, BCL11a has been implicated in the development of B cell progenitors in mouse. When BCL11a was knocked down in cord blood derived hematopoietic stem cells and early progenitors, we observed reduced formation of cells committed to the B cell fate both in vitro and in an in vivo xenograft assay. BCL11a knock-down resulted in a partial block of B cell maturation at the proB to preB cell transition, that was accompanied by a decrease in the key B cell maturation transcription factor, Pax5. These preliminary results suggest that BCL11a directs B cell specification in human and that our genome-wide strategy not only provides a valuable resource for the hematology community but also allows identification of key regulators of early human lineage commitment. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Mapping single-cell atlases throughout Metazoa unravels cell type evolution

2020 ◽  
Author(s):  
Alexander J. Tarashansky ◽  
Jacob M. Musser ◽  
Margarita Khariton ◽  
Pengyang Li ◽  
Detlev Arendt ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Single Cell ◽  
Cell Types ◽  
Tree Of Life ◽  
Transfer Cell ◽  
Cell Type ◽  
Germ Layers ◽  
Animal Evolution ◽  
Definition Of

AbstractComparing single-cell transcriptomic atlases from diverse organisms can provide evolutionary definition of cell types, elucidate the origins of cellular diversity, and transfer cell type knowledge between species. Yet, comparison among distant relatives, especially beyond a single phylum, is hindered by complex gene histories, lineage-specific inventions, and cell type evolutionary diversifications. Here, we develop a method to enable mapping cell atlases throughout Metazoa spanning sponge to mouse. Within phyla, we identify homologous cell types, even between distant species, with some even emerging from distinct germ layers. Across phyla, we find ancient cell type families that form densely interconnected groups, including contractile and stem cells, indicating they likely arose early in animal evolution through hierarchical diversifications. These homologous cell types often substitute paralog expressions at surprising prevalence. Our findings advance the understanding of cell type diversity across the tree of life and the evolution of associated gene expression programs.

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