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1.
Nat Commun ; 14(1): 6062, 2023 09 28.
Article in English | MEDLINE | ID: mdl-37770432

ABSTRACT

Hematopoietic stem cells (HSCs) residing in specialized niches in the bone marrow are responsible for the balanced output of multiple short-lived blood cell lineages in steady-state and in response to different challenges. However, feedback mechanisms by which HSCs, through their niches, sense acute losses of specific blood cell lineages remain to be established. While all HSCs replenish platelets, previous studies have shown that a large fraction of HSCs are molecularly primed for the megakaryocyte-platelet lineage and are rapidly recruited into proliferation upon platelet depletion. Platelets normally turnover in an activation-dependent manner, herein mimicked by antibodies inducing platelet activation and depletion. Antibody-mediated platelet activation upregulates expression of Interleukin-1 (IL-1) in platelets, and in bone marrow extracellular fluid in vivo. Genetic experiments demonstrate that rather than IL-1 directly activating HSCs, activation of bone marrow Lepr+ perivascular niche cells expressing IL-1 receptor is critical for the optimal activation of quiescent HSCs upon platelet activation and depletion. These findings identify a feedback mechanism by which activation-induced depletion of a mature blood cell lineage leads to a niche-dependent activation of HSCs to reinstate its homeostasis.


Subject(s)
Interleukin-1 , Thrombocytopenia , Humans , Interleukin-1/metabolism , Hematopoietic Stem Cells/metabolism , Bone Marrow/metabolism , Megakaryocytes , Thrombocytopenia/metabolism
2.
Blood Adv ; 7(9): 1725-1738, 2023 05 09.
Article in English | MEDLINE | ID: mdl-36453632

ABSTRACT

We recently described a low-affinity second-generation CD19 chimeric antigen receptor (CAR) CAT that showed enhanced expansion, cytotoxicity, and antitumor efficacy compared with the high-affinity (FMC63-based) CAR used in tisagenlecleucel, in preclinical models. Furthermore, CAT demonstrated an excellent toxicity profile, enhanced in vivo expansion, and long-term persistence in a phase 1 clinical study. To understand the molecular mechanisms behind these properties of CAT CAR T cells, we performed a systematic in vitro characterization of the transcriptomic (RNA sequencing) and protein (cytometry by time of flight) changes occurring in T cells expressing low-affinity vs high-affinity CD19 CARs following stimulation with CD19-expressing cells. Our results show that CAT CAR T cells exhibit enhanced activation to CD19 stimulation and a distinct transcriptomic and protein profile, with increased activation and cytokine polyfunctionality compared with FMC63 CAR T cells. We demonstrate that the enhanced functionality of low-affinity CAT CAR T cells is a consequence of an antigen-dependent priming induced by residual CD19-expressing B cells present in the manufacture.


Subject(s)
Cytokines , Receptors, Chimeric Antigen , Cytokines/metabolism , Immunotherapy, Adoptive/methods , T-Lymphocytes , Receptors, Chimeric Antigen/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Antigens, CD19
3.
STAR Protoc ; 3(1): 101174, 2022 03 18.
Article in English | MEDLINE | ID: mdl-35199038

ABSTRACT

Here, we present a comprehensive protocol for the generation and functional characterization of chimeric antigen receptor (CAR) T cells and their products by mass cytometry in a reproducible and scalable manner. We describe the production of CAR T cells from human peripheral blood mononuclear cells. We then detail a three-step staining protocol with metal-labeled antibodies and the subsequent mass cytometry analysis. This protocol allows simultaneous characterization of CAR T cell intracellular signaling, activation, proliferation, cytokine production, and phenotype in a single assay.


Subject(s)
Leukocytes, Mononuclear , T-Lymphocytes , Antibodies , Humans
4.
Cancers (Basel) ; 13(11)2021 Jun 05.
Article in English | MEDLINE | ID: mdl-34198742

ABSTRACT

Current treatment outcome for acute myeloid leukemia (AML) patients is unsatisfactory and characterized by high rates of relapse and poor overall survival. Increasing evidence points to a crucial role of leukemic stem cells (LSC) and the bone marrow (BM) leukemic niche, in which they reside, in AML evolution and chemoresistance. Thus, future strategies aiming at improving AML therapeutic protocols are likely to be directed against LSC and their niche. Chimeric antigen receptor (CAR) T-cells have been extremely successful in the treatment of relapsed/refractory acute lymphoblastic leukemia and B-cell non-Hodgkin lymphoma and comparable results in AML are highly desirable. At present, we are at the dawn of CAR T-cell application in AML, with several preclinical studies and few early phase clinical trials. However, the lack of leukemia-specific targets and the genetic and phenotypic heterogeneity of the disease combined with the leukemia-induced remodeling of the BM microenvironment are limiting CAR T-cell exploitation in AML. Here, we reviewed AML-LSC and AML-BM niche features in the context of their therapeutic targeting using CAR T-cells. We summarized recent progress in CAR T-cell application to the treatment of AML, and we discussed the remaining therapeutic challenges and promising novel strategies to overcome them.

5.
Front Oncol ; 11: 666829, 2021.
Article in English | MEDLINE | ID: mdl-33996595

ABSTRACT

Acute Myeloid Leukaemia (AML) is a phenotypically and genetically heterogenous blood cancer characterised by very poor prognosis, with disease relapse being the primary cause of treatment failure. AML heterogeneity arise from different genetic and non-genetic sources, including its proposed hierarchical structure, with leukemic stem cells (LSCs) and progenitors giving origin to a variety of more mature leukemic subsets. Recent advances in single-cell molecular and phenotypic profiling have highlighted the intra and inter-patient heterogeneous nature of AML, which has so far limited the success of cell-based immunotherapy approaches against single targets. Machine Learning (ML) can be uniquely used to find non-trivial patterns from high-dimensional datasets and identify rare sub-populations. Here we review some recent ML tools that applied to single-cell data could help disentangle cell heterogeneity in AML by identifying distinct core molecular signatures of leukemic cell subsets. We discuss the advantages and limitations of unsupervised and supervised ML approaches to cluster and classify cell populations in AML, for the identification of biomarkers and the design of personalised therapies.

6.
Cell Stem Cell ; 27(3): 359-360, 2020 09 03.
Article in English | MEDLINE | ID: mdl-32888424

ABSTRACT

COVID-19 has unfortunately halted lab work, conferences, and in-person networking, which is especially detrimental to researchers just starting their labs. Through social media and our reviewer networks, we met some early-career stem cell investigators impacted by the closures. Here, they introduce themselves and their research to our readers.


Subject(s)
Betacoronavirus/physiology , Coronavirus Infections/virology , Pneumonia, Viral/virology , Research Personnel , Animals , COVID-19 , Humans , Pandemics , SARS-CoV-2 , Stem Cells/cytology
7.
Mol Cell ; 73(6): 1292-1305.e8, 2019 03 21.
Article in English | MEDLINE | ID: mdl-30765193

ABSTRACT

Single-cell RNA sequencing (scRNA-seq) has emerged as a powerful tool for resolving transcriptional heterogeneity. However, its application to studying cancerous tissues is currently hampered by the lack of coverage across key mutation hotspots in the vast majority of cells; this lack of coverage prevents the correlation of genetic and transcriptional readouts from the same single cell. To overcome this, we developed TARGET-seq, a method for the high-sensitivity detection of multiple mutations within single cells from both genomic and coding DNA, in parallel with unbiased whole-transcriptome analysis. Applying TARGET-seq to 4,559 single cells, we demonstrate how this technique uniquely resolves transcriptional and genetic tumor heterogeneity in myeloproliferative neoplasms (MPN) stem and progenitor cells, providing insights into deregulated pathways of mutant and non-mutant cells. TARGET-seq is a powerful tool for resolving the molecular signatures of genetically distinct subclones of cancer cells.


Subject(s)
Biomarkers, Tumor/genetics , DNA Mutational Analysis/methods , Genetic Heterogeneity , High-Throughput Nucleotide Sequencing , Leukemia/genetics , Mutation , Sequence Analysis, RNA , Single-Cell Analysis , Humans , Jurkat Cells , K562 Cells , Reproducibility of Results , Schizosaccharomyces/genetics
8.
Cancer Cell ; 33(2): 274-291.e8, 2018 02 12.
Article in English | MEDLINE | ID: mdl-29438697

ABSTRACT

Lympho-myeloid restricted early thymic progenitors (ETPs) are postulated to be the cell of origin for ETP leukemias, a therapy-resistant leukemia associated with frequent co-occurrence of EZH2 and RUNX1 inactivating mutations, and constitutively activating signaling pathway mutations. In a mouse model, we demonstrate that Ezh2 and Runx1 inactivation targeted to early lymphoid progenitors causes a marked expansion of pre-leukemic ETPs, showing transcriptional signatures characteristic of ETP leukemia. Addition of a RAS-signaling pathway mutation (Flt3-ITD) results in an aggressive leukemia co-expressing myeloid and lymphoid genes, which can be established and propagated in vivo by the expanded ETPs. Both mouse and human ETP leukemias show sensitivity to BET inhibition in vitro and in vivo, which reverses aberrant gene expression induced by Ezh2 inactivation.


Subject(s)
Core Binding Factor Alpha 2 Subunit/genetics , Enhancer of Zeste Homolog 2 Protein/genetics , Leukemia, Myeloid, Acute/genetics , Mutation/genetics , Animals , Gene Expression Regulation, Leukemic , Mice, Knockout , Myeloid Cells/metabolism , Signal Transduction/genetics , Stem Cells
9.
J Exp Med ; 214(10): 3085-3104, 2017 Oct 02.
Article in English | MEDLINE | ID: mdl-28899870

ABSTRACT

Differentiation of lineage-committed cells from multipotent progenitors requires the establishment of accessible chromatin at lineage-specific transcriptional enhancers and promoters, which is mediated by pioneer transcription factors that recruit activating chromatin remodeling complexes. Here we show that the Mbd3/nucleosome remodeling and deacetylation (NuRD) chromatin remodeling complex opposes this transcriptional pioneering during B cell programming of multipotent lymphoid progenitors by restricting chromatin accessibility at B cell enhancers and promoters. Mbd3/NuRD-deficient lymphoid progenitors therefore prematurely activate a B cell transcriptional program and are biased toward overproduction of pro-B cells at the expense of T cell progenitors. The striking reduction in early thymic T cell progenitors results in compensatory hyperproliferation of immature thymocytes and development of T cell lymphoma. Our results reveal that Mbd3/NuRD can regulate multilineage differentiation by constraining the activation of dormant lineage-specific enhancers and promoters. In this way, Mbd3/NuRD protects the multipotency of lymphoid progenitors, preventing B cell-programming transcription factors from prematurely enacting lineage commitment. Mbd3/NuRD therefore controls the fate of lymphoid progenitors, ensuring appropriate production of lineage-committed progeny and suppressing tumor formation.


Subject(s)
B-Lymphocytes/metabolism , Carcinogenesis/metabolism , Cell Lineage/physiology , DNA-Binding Proteins/physiology , Lymphocytes/physiology , Mi-2 Nucleosome Remodeling and Deacetylase Complex/physiology , Transcription Factors/physiology , Animals , Cell Differentiation/physiology , Gene Expression Regulation/physiology , Lymphoma, T-Cell/etiology , Mice , Mice, Inbred C57BL , Multipotent Stem Cells/physiology , Thymocytes/metabolism , Thymocytes/physiology
10.
J Exp Med ; 214(7): 2005-2021, 2017 Jul 03.
Article in English | MEDLINE | ID: mdl-28637883

ABSTRACT

Although previous studies suggested that the expression of FMS-like tyrosine kinase 3 (Flt3) initiates downstream of mouse hematopoietic stem cells (HSCs), FLT3 internal tandem duplications (FLT3 ITDs) have recently been suggested to intrinsically suppress HSCs. Herein, single-cell interrogation found Flt3 mRNA expression to be absent in the large majority of phenotypic HSCs, with a strong negative correlation between Flt3 and HSC-associated gene expression. Flt3-ITD knock-in mice showed reduced numbers of phenotypic HSCs, with an even more severe loss of long-term repopulating HSCs, likely reflecting the presence of non-HSCs within the phenotypic HSC compartment. Competitive transplantation experiments established that Flt3-ITD compromises HSCs through an extrinsically mediated mechanism of disrupting HSC-supporting bone marrow stromal cells, with reduced numbers of endothelial and mesenchymal stromal cells showing increased inflammation-associated gene expression. Tumor necrosis factor (TNF), a cell-extrinsic potent negative regulator of HSCs, was overexpressed in bone marrow niche cells from FLT3-ITD mice, and anti-TNF treatment partially rescued the HSC phenotype. These findings, which establish that Flt3-ITD-driven myeloproliferation results in cell-extrinsic suppression of the normal HSC reservoir, are of relevance for several aspects of acute myeloid leukemia biology.


Subject(s)
Cell Proliferation/genetics , Hematopoietic Stem Cells/metabolism , Mutation , Stem Cell Niche/genetics , fms-Like Tyrosine Kinase 3/genetics , Animals , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Bone Marrow Cells/drug effects , Bone Marrow Cells/metabolism , Bone Marrow Transplantation , Cells, Cultured , Etanercept/pharmacology , Gene Expression Profiling/methods , Hematopoietic Stem Cells/drug effects , Mesenchymal Stem Cells/drug effects , Mesenchymal Stem Cells/metabolism , Mice, Inbred C57BL , Mice, Transgenic , Reverse Transcriptase Polymerase Chain Reaction , Single-Cell Analysis/methods , Tandem Repeat Sequences/genetics , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolism
11.
Nat Med ; 23(6): 692-702, 2017 Jun.
Article in English | MEDLINE | ID: mdl-28504724

ABSTRACT

Recent advances in single-cell transcriptomics are ideally placed to unravel intratumoral heterogeneity and selective resistance of cancer stem cell (SC) subpopulations to molecularly targeted cancer therapies. However, current single-cell RNA-sequencing approaches lack the sensitivity required to reliably detect somatic mutations. We developed a method that combines high-sensitivity mutation detection with whole-transcriptome analysis of the same single cell. We applied this technique to analyze more than 2,000 SCs from patients with chronic myeloid leukemia (CML) throughout the disease course, revealing heterogeneity of CML-SCs, including the identification of a subgroup of CML-SCs with a distinct molecular signature that selectively persisted during prolonged therapy. Analysis of nonleukemic SCs from patients with CML also provided new insights into cell-extrinsic disruption of hematopoiesis in CML associated with clinical outcome. Furthermore, we used this single-cell approach to identify a blast-crisis-specific SC population, which was also present in a subclone of CML-SCs during the chronic phase in a patient who subsequently developed blast crisis. This approach, which might be broadly applied to any malignancy, illustrates how single-cell analysis can identify subpopulations of therapy-resistant SCs that are not apparent through cell-population analysis.


Subject(s)
Blast Crisis/genetics , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Neoplastic Stem Cells/metabolism , Single-Cell Analysis , Adult , Aged , Chromatin Immunoprecipitation , Core Binding Factor Alpha 2 Subunit/genetics , Female , Flow Cytometry , Gene Library , Genes, abl/genetics , Humans , In Situ Hybridization, Fluorescence , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , Male , Middle Aged , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis, DNA , Sequence Analysis, RNA , Transcriptome , Young Adult
12.
Nat Immunol ; 17(12): 1424-1435, 2016 Dec.
Article in English | MEDLINE | ID: mdl-27695000

ABSTRACT

The final stages of restriction to the T cell lineage occur in the thymus after the entry of thymus-seeding progenitors (TSPs). The identity and lineage potential of TSPs remains unclear. Because the first embryonic TSPs enter a non-vascularized thymic rudiment, we were able to directly image and establish the functional and molecular properties of embryonic thymopoiesis-initiating progenitors (T-IPs) before their entry into the thymus and activation of Notch signaling. T-IPs did not include multipotent stem cells or molecular evidence of T cell-restricted progenitors. Instead, single-cell molecular and functional analysis demonstrated that most fetal T-IPs expressed genes of and had the potential to develop into lymphoid as well as myeloid components of the immune system. Moreover, studies of embryos deficient in the transcriptional regulator RBPJ demonstrated that canonical Notch signaling was not involved in pre-thymic restriction to the T cell lineage or the migration of T-IPs.


Subject(s)
Immunoglobulin J Recombination Signal Sequence-Binding Protein/metabolism , Lymphoid Progenitor Cells/physiology , Myeloid Progenitor Cells/physiology , Receptors, Notch/metabolism , T-Lymphocytes/physiology , Thymus Gland/immunology , Animals , Cell Differentiation , Cell Lineage , Cell Movement , Cells, Cultured , Fetus , Gene Expression Regulation, Developmental , Immunoglobulin J Recombination Signal Sequence-Binding Protein/genetics , Mice , Mice, Inbred C57BL , Mice, Transgenic , Signal Transduction
13.
Cancer Cell ; 29(6): 905-921, 2016 06 13.
Article in English | MEDLINE | ID: mdl-27300437

ABSTRACT

MicroRNA (miRNA)-126 is a known regulator of hematopoietic stem cell quiescence. We engineered murine hematopoiesis to express miRNA-126 across all differentiation stages. Thirty percent of mice developed monoclonal B cell leukemia, which was prevented or regressed when a tetracycline-repressible miRNA-126 cassette was switched off. Regression was accompanied by upregulation of cell-cycle regulators and B cell differentiation genes, and downregulation of oncogenic signaling pathways. Expression of dominant-negative p53 delayed blast clearance upon miRNA-126 switch-off, highlighting the relevance of p53 inhibition in miRNA-126 addiction. Forced miRNA-126 expression in mouse and human progenitors reduced p53 transcriptional activity through regulation of multiple p53-related targets. miRNA-126 is highly expressed in a subset of human B-ALL, and antagonizing miRNA-126 in ALL xenograft models triggered apoptosis and reduced disease burden.


Subject(s)
Hematopoietic Stem Cells/metabolism , MicroRNAs/genetics , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/pathology , Tumor Suppressor Protein p53/genetics , Animals , Apoptosis , Cell Cycle , Cell Differentiation , Gene Expression Regulation, Neoplastic , Hematopoietic Stem Cell Transplantation , Humans , Mice , MicroRNAs/metabolism , Neoplasms, Experimental , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Signal Transduction , Up-Regulation
14.
Nat Immunol ; 17(6): 666-676, 2016 06.
Article in English | MEDLINE | ID: mdl-27043410

ABSTRACT

According to current models of hematopoiesis, lymphoid-primed multi-potent progenitors (LMPPs) (Lin(-)Sca-1(+)c-Kit(+)CD34(+)Flt3(hi)) and common myeloid progenitors (CMPs) (Lin(-)Sca-1(+)c-Kit(+)CD34(+)CD41(hi)) establish an early branch point for separate lineage-commitment pathways from hematopoietic stem cells, with the notable exception that both pathways are proposed to generate all myeloid innate immune cell types through the same myeloid-restricted pre-granulocyte-macrophage progenitor (pre-GM) (Lin(-)Sca-1(-)c-Kit(+)CD41(-)FcγRII/III(-)CD150(-)CD105(-)). By single-cell transcriptome profiling of pre-GMs, we identified distinct myeloid differentiation pathways: a pathway expressing the gene encoding the transcription factor GATA-1 generated mast cells, eosinophils, megakaryocytes and erythroid cells, and a pathway lacking expression of that gene generated monocytes, neutrophils and lymphocytes. These results identify an early hematopoietic-lineage bifurcation that separates the myeloid lineages before their segregation from other hematopoietic-lineage potential.


Subject(s)
Cell Differentiation , Cell Lineage , Lymphocytes/physiology , Myeloid Cells/physiology , Myeloid Progenitor Cells/physiology , Animals , Antigens, CD/metabolism , Cells, Cultured , Computational Biology , GATA1 Transcription Factor/genetics , GATA1 Transcription Factor/metabolism , Hematopoiesis , Immunity, Innate , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Sequence Analysis, RNA , Single-Cell Analysis , Tissue Array Analysis , fms-Like Tyrosine Kinase 3/genetics , fms-Like Tyrosine Kinase 3/metabolism
15.
Nat Commun ; 7: 11075, 2016 Mar 24.
Article in English | MEDLINE | ID: mdl-27009448

ABSTRACT

Aged haematopoietic stem cells (HSCs) generate more myeloid cells and fewer lymphoid cells compared with young HSCs, contributing to decreased adaptive immunity in aged individuals. However, it is not known how intrinsic changes to HSCs and shifts in the balance between biased HSC subsets each contribute to the altered lineage output. Here, by analysing HSC transcriptomes and HSC function at the single-cell level, we identify increased molecular platelet priming and functional platelet bias as the predominant age-dependent change to HSCs, including a significant increase in a previously unrecognized class of HSCs that exclusively produce platelets. Depletion of HSC platelet programming through loss of the FOG-1 transcription factor is accompanied by increased lymphoid output. Therefore, increased platelet bias may contribute to the age-associated decrease in lymphopoiesis.


Subject(s)
Blood Platelets/metabolism , Cellular Senescence , Hematopoietic Stem Cells/cytology , Sequence Analysis, RNA/methods , Single-Cell Analysis/methods , Animals , Cell Lineage/genetics , Cell Proliferation , Female , Gene Expression Profiling , Hematopoietic Stem Cells/metabolism , Male , Mice , Myeloid Cells/cytology , Nuclear Proteins/metabolism , Phenotype , Transcription Factors/metabolism
16.
Nat Cell Biol ; 18(2): 157-67, 2016 Feb.
Article in English | MEDLINE | ID: mdl-26780297

ABSTRACT

Thymic T cell development is initiated from bone-marrow-derived multi potent thymus-seeding progenitors. During the early stages of thymocyte differentiation, progenitors become T cell restricted. However, the cellular environments supporting these critical initial stages of T cell development within the thymic cortex are not known. Here we use the dependence of early, c-Kit-expressing thymic progenitors on Kit ligand (KitL) to show that CD4(-)CD8(-)c-Kit(+)CD25(-) DN1-stage progenitors associate with, and depend on, the membrane-bound form of KitL (mKitL) provided by a cortex-specific KitL-expressing vascular endothelial cell (VEC) population. In contrast, the subsequent CD4(-)CD8(-)c-Kit(+)CD25(+) DN2-stage progenitors associate selectively with cortical thymic epithelial cells (cTECs) and depend on cTEC-presented mKitL. These results show that the dynamic process of early thymic progenitor differentiation is paralleled by migration-dependent change to the supporting niche, and identify VECs as a thymic niche cell, with mKitL as a critical ligand.


Subject(s)
Cell Differentiation , Cell Movement , Endothelial Cells/metabolism , Multipotent Stem Cells/metabolism , Paracrine Communication , Stem Cell Factor/metabolism , Stem Cell Niche , Thymocytes/metabolism , Animals , Cell Differentiation/genetics , Cell Lineage , Cell Movement/genetics , Cells, Cultured , Coculture Techniques , Gene Expression Regulation, Developmental , Mice, Transgenic , Paracrine Communication/genetics , Phenotype , Signal Transduction , Stem Cell Factor/genetics
17.
Hum Mol Genet ; 25(5): 989-1000, 2016 Mar 01.
Article in English | MEDLINE | ID: mdl-26740550

ABSTRACT

Induced pluripotent stem cell (iPSC)-derived cortical neurons potentially present a powerful new model to understand corticogenesis and neurological disease. Previous work has established that differentiation protocols can produce cortical neurons, but little has been done to characterize these at cellular resolution. In particular, it is unclear to what extent in vitro two-dimensional, relatively disordered culture conditions recapitulate the development of in vivo cortical layer identity. Single-cell multiplex reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) was used to interrogate the expression of genes previously implicated in cortical layer or phenotypic identity in individual cells. Totally, 93.6% of single cells derived from iPSCs expressed genes indicative of neuronal identity. High proportions of single neurons derived from iPSCs expressed glutamatergic receptors and synaptic genes. And, 68.4% of iPSC-derived neurons expressing at least one layer marker could be assigned to a laminar identity using canonical cortical layer marker genes. We compared single-cell RNA-seq of our iPSC-derived neurons to available single-cell RNA-seq data from human fetal and adult brain and found that iPSC-derived cortical neurons closely resembled primary fetal brain cells. Unexpectedly, a subpopulation of iPSC-derived neurons co-expressed canonical fetal deep and upper cortical layer markers. However, this appeared to be concordant with data from primary cells. Our results therefore provide reassurance that iPSC-derived cortical neurons are highly similar to primary cortical neurons at the level of single cells but suggest that current layer markers, although effective, may not be able to disambiguate cortical layer identity in all cells.


Subject(s)
Cerebral Cortex/metabolism , Induced Pluripotent Stem Cells/metabolism , Nerve Tissue Proteins/metabolism , Neurogenesis/genetics , Neurons/metabolism , Transcriptome , Adult , Aged , Biomarkers/metabolism , Cell Differentiation , Cell Line , Cerebral Cortex/cytology , Female , Fetus , Fibroblasts/cytology , Fibroblasts/metabolism , Gene Expression Profiling , Gene Expression Regulation, Developmental , Humans , Induced Pluripotent Stem Cells/cytology , Nerve Tissue Proteins/genetics , Neurons/cytology , Real-Time Polymerase Chain Reaction , Receptors, Glutamate/genetics , Receptors, Glutamate/metabolism , Sequence Analysis, RNA , Single-Cell Analysis
18.
Cancer Cell ; 25(6): 794-808, 2014 Jun 16.
Article in English | MEDLINE | ID: mdl-24835589

ABSTRACT

Evidence for distinct human cancer stem cells (CSCs) remains contentious and the degree to which different cancer cells contribute to propagating malignancies in patients remains unexplored. In low- to intermediate-risk myelodysplastic syndromes (MDS), we establish the existence of rare multipotent MDS stem cells (MDS-SCs), and their hierarchical relationship to lineage-restricted MDS progenitors. All identified somatically acquired genetic lesions were backtracked to distinct MDS-SCs, establishing their distinct MDS-propagating function in vivo. In isolated del(5q)-MDS, acquisition of del(5q) preceded diverse recurrent driver mutations. Sequential analysis in del(5q)-MDS revealed genetic evolution in MDS-SCs and MDS-progenitors prior to leukemic transformation. These findings provide definitive evidence for rare human MDS-SCs in vivo, with extensive implications for the targeting of the cells required and sufficient for MDS-propagation.


Subject(s)
Myelodysplastic Syndromes/genetics , Myelodysplastic Syndromes/pathology , Neoplastic Stem Cells/physiology , Animals , Antigens, CD/biosynthesis , Antigens, CD/immunology , Chromosome Deletion , Chromosomes, Human, Pair 5 , Flow Cytometry , Gene Expression Profiling , Genetic Predisposition to Disease , Heterografts , Humans , Mice , Mice, Inbred NOD , Mutation , Myelodysplastic Syndromes/immunology , Neoplastic Stem Cells/immunology , Neoplastic Stem Cells/pathology , Prognosis
19.
Mol Ther ; 21(5): 934-46, 2013 May.
Article in English | MEDLINE | ID: mdl-23439497

ABSTRACT

To better understand and exploit microRNA (miR) regulation, a more precise characterization of miR expression patterns within a tissue or a lineage during development, differentiation, and homeostasis is needed. We previously showed that lentiviral vectors (LV) can be made responsive to miR to stringently control transgene expression as well as to report miR activity "live" and at the single-cell level. Although very useful, this approach reports miR activity by transgene suppression, hampering the direct identification and selection of miR-expressing cells. Here, we describe a strategy to couple transgene expression to the activity of the miR of interest. To this aim, we generated LV encoding two in-series OFF switches: a transcriptional repressor tagged with miR target sequences and a reporter cassette under the control of the repressor. Reporter expression is ON only when the miR is active and represses translation of the transcriptional repressor. We successfully applied this design to different types of repressors, multiple gene encoding vectors and delivered the system either by two separate or a self-contained vector. We demonstrated its performance by live monitoring of two miRs in different stages of human primary hematopoietic stem/progenitor cell differentiation in vivo. Further applications of this approach include imaging of rare miR-expressing cells and positive regulation of a therapeutic or selector gene in target cells identified by the expression of selected miRs.


Subject(s)
Gene Expression Regulation , Genetic Vectors/genetics , Lentivirus/genetics , MicroRNAs , Transgenes , Binding Sites , Cell Line , Gene Expression , Gene Order , Gene Transfer Techniques , Genes, Reporter , Hematopoietic Stem Cells/metabolism , Humans , Protein Binding , Trans-Activators/genetics , Trans-Activators/metabolism , Transcription, Genetic
20.
Cell Stem Cell ; 11(6): 799-811, 2012 Dec 07.
Article in English | MEDLINE | ID: mdl-23142521

ABSTRACT

Lifelong blood cell production is governed through the poorly understood integration of cell-intrinsic and -extrinsic control of hematopoietic stem cell (HSC) quiescence and activation. MicroRNAs (miRNAs) coordinately regulate multiple targets within signaling networks, making them attractive candidate HSC regulators. We report that miR-126, a miRNA expressed in HSC and early progenitors, plays a pivotal role in restraining cell-cycle progression of HSC in vitro and in vivo. miR-126 knockdown by using lentiviral sponges increased HSC proliferation without inducing exhaustion, resulting in expansion of mouse and human long-term repopulating HSC. Conversely, enforced miR-126 expression impaired cell-cycle entry, leading to progressively reduced hematopoietic contribution. In HSC/early progenitors, miR-126 regulates multiple targets within the PI3K/AKT/GSK3ß pathway, attenuating signal transduction in response to extrinsic signals. These data establish that miR-126 sets a threshold for HSC activation and thus governs HSC pool size, demonstrating the importance of miRNA in the control of HSC function.


Subject(s)
Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , MicroRNAs/metabolism , Animals , Cell Line , Cell Proliferation , Gene Knockdown Techniques , Glycogen Synthase Kinase 3/metabolism , Glycogen Synthase Kinase 3 beta , Hematopoiesis/genetics , Hematopoietic Stem Cells/enzymology , Humans , Mice , MicroRNAs/genetics , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction , Transplantation, Heterologous
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