Project description:Background: Recent advances in single-cell techniques have provided the opportunity to finely dissect cellular heterogeneity within populations previously defined by âbulkâ assays and to uncover rare cell types. In human hematopoiesis, megakaryocytes and erythroid cells differentiate from a shared precursor, the megakaryocyte-erythroid progenitor (MEP), which remains poorly defined.Results: To clarify the cellular pathway in erythro-megakaryocyte differentiation, we correlated the surface immunophenotype, transcriptional profile and differentiation potential of individual MEP cells. Highly purified, single MEP cells (n=681) were analyzed using index fluorescence-activated cell sorting with parallel targeted transcriptional profiling of the same cells performed using a specifically designed panel of 87 genes. Differentiation potential was tested in novel, single-cell differentiation assays. Our results demonstrated that immunophenotypic MEP in fact comprise three distinct subpopulations: (1) âPre-MEPâ, enriched for erythroid/megakaryocyte progenitors but with residual myeloid differentiation capacity (2) âE-MEPâ, strongly biased towards erythroid differentiation, and (3) âMK-MEPâ, a previously undescribed, rare population of cells that are bipotent but primarily generate megakaryocytic progeny. Therefore, conventionally-defined MEP are in fact a mixed population: a minority give rise to mixed-lineage colonies while the majority of cells are transcriptionally-primed to generate exclusively single-lineage output. Conclusions: Our study clarifies the cellular hierarchy in human megakaryocyte/erythroid lineage commitment and highlights the importance of using a combination of single-cell approaches to dissect cellular heterogeneity and identify rare cell types within a population. We present a novel immunophenotyping strategy that enables the prospective identification of specific intermediate progenitor populations in erythro-megakaryopoiesis, allowing for in-depth study of disorders including inherited cytopenias, myeloproliferative disorders and erythromegakaryocytic leukemias. Multiplex RT-PCR gene expression profiling of 807 human megakaryocyte-erythroid progenitor cells (MEP) isolated from three healthy donors by apheresis following G-CSF treatment. Cells were excluded if more than 70 assays did not result in amplification or displayed Ct higer than 13 for B2M or higher than 15 for GAPDH. Furthermore cells with a mean non-dropout Ct value greater than 20 were removed. This resulted in a dataset of 681 cells, which were subsequently normalised to the mean of B2M and GAPDH expression.
Project description:Background: Recent advances in single-cell techniques have provided the opportunity to finely dissect cellular heterogeneity within populations previously defined by “bulk” assays and to uncover rare cell types. In human hematopoiesis, megakaryocytes and erythroid cells differentiate from a shared precursor, the megakaryocyte-erythroid progenitor (MEP), which remains poorly defined.Results: To clarify the cellular pathway in erythro-megakaryocyte differentiation, we correlated the surface immunophenotype, transcriptional profile and differentiation potential of individual MEP cells. Highly purified, single MEP cells (n=681) were analyzed using index fluorescence-activated cell sorting with parallel targeted transcriptional profiling of the same cells performed using a specifically designed panel of 87 genes. Differentiation potential was tested in novel, single-cell differentiation assays. Our results demonstrated that immunophenotypic MEP in fact comprise three distinct subpopulations: (1) “Pre-MEP”, enriched for erythroid/megakaryocyte progenitors but with residual myeloid differentiation capacity (2) “E-MEP”, strongly biased towards erythroid differentiation, and (3) “MK-MEP”, a previously undescribed, rare population of cells that are bipotent but primarily generate megakaryocytic progeny. Therefore, conventionally-defined MEP are in fact a mixed population: a minority give rise to mixed-lineage colonies while the majority of cells are transcriptionally-primed to generate exclusively single-lineage output. Conclusions: Our study clarifies the cellular hierarchy in human megakaryocyte/erythroid lineage commitment and highlights the importance of using a combination of single-cell approaches to dissect cellular heterogeneity and identify rare cell types within a population. We present a novel immunophenotyping strategy that enables the prospective identification of specific intermediate progenitor populations in erythro-megakaryopoiesis, allowing for in-depth study of disorders including inherited cytopenias, myeloproliferative disorders and erythromegakaryocytic leukemias.
Project description:We leverage the extraordinary molecular diversity of modified heparan sulfate (HS) glycans 8 to establish cellular glycotypes, defined by binding patterns of a panel of flow-cytometry compatible single-chain variable fragment antibodies (scFvs) specific for differentially modified HS. We find distinct glycotypes between closely related hematopoietic progenitors and lineages. The glycotypes of murine and human hematopoietic stem and progenitor cells (HSPCs) reveal dynamic yet similar HS modification patterns in vivo and in vitro, including along megakaryocyte and erythrocyte differentiation. Prospective HS scFv-based sorting identifies new cellular subtypes from both immunophenotypic megakaryocyte-erythrocyte progenitors and heterogeneous pools of HSPCs, thus offering additional discriminative power beyond conventional CD markers. Mechanistically, single-cell RNAseq revealed that a heptad of HS-related genes participate in megakaryocyte-erythrocyte fate determination and are reflective of the HS epitope recognized by specific HS scFvs. In summary, HS glycotyping establishes a role for HS modification patterns in hematopoietic lineage differentiation in mouse and human, and provides an orthogonal approach to define and isolate viable cell types across different cell lineages and species at unprecedented resolution.
Project description:We leverage the extraordinary molecular diversity of modified heparan sulfate (HS) glycans to establish cellular glycotypes, defined by binding patterns of a panel of flow-cytometry compatible single-chain variable fragment antibodies (scFvs) specific for differentially modified HS. We find distinct glycotypes between closely related hematopoietic progenitors and lineages. The glycotypes of murine and human hematopoietic stem and progenitor cells (HSPCs) reveal dynamic yet similar HS modification patterns in vivo and in vitro, including along megakaryocyte and erythrocyte differentiation. Prospective HS scFv-based sorting identifies new cellular subtypes from both immunophenotypic megakaryocyte-erythrocyte progenitors and heterogeneous pools of HSPCs, thus offering additional discriminative power beyond conventional CD markers. Mechanistically, single-cell RNAseq revealed that a heptad of HS-related genes participate in megakaryocyte-erythrocyte fate determination and are reflective of the HS epitope recognized by specific HS scFvs. In summary, HS glycotyping establishes a role for HS modification patterns in hematopoietic lineage differentiation in mouse and human, and provides an orthogonal approach to define and isolate viable cell types across different cell lineages and species at unprecedented resolution.
Project description:MEIS1 is a transcription factor expressed in hematopoietic stem and progenitor cells (HSPC) and in mature megakaryocytes. In contrast to its role in leukemogenesis, the role of MEIS1 in normal hematopoiesis is largely unknown. We show that MEIS1 can direct human hematopoietic progenitors towards a megakaryocyte-erythroid progenitor (MEP) fate. Ectopoic expression of MEIS1 in CD34+ cells resulted in increased erythroid differentiation at the expense of granulocyte and monocyte (GM) differentiation. MEIS1 overexpression not only skewed differentiation of CMPs towards the erythroid lineage but also reprogrammed GM progenitors towards erythrocyte differentiation. Expression profiling was used to determine the transcriptional changes induced by MEIS1 that lead to the oberved phenotype. A transcriptional program enriched for erythrocytic and megakaryocytic genes was detected.
Project description:Expression data from FACS-purified hematopoietic stem cells, common myeloid progenitors, granulocyte-macrophage progenitors, and megakaryocyte-erythroid progenitors from human bone marrow samples of elderly anemic patients
Project description:To clarigy the molecular mechanism underlying the IMiD-induced megakaryocytic lineage commitment in human megakaryocyte/erythrocyte progenitors, we performed transcriptome analysis of CD41-CD105+ and CD41+CD105- cells obtained from megakaryocyte/erythrocyte progenitors treated with lenalidomide for 72 hours. Differentially expressed genes were comprehensively evaluated by gene set enrichment analysis. CD105-CD41+ cells showed the enrichment in the expression of genes related to megakaryocytes and platelet, whereas genes related to erythroid lineage were negatively enriched. CD41+CD105- cells expressed higher levels of transcription factors required for megakaryocyte differentiation including FLI1, RUNX1, and MEIS1. Furthermore, we performed a knockdown strategy with small interfering RNA against MEIS1 mRNA, confirming that lenalidomide-induced megakaryocyte commitment should be due to the up-regulation of MEIS1 in megakaryocyte/erythrocyte progenitors.
Project description:Embryonic stem (ES) and induced pluripotent stem (iPS) cells represent a potential source of megakaryocytes and platelets for transfusion therapies. However, most current ES/iPS cell differentiation protocols are limited by low yields of hematopoietic progeny. Mutations in the mouse and human genes encoding transcription factor GATA1 cause accumulation of proliferating, developmentally arrested megakaryocytes. To exploit this clinical observation, we engineered wildtype (WT) murine ES cells to express doxycycline (dox)-regulated Gata1 short hairpin (sh) RNAs. In vitro differentiation with dox and thrombopoietin (Tpo) resulted in approximately 1013-fold expansion of immature hematopoietic progenitors. Upon dox withdrawal with multilineage cytokines, GATA1 expression was restored and the cells differentiated into erythroblasts and megakaryocytes. With Tpo alone, dox-deprived progenitors formed mainly mature megakaryocytes that generated functional platelets in vivo. Our findings provide a novel, readily reproducible strategy to expand ES-cell derived megakaryocyte-erythroid progenitors and direct their differentiation into megakaryocytes producing functional platelets in clinically relevant numbers. 3 classes of samples were compared 1) fetal liver derived megkaryocytes 2) G1ME (Gata1â megakaryocyte-erythroid) 3) G1ME2 (engineered wildtype (WT) murine ES cells to express doxycycline (dox)-regulated Gata1 short hairpin (sh) RNAs)
Project description:Hamey2017 - Blood stem cell regulatory
network (LMPP network)
This model is described in the article:
Reconstructing blood stem
cell regulatory network models from single-cell molecular
profiles
Fiona K. Hamey, Sonia Nestorowa,
Sarah J. Kinston, David G. Kent, Nicola K. Wilson, and Berthold
Göttgens
Proceedings of the National Academy of
Sciences of the United States of America
Abstract:
Adult blood contains a mixture of mature cell types, each
with specialized functions. Single hematopoietic stem cells
(HSCs) have been functionally shown to generate all mature cell
types for the lifetime of the organism. Differentiation of HSCs
toward alternative lineages must be balanced at the population
level by the fate decisions made by individual cells.
Transcription factors play a key role in regulating these
decisions and operate within organized regulatory programs that
can be modeled as transcriptional regulatory networks. As
dysregulation of single HSC fate decisions is linked to fatal
malignancies such as leukemia, it is important to understand
how these decisions are controlled on a cell-by-cell basis.
Here we developed and applied a network inference method,
exploiting the ability to infer dynamic information from
single-cell snapshot expression data based on expression
profiles of 48 genes in 2,167 blood stem and progenitor cells.
This approach allowed us to infer transcriptional regulatory
network models that recapitulated differentiation of HSCs into
progenitor cell types, focusing on trajectories toward
megakaryocyte–erythrocyte progenitors and lymphoid-primed
multipotent progenitors. By comparing these two models, we
identified and subsequently experimentally validated a
difference in the regulation of nuclear factor, erythroid 2
(Nfe2) and core-binding factor, runt domain, alpha subunit 2,
translocated to, 3 homolog (Cbfa2t3h) by the transcription
factor Gata2. Our approach confirms known aspects of
hematopoiesis, provides hypotheses about regulation of HSC
differentiation, and is widely applicable to other hierarchical
biological systems to uncover regulatory relationships.
This model is hosted on
BioModels Database
and identified by:
MODEL1610060001.
To cite BioModels Database, please use:
BioModels Database:
An enhanced, curated and annotated resource for published
quantitative kinetic models.
To the extent possible under law, all copyright and related or
neighbouring rights to this encoded model have been dedicated to
the public domain worldwide. Please refer to
CC0
Public Domain Dedication for more information.
Project description:Hamey2017 - Blood stem cell regulatory
network
This model is described in the article:
Reconstructing blood stem
cell regulatory network models from single-cell molecular
profiles
Fiona K. Hamey, Sonia Nestorowa,
Sarah J. Kinston, David G. Kent, Nicola K. Wilson, and Berthold
Göttgens
Proceedings of the National Academy of
Sciences of the United States of America
Abstract:
Adult blood contains a mixture of mature cell types, each
with specialized functions. Single hematopoietic stem cells
(HSCs) have been functionally shown to generate all mature cell
types for the lifetime of the organism. Differentiation of HSCs
toward alternative lineages must be balanced at the population
level by the fate decisions made by individual cells.
Transcription factors play a key role in regulating these
decisions and operate within organized regulatory programs that
can be modeled as transcriptional regulatory networks. As
dysregulation of single HSC fate decisions is linked to fatal
malignancies such as leukemia, it is important to understand
how these decisions are controlled on a cell-by-cell basis.
Here we developed and applied a network inference method,
exploiting the ability to infer dynamic information from
single-cell snapshot expression data based on expression
profiles of 48 genes in 2,167 blood stem and progenitor cells.
This approach allowed us to infer transcriptional regulatory
network models that recapitulated differentiation of HSCs into
progenitor cell types, focusing on trajectories toward
megakaryocyte–erythrocyte progenitors and lymphoid-primed
multipotent progenitors. By comparing these two models, we
identified and subsequently experimentally validated a
difference in the regulation of nuclear factor, erythroid 2
(Nfe2) and core-binding factor, runt domain, alpha subunit 2,
translocated to, 3 homolog (Cbfa2t3h) by the transcription
factor Gata2. Our approach confirms known aspects of
hematopoiesis, provides hypotheses about regulation of HSC
differentiation, and is widely applicable to other hierarchical
biological systems to uncover regulatory relationships.
This model is hosted on
BioModels Database
and identified by:
MODEL1610060000.
To cite BioModels Database, please use:
BioModels Database:
An enhanced, curated and annotated resource for published
quantitative kinetic models.
To the extent possible under law, all copyright and related or
neighbouring rights to this encoded model have been dedicated to
the public domain worldwide. Please refer to
CC0
Public Domain Dedication for more information.