A NOVEL MODEL OF HUMAN LYMPHO-MYELOID PROGENITOR HIERARCHY BASED ON SINGLE CELL FUNCTIONAL AND TRANSCRIPTIONAL ANALYSIS
(Abstract release date: 05/18/17)
EHA Library. Karamitros D. 06/23/17; 181427; S140
Dr. Dimitrios Karamitros
Contributions
Contributions
Abstract
Abstract: S140
Type: Oral Presentation
Presentation during EHA22: On Friday, June 23, 2017 from 12:30 - 12:45
Location: Room N104
Background
Human hemopoiesis produces 10 billion new, terminally mature, blood cells daily; a production that is also rapidly responsive to external change. Dysregulation of this complex process can lead to hemopoietic and immune deficiencies and blood cancers. In humans the hemopoietic progenitor hierarchy producing lymphoid and granulocytic-monocytic (myeloid) lineages is unclear. Multiple progenitor populations give rise to lymphoid and myeloid cells but they remain incompletely characterized at the immunophenotypic, transcriptional and functional level.
Aims
Here, we aim to understand the clonal functional output and transcriptional programs of current primary human lympho-myeloid containing progenitor populations - the lymphoid-primed multi-potential progenitor (LMPP)1, multilymphoid progenitor (MLP)2 and granulocyte-macrophage progenitor (GMP).
Methods
We devised a FACS-staining and sorting strategy to prospectively purify eight human hematopoietic stem and progenitor cell (HSPC) populations. We compared function of LMPP, MLP and GMP in vitro by quantitative CFU assays, single cell liquid cultures or limit dilution analysis and in vivo by transplantation into humanized ossciles. We performed population RNA sequencing and single cell RT-PCR analysis to understand the relationship between functional and transcriptional heterogeneity.
Results
Our study comprehensively characterized the LMPP, MLP and GMP lympho-myeloid populations. Both LMPP and MLP are very rare within the mononuclear fraction (1 in 104 to 1 in 105). We cultured 3806 single LMPP, GMP and MLP cells (isolated from 21 cord blood units and equivalent to ~1011 mononuclear cells) under three different culture conditions. We observed marked functional heterogeneity in the three lympho-myeloid progenitor populations. Focusing on the wells that gave single cell initiated cultures the majority of cells from LMPP, MLP and GMP gave unilineage output (50-80%). Bi-lineage output was the next most common output, while multilineage output was rare and only seen from LMPP and GMP cells (9-15%). In vivo transplantation using a novel humanized ossicle assay increased the engraftment of lympho-myeloid progenitors 10-fold compared to previous reports. In vivo, the LMPP and GMP gave robust engraftment but the MLP substantially less engraftment. The LMPP gave rise to both GM and B cell engraftment, the GMP myeloid engraftment and the MLP mainly B cell output. RNA-Seq revealed distinct transcriptional signatures of these populations: MLP signature enriched for lymphoid-affiliated genes and transcription factors (TF), GMP gene signature enriched for myeloid-affiliated genes and TF and LMPP a hybrid lympho-myeloid signature. Analysis of the expression of 72 genes in 919 single LMPP, MLP and GMP shows that the 3 populations form a transcriptional continuum. Moreover, this analysis allowed us to further purify the myeloid potential of GMP population with the use of an alternative sorting strategy.
Conclusion
These data change our understanding of human hematopoiesis and propose a radically new model of lympho-myeloid progenitor specification. This model has important implications for human immune deficiencies and hemopoietic malignancies.
1. Goardon, N. et al. Coexistence of LMPP-like and GMP-like leukemia stem cells in acute myeloid leukemia. Cancer Cell 19, 138-152, (2011).
2. Doulatov, S. et al. Revised map of the human progenitor hierarchy shows the origin of macrophages and dendritic cells in early lymphoid development. Nat Immunol 11, (2010).
DK and BS are equally contributing authors
Session topic: 23. Hematopoiesis, stem cells and microenvironment
Keyword(s): Progenitor cell, Human CD34+ cell, Hematopoietic stem and progenitor cells, Hematopoiesis
Abstract: S140
Type: Oral Presentation
Presentation during EHA22: On Friday, June 23, 2017 from 12:30 - 12:45
Location: Room N104
Background
Human hemopoiesis produces 10 billion new, terminally mature, blood cells daily; a production that is also rapidly responsive to external change. Dysregulation of this complex process can lead to hemopoietic and immune deficiencies and blood cancers. In humans the hemopoietic progenitor hierarchy producing lymphoid and granulocytic-monocytic (myeloid) lineages is unclear. Multiple progenitor populations give rise to lymphoid and myeloid cells but they remain incompletely characterized at the immunophenotypic, transcriptional and functional level.
Aims
Here, we aim to understand the clonal functional output and transcriptional programs of current primary human lympho-myeloid containing progenitor populations - the lymphoid-primed multi-potential progenitor (LMPP)1, multilymphoid progenitor (MLP)2 and granulocyte-macrophage progenitor (GMP).
Methods
We devised a FACS-staining and sorting strategy to prospectively purify eight human hematopoietic stem and progenitor cell (HSPC) populations. We compared function of LMPP, MLP and GMP in vitro by quantitative CFU assays, single cell liquid cultures or limit dilution analysis and in vivo by transplantation into humanized ossciles. We performed population RNA sequencing and single cell RT-PCR analysis to understand the relationship between functional and transcriptional heterogeneity.
Results
Our study comprehensively characterized the LMPP, MLP and GMP lympho-myeloid populations. Both LMPP and MLP are very rare within the mononuclear fraction (1 in 104 to 1 in 105). We cultured 3806 single LMPP, GMP and MLP cells (isolated from 21 cord blood units and equivalent to ~1011 mononuclear cells) under three different culture conditions. We observed marked functional heterogeneity in the three lympho-myeloid progenitor populations. Focusing on the wells that gave single cell initiated cultures the majority of cells from LMPP, MLP and GMP gave unilineage output (50-80%). Bi-lineage output was the next most common output, while multilineage output was rare and only seen from LMPP and GMP cells (9-15%). In vivo transplantation using a novel humanized ossicle assay increased the engraftment of lympho-myeloid progenitors 10-fold compared to previous reports. In vivo, the LMPP and GMP gave robust engraftment but the MLP substantially less engraftment. The LMPP gave rise to both GM and B cell engraftment, the GMP myeloid engraftment and the MLP mainly B cell output. RNA-Seq revealed distinct transcriptional signatures of these populations: MLP signature enriched for lymphoid-affiliated genes and transcription factors (TF), GMP gene signature enriched for myeloid-affiliated genes and TF and LMPP a hybrid lympho-myeloid signature. Analysis of the expression of 72 genes in 919 single LMPP, MLP and GMP shows that the 3 populations form a transcriptional continuum. Moreover, this analysis allowed us to further purify the myeloid potential of GMP population with the use of an alternative sorting strategy.
Conclusion
These data change our understanding of human hematopoiesis and propose a radically new model of lympho-myeloid progenitor specification. This model has important implications for human immune deficiencies and hemopoietic malignancies.
1. Goardon, N. et al. Coexistence of LMPP-like and GMP-like leukemia stem cells in acute myeloid leukemia. Cancer Cell 19, 138-152, (2011).
2. Doulatov, S. et al. Revised map of the human progenitor hierarchy shows the origin of macrophages and dendritic cells in early lymphoid development. Nat Immunol 11, (2010).
DK and BS are equally contributing authors
Session topic: 23. Hematopoiesis, stem cells and microenvironment
Keyword(s): Progenitor cell, Human CD34+ cell, Hematopoietic stem and progenitor cells, Hematopoiesis
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