Concurrent stem- and lineage-affiliated chromatin programs precede hematopoietic lineage restriction

The emerging notion of hematopoietic stem- and progenitor cells (HSPCs) as a low-primed cloud without sharply demarcated gene expression programs raises the question on how cellular fate options emerge, and at which stem-like stage lineage priming is initiated. Here we investigated single-cell chromatin accessibility of Lineage−, cKit+, Sca1+ (LSK) HSPCs spanning the early differentiation landscape. Application of a signal-processing algorithm to detect transition points corresponding to massive alterations in accessibility of 571 transcription factor-motifs revealed a population of LSK FMS-like tyrosine kinase 3(Flt3)intCD9high cells that concurrently display stem-like and lineage-affiliated chromatin signatures pointing to a simultaneous gain of both Lympho-Myeloid and Megakaryocyte-Erythroid programs. Molecularly and functionally, these cells position between stem cells and committed progenitors, display multi-lineage capacity in vitro and in vivo, but lack self-renewal activity. This integrative molecular analysis resolves the heterogeneity of cells along hematopoietic differentiation and permits investigation of chromatin-mediated transition between multipotency and lineage restriction.

[1]  G. Marguerie,et al.  Serum‐free medium allows the optimal growth of human megakaryocyte progenitors compared with human plasma supplemented cultures: Role of TGF β , 1993, Stem cells.

[2]  C. Bonifer,et al.  Chromatin priming of genes in development: Concepts, mechanisms and consequences. , 2017, Experimental hematology.

[3]  Martin J. Aryee,et al.  Integrated Single-Cell Analysis Maps the Continuous Regulatory Landscape of Human Hematopoietic Differentiation , 2018, Cell.

[4]  L. Zon,et al.  Hematopoiesis: An Evolving Paradigm for Stem Cell Biology , 2008, Cell.

[5]  Howard Y. Chang,et al.  Single-cell lineage tracing by endogenous mutations enriched in transposase accessible mitochondrial DNA , 2018, bioRxiv.

[6]  P. Woll,et al.  FLT3 expression initiates in fully multipotent mouse hematopoietic progenitor cells. , 2011, Blood.

[7]  S. Chiba,et al.  The Road Map for Megakaryopoietic Lineage from Hematopoietic Stem/Progenitor Cells , 2017, Stem cells translational medicine.

[8]  S. Morrison,et al.  SLAM family markers are conserved among hematopoietic stem cells from old and reconstituted mice and markedly increase their purity. , 2006, Blood.

[9]  Lina A. Thoren,et al.  Identification of Flt3+ Lympho-Myeloid Stem Cells Lacking Erythro-Megakaryocytic Potential A Revised Road Map for Adult Blood Lineage Commitment , 2005, Cell.

[10]  Yaniv Lubling,et al.  Single-cell characterization of haematopoietic progenitors and their trajectories in homeostasis and perturbed haematopoiesis , 2018, Nature Cell Biology.

[11]  Allon M. Klein,et al.  Lineage tracing on transcriptional landscapes links state to fate during differentiation , 2020, Science.

[12]  I. Macaulay,et al.  Single-cell RNA sequencing reveals molecular and functional platelet bias of aged haematopoietic stem cells , 2016, Nature Communications.

[13]  G. Sanguinetti,et al.  Multi-omics profiling of mouse gastrulation at single cell resolution , 2019, Nature.

[14]  Phillip A. Richmond,et al.  JASPAR 2020: update of the open-access database of transcription factor binding profiles , 2019, Nucleic Acids Res..

[15]  Ross C. Hardison,et al.  Dynamic shifts in occupancy by TAL1 are guided by GATA factors and drive large-scale reprogramming of gene expression during hematopoiesis , 2014, Genome research.

[16]  L. Steinmetz,et al.  Human haematopoietic stem cell lineage commitment is a continuous process , 2017, Nature Cell Biology.

[17]  Berthold Göttgens,et al.  Functionally Distinct Subsets of Lineage-Biased Multipotent Progenitors Control Blood Production in Normal and Regenerative Conditions. , 2015, Cell stem cell.

[18]  T. Honjo,et al.  Generation of lymphohematopoietic cells from embryonic stem cells in culture. , 1994, Science.

[19]  E. Scott,et al.  Role of PU.1 in Hematopoiesis , 1998, Stem cells.

[20]  H. Stunnenberg,et al.  The tetraspanin CD9 affords high-purity capture of all murine hematopoietic stem cells. , 2013, Cell reports.

[21]  E. Forsberg,et al.  Flk2/Flt3 promotes both myeloid and lymphoid development by expanding non-self-renewing multipotent hematopoietic progenitor cells. , 2014, Experimental hematology.

[22]  I. Weissman,et al.  A clonogenic common myeloid progenitor that gives rise to all myeloid lineages , 2000, Nature.

[23]  I. Weissman,et al.  Flk-2 is a marker in hematopoietic stem cell differentiation: A simple method to isolate long-term stem cells , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[24]  Samuel L. Wolock,et al.  Clonal analysis of lineage fate in native hematopoiesis , 2017, Nature.

[25]  Berthold Göttgens,et al.  Myelo-lymphoid lineage restriction occurs in the human haematopoietic stem cell compartment before lymphoid-primed multipotent progenitors , 2018, Nature Communications.

[26]  Allon M. Klein,et al.  Lineage tracing on transcriptional landscapes links state to fate during differentiation , 2018, Science.

[27]  Hannah A. Pliner,et al.  Reversed graph embedding resolves complex single-cell trajectories , 2017, Nature Methods.

[28]  Michael J. Ziller,et al.  Epigenetic Memory Underlies Cell-Autonomous Heterogeneous Behavior of Hematopoietic Stem Cells , 2016, Cell.

[29]  T. Meehan,et al.  An atlas of active enhancers across human cell types and tissues , 2014, Nature.

[30]  Howard Y. Chang,et al.  Massively parallel single-cell chromatin landscapes of human immune cell development and intratumoral T cell exhaustion , 2019, Nature Biotechnology.

[31]  Lai Guan Ng,et al.  Dimensionality reduction for visualizing single-cell data using UMAP , 2018, Nature Biotechnology.

[32]  Peter J. Campbell,et al.  Population dynamics of normal human blood inferred from somatic mutations , 2018, Nature.

[33]  V. Beneš,et al.  Vitamin A-Retinoic Acid Signaling Regulates Hematopoietic Stem Cell Dormancy , 2017, Cell.

[34]  B. Göttgens,et al.  From haematopoietic stem cells to complex differentiation landscapes , 2018, Nature.

[35]  I. Weissman,et al.  Stem cells, cancer, and cancer stem cells , 2001, Nature.

[36]  Frederik Otzen Bagger,et al.  BloodSpot: a database of healthy and malignant haematopoiesis updated with purified and single cell mRNA sequencing profiles , 2018, Nucleic Acids Res..

[37]  I. Weissman,et al.  Identification of Clonogenic Common Lymphoid Progenitors in Mouse Bone Marrow , 1997, Cell.

[38]  Single-cell lineage tracing by endogenous mutations enriched in transposase accessible mitochondrial DNA , 2018, bioRxiv.

[39]  S. E. Jacobsen,et al.  Identification of Lin(-)Sca1(+)kit(+)CD34(+)Flt3- short-term hematopoietic stem cells capable of rapidly reconstituting and rescuing myeloablated transplant recipients. , 2005, Blood.

[40]  R. Hoffman,et al.  Effect of recombinant and purified hematopoietic growth factors on human megakaryocyte colony formation. , 1988, Experimental hematology.

[41]  Hiromitsu Nakauchi,et al.  Long-Term Lymphohematopoietic Reconstitution by a Single CD34-Low/Negative Hematopoietic Stem Cell , 1996, Science.

[42]  I. Weissman,et al.  Characterization of mouse clonogenic megakaryocyte progenitors , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[43]  T. Cheng,et al.  New paradigms on hematopoietic stem cell differentiation , 2019, Protein & Cell.

[44]  Charles P. Lin,et al.  Epigenetic Memory Underlies Cell-Autonomous Heterogeneous Behavior of Hematopoietic Stem Cells , 2016, Cell.

[45]  V. Yang,et al.  Mammalian Krüppel-like factors in health and diseases. , 2010, Physiological reviews.

[46]  Shamit Soneji,et al.  Molecular evidence for hierarchical transcriptional lineage priming in fetal and adult stem cells and multipotent progenitors. , 2007, Immunity.

[47]  S. Jacobsen,et al.  Upregulation of Flt3 expression within the bone marrow Lin(-)Sca1(+)c-kit(+) stem cell compartment is accompanied by loss of self-renewal capacity. , 2001, Immunity.

[48]  Patrick S. Stumpf,et al.  Thrombopoietin Metabolically Primes Hematopoietic Stem Cells to Megakaryocyte-Lineage Differentiation. , 2018, Cell reports.

[49]  E. Forsberg,et al.  All hematopoietic cells develop from hematopoietic stem cells through Flk2/Flt3-positive progenitor cells. , 2011, Cell stem cell.

[50]  A. Regev,et al.  Spatial reconstruction of single-cell gene expression , 2015, Nature Biotechnology.

[51]  M. Sigvardsson,et al.  Shaping up a lineage--lessons from B lymphopoesis. , 2010, Current opinion in immunology.

[52]  C. Woolthuis,et al.  Hematopoietic stem/progenitor cell commitment to the megakaryocyte lineage. , 2016, Blood.

[53]  Andrew W. Murray,et al.  Association of Spindle Assembly Checkpoint Component XMAD2 with Unattached Kinetochores , 1996, Science.

[54]  Ole Winther,et al.  BloodSpot: a database of gene expression profiles and transcriptional programs for healthy and malignant haematopoiesis , 2015, Nucleic Acids Res..

[55]  Shamit Soneji,et al.  SCExV: a webtool for the analysis and visualisation of single cell qRT-PCR data , 2015, BMC Bioinformatics.

[56]  I. Weissman,et al.  Identification of a lineage of multipotent hematopoietic progenitors. , 1997, Development.

[57]  I. Amit,et al.  Transcriptional Heterogeneity and Lineage Commitment in Myeloid Progenitors , 2016, Cell.

[58]  Martin J. Aryee,et al.  Lineage Tracing in Humans Enabled by Mitochondrial Mutations and Single-Cell Genomics , 2019, Cell.

[59]  S. Morrison,et al.  CD150- cells are transiently reconstituting multipotent progenitors with little or no stem cell activity. , 2008, Blood.

[60]  Bruce J. Aronow,et al.  Single-cell analysis of mixed-lineage states leading to a binary cell fate choice , 2016, Nature.

[61]  J. Wysocka,et al.  Modification of enhancer chromatin: what, how, and why? , 2013, Molecular cell.

[62]  Russell B. Fletcher,et al.  Slingshot: cell lineage and pseudotime inference for single-cell transcriptomics , 2017, BMC Genomics.

[63]  M. Sommarin,et al.  A Combinatorial Single-cell Approach to Characterize the Molecular and Immunophenotypic Heterogeneity of Human Stem and Progenitor Populations. , 2018, Journal of visualized experiments : JoVE.