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Hematopoiesis
Clonal tracking of erythropoiesis in rhesus macaques
Ferrata Storti Foundation
Haematologica 2020 Volume 105(7):1813-1824
Xing Fan,1 Chuanfeng Wu,1 Lauren L. Truitt,1 Diego A. Espinoza,1,2 Stephanie Sellers,1 Aylin Bonifacino,1 Yifan Zhou,1,3 Stefan F. Cordes,1 Allen Krouse,1 Mark Metzger,1 Robert E. Donahue,1 Rong Lu4
and Cynthia E. Dunbar1
1Translational Stem Cell Biology Branch, National Heart, Lung, and Blood Institute, National Institute of Health, Bethesda, MA, USA; 2Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA; 3Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, UK and 4Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, University of Southern California, Los Angeles, CA, USA
ABSTRACT
The classical model of hematopoietic hierarchies is being reconsidered on the basis of data from in vitro assays and single cell expression pro- filing. Recent experiments suggested that the erythroid lineage might differentiate directly from multipotent hematopoietic stem cells / progeni- tors or from a highly biased subpopulation of stem cells, rather than tran- siting through common myeloid progenitors or megakaryocyte-erythro- cyte progenitors. We genetically barcoded autologous rhesus macaque stem and progenitor cells, allowing quantitative tracking of the in vivo clonal out- put of thousands of individual cells over time following transplantation. CD34+ cells were lentiviral-transduced with a high diversity barcode library, with the barcode in an expressed region of the provirus, allowing barcode retrieval from DNA or RNA, with each barcode representing an individual stem or progenitor cell clone. Barcode profiles from bone marrow CD45–CD71+ maturing nucleated red blood cells were compared with other lineages purified from the same bone marrow sample. There was very high correlation of barcode contributions between marrow nucleated red blood cells and other lineages, with the highest correlation between nucleated red blood cells and myeloid lineages, whether at earlier or later time points post transplantation, without obvious clonal contributions from highly ery- throid-biased or restricted clones. A similar profile occurred even under stressors such as aging or erythropoietin stimulation. RNA barcode analysis on circulating mature red blood cells followed over long time periods demonstrated stable erythroid clonal contributions. Overall, in this non- human primate model with great relevance to human hematopoiesis, we documented continuous production of erythroid cells from multipotent, non-biased hematopoietic stem cell clones at steady-state or under stress.
Introduction
In the classical model of hematopoiesis, initially constructed from data obtained via in vitro colony assays and transplantation of populations of flow-sorted phenotypically-defined murine bone marrow (BM) cells, the top of the hematopo- ietic hierarchy is comprised of a pool of homogenous, self-renewing and always multipotent long-term hematopoietic stem cells (LT-HSC), producing downstream stem and progenitor cells via branching pathways passing through discrete interme- diate stages. These processes were characterized by stepwise restriction of self-renewal and lineage potential, passing through short-term multipotent HSC (ST-HSC), multipotent progenitors (MPP), and lineage-restricted progenitors, bifur- cating first into lymphoid versus myeloid progenitors, followed by common myeloid progenitors (CMP) branching towards granulocyte-monocyte progenitors (GMP) and megakaryocyte-erythrocyte progenitors (MEP) in both murine and human studies.1-3 Optimized in vitro clonal assays, large-scale single cell murine transplantation assays, in vivo clonal tracking via genetic tags and single cell gene
Correspondence:
CYNTHIA DUNBAR
dunbarc@nhlbi.nih.gov
Received: July 8, 2019. Accepted: October 3, 2019. Pre-published: October 3, 2019.
doi:10.3324/haematol.2019.231811
Check the online version for the most updated information on this article, online supplements, and information on authorship & disclosures: www.haematologica.org/content/105/7/1813
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