Cellular and Molecular Architecture of the Human Hematopoietic Hierarchy by Sergei Doulatov A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Molecular and Medical Genetics University of Toronto © Copyright by Sergei Doulatov 2010 ii Cellular and Molecular Architecture of the Human Hematopoietic Hierarchy For the degree of Doctor of Philosophy, 2010 By Sergei Doulatov Graduate Department of Molecular Genetics University of Toronto ABSTRACT The blood system is organized as a developmental hierarchy in which rare hematopoietic stem cells (HSCs) generate large numbers of immature progenitors and differentiated mature blood cells. In this process, at least ten distict lineages are specified from multipotent stem cells, however the cellular and molecular organization of the hematopoietic hierarchy is a topic of intense investigation. While much has been learned from mouse models, there is also an appreciation for species-specific differences and the need for human studies. Blood lineages have been traditionally grouped into myeloid and lymphoid branches, and the long- standing dogma has been that the separation between these branches is the earliest event in fate specification. However, recent murine studies indicate that the progeny of initial specification retain the more ancestral myeloid potential. By contrast, much less is known about the progenitor hierarchy in human hematopoiesis. To dissect human hematopoiesis, we developed a novel sorting scheme to isolate human stem and progenitor cells from neonatal cord blood and adult bone marrow. As few as one in five single sorted HSCs efficiently repopulated immunodeficient mice enabling interrogation of homogeneous human stem cells. By analyzing the developmental potential of sorted progenitors at a single-cell level we iii showed that earliest human lymphoid progenitors (termed LMPs) possess myelo-monocytic potential. In addition to B-, T-, and natural killer cells, LMPs gave rise to dendritic cells and macrophages indicating that these closely related myeloid lineages also remain entangled in lymphoid development. These studies provide systematic insight into the organization of the human hematopoietic hierarchy, which provides the basis for detailed genetic analysis of molecular regulation in defined cell populations. In a pilot study, we investigated the role of a zinc finger transcription factor (ZNF145), PLZF, in myeloid development. We found that PLZF restrained proliferation and differentiation of myeloid progenitors and maintained the progenitor pool. Induction of ERK1/2 by myeloid cytokines, reflective of a stress response, leads to nuclear export and inactivation of PLZF, which augments mature cell production. Thus, negative regulators of differentiation can serve to maintain developmental systems in a primed state, so that their inactivation by extrinsic signals can induce proliferation and differentiation to rapidly satisfy increased demand for mature cells. Taken together, these studies advance our understanding of the cellular and molecular architecture of human hematopoiesis. iv Acknowledgements There are a number of people who have made this thesis possible. First of all, I want to thank my mentor, John. It’s not easy to be at the helm of a large successful lab, and for that matter, many stem-cell-related groups and consortia in Toronto and all over the country. During PhD, one defines themselves as a scientist, and that’s exactly what John did for all of his students – he let us define ourselves. Instead of imposing his own ideas of how science should be done, as do many others, he guided us to formulate our own ways of thinking and approaching problems. Never once has he told me that I couldn’t or shouldn’t do a particular experiment or pursue some flight of fancy idea, I suspect knowing full well that many of them were doomed to failure causing much pain and mental anguish. But he let us explore these wasteful directions anyways, so that we could learn from them, work through the anguish, and in the end become successful, productive, and independent scientists. I am indebted to him for guiding me through these difficult and exciting times. Science should never be done alone, if only because sometimes the sheer pain or joy that comes with this work has to be shared with someone. In this, I’ve been fortunate to have found a great partner, Faiyaz Notta, with whom we’ve split the scientific ‘bread’ every day. I know no one else who has such intelligence, tenacity and integrity, to which I credit so many of our successes. I also want to thank Jennifer Warner, for helping me get on my feet when I was starting in the lab. It’s been such a long time, but I will always be grateful to her for supporting me during that vulnerable time. I’ve also been lucky to have so many good friends from among colleagues in the lab, especially Antonija and Peter. v Luck plays a big part in our lives, on and off the bench. I’ve had the greatest luck of my life during my PhD to have met my fiancee, Nastasia, and after one amazing year know that I want to spend all of them with her. The least likely of chances have brought us together and I remember as clearly as it was yesterday the day I went to meet a total stranger, only to have met my sweetheart. There hasn’t been a day since that I have reminded myself of how incredible life’s ways can be. My sister, Masha, who will be starting on her own graduate path in philosophy soon, and whom I love and am so proud of. Lastly, my parents, to whom I owe everything that I am. No words that I have can express the gratitude and debt that I have to them. vi Table of Contents ABSTRACT.........................................................................................................ii Acknowledgements.........................................................................................iv Table of Contents ...........................................................................................vi LIST OF TABLES ..............................................................................................xii LIST OF FIGURES ...........................................................................................xiii LIST OF ABBREVIATIONS ................................................................................ xv 1. Introduction................................................................................................. 1 1.1. Models of hematopoiesis............................................................................................1 1.1.1 The structure of hematopoietic hierarchies: basic principles.................................2 1.1.2 Hematopoietic stem cells ......................................................................................3 1.1.3 The classical model of hematopoiesis....................................................................6 1.1.4 Towards a revised model of hematopoiesis ...........................................................9 1.1.4.1 The diversity of lymphoid progenitors ...........................................................................................10 1.1.4.2 Developmental flexibility of lymphoid progenitors.......................................................................13 1.1.5 Xenotransplantation of human cells....................................................................15 1.1.6 A model for human hematopoiesis ......................................................................16 1.1.7 Progenitor origins of dendritic cells ...................................................................18 vii 1.1.8 Conclusions 1 .....................................................................................................20 1.2 Transcription factor networks that control self-renewal and lineage choice................21 1.2.1 Lineage commitment: basic principles ................................................................21 1.2.2 Stages of lineage commitment.............................................................................22 1.2.2.1 Lineage priming ...............................................................................................................................22 1.2.2.2 Lineage reinforcement .....................................................................................................................23 1.2.2.3 Lineage commitment........................................................................................................................25 1.2.3 Mechanisms of lymphoid lineage commitment.....................................................26 1.2.4 Mechanisms of myeloid differentiation................................................................27 1.2.5 Mechanisms of progenitor homeostasis...............................................................31 1.2.6 Conclusions II.....................................................................................................33 2. Isolation of single human HSCs............................................................... 37 2.1 Abstract.....................................................................................................................38 2.2 Matherials and Methods ............................................................................................38 2.3 Results and Discussion..............................................................................................41 2.4 Tables and Figures ....................................................................................................49