In Vivo Tracking of Human Hematopoiesis Reveals Patterns of Clonal Dynamics during Early and Steady-State Reconstitution Phases

摘要

class="head no_bottom_margin" id="sec1title">IntroductionThe hematopoietic system is a complex hierarchical structure that produces several different types of specialized blood cells, most of which are short-lived and thereby require continuous replenishment with hematopoietic stem/progenitor cells (HSPCs). Autologous or allogeneic transplantation of HSPCs is widely used to reconstitute functional hematopoiesis in patients with hematological diseases (, , , , , ). Despite the well-established clinical use of HSPCs, their short- and long-term fate after transplantation and the clonal dynamics of hematopoietic reconstitution in humans remain poorly understood. Over the past few years, a series of phenotypic and functional characterization studies have identified various HSPC subpopulations within cells expressing the CD34 antigen, including hematopoietic stem cells (HSCs), which are the most undifferentiated stem cell type, and multipotent progenitors (MPPs), which are downstream of the differentiation hierarchy but still capable of multilineage output (). Different cell hierarchies of human hematopoiesis have been proposed, including the early branching of myeloid and lymphoid lineages (, ) or the ontological proximity of lymphoid lineages to myeloid compartments due to the existence of a myeloid-primed lymphoid progenitor that is distinct from HSC (, ). Data on HSPC activity have been collected mainly through in vitro assays or using humanized, wild-type animal models (, , , , href="#bib41" rid="bib41" class=" bibr popnode">Notta et al., 2011, href="#bib50" rid="bib50" class=" bibr popnode">Wright et al., 2001). Barcoded vector libraries and retroviral integration sites (ISs) have been used to track HSPCs upon transplantation in small animal models and in non-human primates (href="#bib18" rid="bib18" class=" bibr popnode">Dykstra and Bystrykh, 2014, href="#bib21" rid="bib21" class=" bibr popnode">Gerrits et al., 2010, href="#bib29" rid="bib29" class=" bibr popnode">Kim et al., 2014, href="#bib37" rid="bib37" class=" bibr popnode">Naik et al., 2013, href="#bib43" rid="bib43" class=" bibr popnode">Perié et al., 2014, href="#bib51" rid="bib51" class=" bibr popnode">Wu et al., 2014). Additionally, recent mouse studies marking HSPCs in vivo suggest that unperturbed hematopoiesis may be driven more substantially by MPPs rather than by HSCs (href="#bib46" rid="bib46" class=" bibr popnode">Sun et al., 2014). Ideally, hematopoietic clonal dynamics should be studied by tracking the fate of individual clones in humans, revealing the rate and extent of hematopoietic recovery after transplant, and evaluating the possibility of long-term exhaustion due to in vitro cell manipulation. Such a study would have highly relevant implications for the broad clinical use of HSPCs and the long-term prognosis of treated patients.Ex vivo gene therapy (GT), based on the permanent gene correction of human HSPCs through the transfer of a therapeutic gene using retroviral (RV) or lentiviral (LV) vectors, has recently provided preliminary evidence of safety and efficacy for the treatment of various blood-borne genetic disorders (href="#bib2" rid="bib2" class=" bibr popnode">Aiuti et al., 2009, href="#bib3" rid="bib3" class=" bibr popnode">Aiuti et al., 2013, href="#bib10" rid="bib10" class=" bibr popnode">Biffi et al., 2013, href="#bib11" rid="bib11" class=" bibr popnode">Candotti et al., 2012, href="#bib20" rid="bib20" class=" bibr popnode">Gaspar et al., 2011, href="#bib25" rid="bib25" class=" bibr popnode">Hacein-Bey Abina et al., 2015, href="#bib24" rid="bib24" class=" bibr popnode">Hacein-Bey-Abina et al., 2010, href="#bib38" rid="bib38" class=" bibr popnode">Naldini, 2011, href="#bib39" rid="bib39" class=" bibr popnode">Naldini, 2015, href="#bib49" rid="bib49" class=" bibr popnode">Williams, 2013). Following GT, each vector-marked cell is univocally barcoded by a vector IS, providing an ideal setting for the study of human hematopoiesis (href="#bib39" rid="bib39" class=" bibr popnode">Naldini, 2015). We and others have already shown that IS-based tracking can be exploited to study the clonal composition of engineered cells and to assess the safety of gene transfer as well as the in vivo engraftment of marked HSPCs (href="#bib1" rid="bib1" class=" bibr popnode">Aiuti et al., 2007, href="#bib3" rid="bib3" class=" bibr popnode">Aiuti et al., 2013, href="#bib9" rid="bib9" class=" bibr popnode">Biasco et al., 2015, href="#bib25" rid="bib25" class=" bibr popnode">Hacein-Bey Abina et al., 2015, href="#bib47" rid="bib47" class=" bibr popnode">Tey and Brenner, 2007, href="#bib48" rid="bib48" class=" bibr popnode">Wang et al., 2010).In the present study, we used IS-based clonal tracking on individually purified lineages to examine early and late human hematopoiesis up to 4 years after transplant in the context of LV GT for Wiskott-Aldrich syndrome (WAS), an inherited disorder characterized by thrombocytopenia, bleeding episodes, eczema, and immunodeficiency (href="#bib3" rid="bib3" class=" bibr popnode">Aiuti et al., 2013). We measured, at qualitative and quantitative levels, the contribution of progenitors to an extensively engineered hematopoietic system and assessed over time the in vivo clonal relationships among blood cells, providing crucial information on human hematopoietic dynamics.