Lineage commitment of hematopoietic stem cells and progenitors: insights from recent single cell and lineage tracing technologies
2020; Elsevier BV; Volume: 88; Linguagem: Inglês
10.1016/j.exphem.2020.07.002
ISSN1873-2399
AutoresStephen J. Loughran, Simon Haas, Adam C. Wilkinson, Allon M. Klein, Marjorie Brand,
Tópico(s)Cancer Genomics and Diagnostics
Resumo•Cellular lineage commitment within hematopoiesis is reviewed.•Insights from novel lineage tracing and single-cell transcriptomics studies are discussed.•How single cell proteomics has resolved cell state transitions in hematopoiesis is described. Blood production is essential to maintain human health, and even small perturbations in hematopoiesis can cause disease. Hematopoiesis has therefore been the focus of much research for many years. Experiments determining the lineage potentials of hematopoietic stem and progenitor cells (HSPCs) in vitro and after transplantation revealed a hierarchy of progenitor cell states, where differentiating cells undergo lineage commitment—a series of irreversible changes that progressively restrict their potential. New technologies have recently been developed that allow for a more detailed analysis of the molecular states and fates of differentiating HSPCs. Proteomic and lineage-tracing approaches, alongside single-cell transcriptomic analyses, have recently helped to reveal the biological complexity underlying lineage commitment during hematopoiesis. Recent insights from these new technologies were presented by Dr. Marjorie Brand and Dr. Allon Klein in the Summer 2019 ISEH Webinar, and are discussed in this Perspective. Blood production is essential to maintain human health, and even small perturbations in hematopoiesis can cause disease. Hematopoiesis has therefore been the focus of much research for many years. Experiments determining the lineage potentials of hematopoietic stem and progenitor cells (HSPCs) in vitro and after transplantation revealed a hierarchy of progenitor cell states, where differentiating cells undergo lineage commitment—a series of irreversible changes that progressively restrict their potential. New technologies have recently been developed that allow for a more detailed analysis of the molecular states and fates of differentiating HSPCs. Proteomic and lineage-tracing approaches, alongside single-cell transcriptomic analyses, have recently helped to reveal the biological complexity underlying lineage commitment during hematopoiesis. Recent insights from these new technologies were presented by Dr. Marjorie Brand and Dr. Allon Klein in the Summer 2019 ISEH Webinar, and are discussed in this Perspective. The hematopoietic system plays a major role in human health and disease, supplying oxygen and nutrients to tissues, supporting healing, and fighting infections. These essential functions are carried out by the various types of mature hematopoietic cells, including red blood cells, platelets, myeloid immune cells (macrophages, neutrophils), and lymphocytes (T cells, B cells, natural killer [NK] cells). Most of the mature cells lack the ability to proliferate and have limited lifespans, so they must be constantly replenished by a process called hematopoiesis [1Eaves CJ Hematopoietic stem cells: concepts, definitions, and the new reality.Blood. 2015; 125: 2605-2613Crossref PubMed Scopus (304) Google Scholar,2Orkin SH Zon LI Hematopoiesis: an evolving paradigm for stem cell biology.Cell. 2008; 132: 631-644Abstract Full Text Full Text PDF PubMed Scopus (1613) Google Scholar]. Disturbance in the homeostasis of this process results in hematological diseases such as leukemias, lymphomas, anemias, thrombocytopenias, and immunodeficiencies. Hematopoiesis has therefore been the focus of considerable experimental research for many years. Hematopoiesis is sustained by rare hematopoietic stem cells (HSCs) that have two definitive characteristics [1Eaves CJ Hematopoietic stem cells: concepts, definitions, and the new reality.Blood. 2015; 125: 2605-2613Crossref PubMed Scopus (304) Google Scholar,2Orkin SH Zon LI Hematopoiesis: an evolving paradigm for stem cell biology.Cell. 2008; 132: 631-644Abstract Full Text Full Text PDF PubMed Scopus (1613) Google Scholar]. HSCs can self-renew, dividing to produce new HSC daughter cells to maintain lifelong hematopoiesis. HSCs are also multipotent; that is, they have the ability to differentiate into any of the adult hematopoietic cell lineages. To produce mature blood cells, the progeny of HSCs undergo lineage commitment, a process of differentiation in which the potential to produce all hematopoietic cell types is progressively lost until they become restricted to forming one type of blood cell. Molecular mechanisms, cellular relationships, and timing of lineage commitment are fundamental to the regulation of blood production in homeostasis and in disease. Recently developed technologies including lineage tracing, single-cell transcriptomics, and proteomics have provided important new insights into lineage commitment during hematopoiesis. "Changing Concepts in Hematopoietic Lineage Commitment" was the focus of the Summer 2019 International Society for Experimental Hematology (ISEH) New Investigator Committee Webinar, presented by Dr. Marjorie Brand and Dr. Allon Klein and moderated by Dr. Stephen Loughran. Dr. Brand discussed recent progress in using proteomic approaches to trace lineage commitment while Dr. Klein described how novel lineage tracing methods in combination with single-cell transcriptomics were uncovering new cell fate trajectories. In this Perspective, we provide a brief summary of the classic view of hematopoiesis and a discussion of the topics covered by this recent webinar, which can also be viewed online (https://www.youtube.com/watch?v=RqUsYsXqFfA). The differentiation potential of various hematopoietic stem and progenitor cells (HSPCs) was determined over many years of experimentation, using in vitro colony assays and transplantation of prospectively isolated cells into myelo-ablated mice [1Eaves CJ Hematopoietic stem cells: concepts, definitions, and the new reality.Blood. 2015; 125: 2605-2613Crossref PubMed Scopus (304) Google Scholar,3Seita J Weissman IL Hematopoietic stem cell: self-renewal versus differentiation.Wiley Interdiscip Rev Syst Biol Med. 2010; 2: 640-653Crossref PubMed Scopus (476) Google Scholar]. This allowed HSPCs with varying potentials to be fitted into a cellular hierarchy with HSCs at the apex and mature blood cell types at the base. Hematopoiesis is therefore often depicted as a process of branching transitions between phenotypically identifiable cell states, with lineage commitment occurring during these transitions [4Weissman IL Anderson DJ Gage F Stem and progenitor cells: origins, phenotypes, lineage commitments, and transdifferentiations.Annu Rev Cell Dev Biol. 2001; 17: 387-403Crossref PubMed Scopus (751) Google Scholar] (Figure 1A). Studies in the early and late 2000s revealed further complexity, reporting considerable functional and molecular variability between cells with similar cell surface marker phenotypes, and alternative lineage commitment pathways. 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It also appears that this fraction expands during aging in mice. Megakaryocyte-primed HSPCs are efficiently driven into maturation by inflammatory signals, resulting in enhanced platelet generation, and it has been suggested that these might serve as an emergency pool for rapid platelet generation in scenarios of high platelet demand, such as upon blood loss or during infection [23Haas S Hansson J Klimmeck D et al.Inflammation-induced emergency megakaryopoiesis driven by hematopoietic stem cell-like megakaryocyte progenitors.Cell Stem Cell. 2015; 17: 422-434Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar,27Boettcher S Manz MG. 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Notably, this is different from the previously observed cell-autonomous lineage bias of HSCs and progenitors, by which cells are predisposed toward one lineage, but not fully committed to it [10Dykstra B Kent D Bowie M et al.Long-term propagation of distinct hematopoietic differentiation programs in vivo.Cell Stem Cell. 2007; 1: 218-229Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar,31Sanjuan-Pla A Macaulay IC Jensen CT et al.Platelet-biased stem cells reside at the apex of the haematopoietic stem-cell hierarchy.Nature. 2013; 502: 232-236Crossref PubMed Scopus (367) Google Scholar]. Recent fate mapping studies of native hematopoiesis have also identified megakaryocyte-restricted output from HSCs [26Rodriguez-Fraticelli AE Wolock SL Weinreb CS et al.Clonal analysis of lineage fate in native haematopoiesis.Nature. 2018; 553: 212-216Crossref PubMed Scopus (243) Google Scholar]. 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In the last 4 years, large-scale single-cell transcriptomics of the mouse, human, and zebrafish HSPC compartments have provided detailed insights into the transcriptomic landscape of hematopoiesis [35Macaulay IC Svensson V Labalette C et al.Single-Cell RNA-sequencing reveals a continuous spectrum of differentiation in hematopoietic cells.Cell Rep. 2016; 14: 966-977Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 36Nestorowa S Hamey FK Pijuan Sala B et al.A single-cell resolution map of mouse hematopoietic stem and progenitor cell differentiation.Blood. 2016; 128: e20-e31Crossref PubMed Scopus (299) Google Scholar, 37Pellin D Loperfido M Baricordi C et al.A comprehensive single cell transcriptional landscape of human hematopoietic progenitors.Nat Commun. 2019; 10: 2395Crossref PubMed Scopus (110) Google Scholar, 38Tusi BK Wolock SL Weinreb C et al.Population snapshots predict early haematopoietic and erythroid hierarchies.Nature. 2018; 555: 54-60Crossref PubMed Scopus (170) Google Scholar, 39Velten L Haas SF Raffel S et al.Human haematopoietic stem cell lineage commitment is a continuous process.Nat Cell Biol. 2017; 19: 271-281Crossref PubMed Scopus (405) Google Scholar, 40Zheng S Papalexi E Butler A Stephenson W Satija R Molecular transitions in early progenitors during human cord blood hematopoiesis.Mol Syst Biol. 2018; 14: 1-20Crossref Scopus (71) Google Scholar]. These studies have suggested that at the mRNA level, hematopoiesis occurs as a continuum rather than by the acquisition of discrete transcriptional states (Figure 1B). With these methods, discrete transcriptional patterns were observed only at the level of mature cell types [35Macaulay IC Svensson V Labalette C et al.Single-Cell RNA-sequencing reveals a continuous spectrum of differentiation in hematopoietic cells.Cell Rep. 2016; 14: 966-977Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar,36Nestorowa S Hamey FK Pijuan Sala B et al.A single-cell resolution map of mouse hematopoietic stem and progenitor cell differentiation.Blood. 2016; 128: e20-e31Crossref PubMed Scopus (299) Google Scholar,38Tusi BK Wolock SL Weinreb C et al.Population snapshots predict early haematopoietic and erythroid hierarchies.Nature. 2018; 555: 54-60Crossref PubMed Scopus (170) Google Scholar,39Velten L Haas SF Raffel S et al.Human haematopoietic stem cell lineage commitment is a continuous process.Nat Cell Biol. 2017; 19: 271-281Crossref PubMed Scopus (405) Google Scholar]. 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These single cell transcriptomic data sets, however, represent only snapshots of distinct stages of HSC commitment rather than a time-resolved picture of hematopoiesis. To overcome this limitation, new tools combining lineage-tracing and single-cell transcriptomics have recently been developed to address the question of how accurately single-cell RNA sequencing (scRNA-Seq)-inferred hierarchies reflect actual fate choices and to determine transcriptional states upstream of lineage commitment branchpoints [44Weinreb C Rodriguez-Fraticelli A Camargo FD Klein AM Lineage tracing on transcriptional landscapes links state to fate during differentiation.Science. 2020; 367 (eaaw3381)Crossref PubMed Scopus (179) Google Scholar]. A new method, developed by Klein and colleagues, to overlay lineage relationships from barcoding analysis with single-cell transcriptomics data is providing a powerful approach to interrogate how single HSCs (and their progeny) move through the continuous lineage differentiation models provided by single-cell transcriptomics [26Rodriguez-Fraticelli AE Wolock SL Weinreb CS et al.Clonal analysis of lineage fate in native haematopoiesis.Nature. 2018; 553: 212-216Crossref PubMed Scopus (243) Google Scholar,44Weinreb C Rodriguez-Fraticelli A Camargo FD Klein AM Lineage tracing on transcriptional landscapes links state to fate during differentiation.Science. 2020; 367 (eaaw3381)Crossref PubMed Scopus (179) Google Scholar]. First, these data reveal that hematopoietic differentiation is not a strict treelike branching process; instead, some cell types appear to reflect more than one possible sequence of molecular events, leading to "loops" as different branches of the tree converge (Figure 1C). This was most apparent for monocytes. Second, sister cell experiments reveal that cells with very similar gene expression profiles can nonetheless be pre-committed to different fates, suggesting that transcriptional circuits alone do not encode the potential of cells toward different fates. Combining CRISPR/Cas9 perturbation with large-scale single readouts [45Dixit A Parnas O Li B et al.Perturb-Seq: dissecting molecular circuits with scalable single-cell RNA profiling of pooled genetic screens.Cell. 2016; 167 (1853–1866.e17)Abstract Full Text Full Text PDF Scopus (571) Google Scholar, 46Gundry MC Dever DP Yudovich D et al.Technical considerations for the use of CRISPR/Cas9 in hematology research.Exp Hematol. 2017; 54: 4-11Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar, 47Jaitin DA Weiner A Yofe I et al.Dissecting immune circuits by linking CRISPR-pooled screens with single-cell RNA-Seq.Cell. 2016; 167 (1883–1896.e15)Abstract Full Text Full Text PDF Scopus (358) Google Scholar] ex vivo and in vivo is also likely to provide novel insights into the molecular mechanisms driving HSC lineage commitment. The studies described above focused on linking cellular differentiation with patterns of mRNA expression. 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Recent studies measuring dynamic changes in transcription factor abundance at the protein level within single cells have challenged these binary fate models. For example, Hoppe et al. [64Hoppe PS Schwarzfischer M Loeffler D et al.Early myeloid lineage choice is not initiated by random PU.1 to GATA1 protein ratios.Nature. 2016; 535: 299-302Crossref PubMed Scopus (131) Google Scholar] quantified fluorescently tagged GATA1 and PU.1 in a large number of single differentiating HSCs and their progeny for several days. PU.1 was detected in all HSCs and uncommitted progenitors. GATA1 expression was not detected at any time during lineage commitment to the granulocyte/macrophage lineage. In granulocyte/macrophage differentiation events, PU.1 protein levels increased steadily during about half of the events, and in the other half, PU.1 levels dipped transiently before undergoing a similar steady increase. Cells in which GATA1 was detected, even at low levels, invariably continued to express GATA1 and differentiated into GATA1+PU.1– megakaryocytic and/or erythroid cells. During the majority of these megakaryocyte/erythroid differentiation events, downregulation of PU.1 occurred before detection of GATA1. These findings are incompatible with an abrupt GATA1-versus-PU.1 binary switching event driving lineage commitment: uncommitted progenitors contained only PU.1, GATA1 detection was always associated with megakaryocyte/erythroid commitment, and changes in GATA1 and PU.1 protein levels during differentiation occurred gradually. Another recent study by Brand and colleagues was the first to quantify the levels of endogenous lineage-specific transcription factors in single cells during hematopoietic differentiation. Mass cytometry time of flight (CyTOF) was used to simultaneously measure 11 cell surface proteins and 16 transcription factors in single human hematopoietic stem and progenitor cells and their progeny at 13 stages of in vitro erythroid development [65Palii CG Cheng Q Gillespie MA et al.Single-cell proteomics reveal that quantitative changes in co-expressed lineage-specific transcription factors determine cell fate.Cell Stem Cell. 2019; 24 (812–820.e5)Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar]. By use of this technique, KLF1 and FLI1 co-expression was detected in the majority of single megakaryocyte-erythroid progenitors (MEPs), and GATA1 and PU.1 co-expression, in the majority of common myeloid progenitors (CMPs). These data conflict with the lack of co-expression reported by Hoppe et al. [64Hoppe PS Schwarzfischer M Loeffler D et al.Early myeloid lineage choice is not initiated by random PU.1 to GATA1 protein ratios.Nature. 2016; 535: 299-302Crossref PubMed Scopus (131) Google Scholar] and highlight potential limitations in the ability of live imaging of fluorescent labels to detect proteins present at low levels. As MEPs underwent erythroid differentiation, KLF1 levels gradually increased, and FLI1 gradually decreased. Co-expression of KLF1 and FLI1 persisted for 14 days, until the pro-erythroblast stage of differentiation. This demonstrates that lineage commitment is not an abrupt transition from a metastable uncommitted state to one of two distinct lineage-committed states initiated by the rapid switching of a pair of cross-antagonistic, autoregulatory transcription factors. Instead, these findings are consistent with lineage commitment occurring during a continuous process of differentiation in which cells gradually transition along an ordered series of states. Changes in lineage-specific transcription factor levels during lineage commitment were gradual and continuous. Under the same conditions, artificially increasing FLI1 protein levels in progenitors was sufficient to divert cells from their preferred erythroid trajectory to take on a megakaryocytic fate [65Palii CG Cheng Q Gillespie MA et al.Single-cell proteomics reveal that quantitative changes in co-expressed lineage-specific transcription factors determine cell fate.Cell Stem Cell. 2019; 24 (812–820.e5)Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar]. This highlights the ability of quantitative changes in transcription factor protein levels to determine cell fate decisions and strongly supports their being the main process that initiates lineage commitment, a finding also compatible with scRNA-Seq data. However, because it is difficult to precisely determine when a cell undergoes lineage commitment, it is difficult to distinguish which changes in transcription factor levels initiate lineage commitment and which are downstream events that reinforce a prior commitment decision. The granulocyte–macrophage versus erythroid–megakaryocyte lineage commitment decision time point, computationally inferred from the genealogy of single cell-tracked differentiating HSCs, occurred much earlier than changes in the level of fluorescently tagged PU.1 [66Strasser MK Hoppe PS Loeffler D et al.Lineage marker synchrony in hematopoietic genealogies refutes the PU.1/GATA1 toggle switch paradigm.Nat Commun. 2018; 9: 2697Crossref PubMed Scopus (18) Google Scholar]. This suggests that PU.1 protein levels do not initiate this lineage commitment decision and indicates that further work is required to determine which transcription factors are responsible. The development and application of new technologies to study hematopoiesis are creating a more complex but more complete understanding of hematopoietic lineage commitment. This hematopoietic lineage commitment landscape has now been mapped by different technologies in several distinct, but related, ways. One type of map depicts the lineage potential of HSPCs: What lineages can isolated cells differentiate into if placed in appropriate conditions? Another is a map of lineage fates: What lineages do HSPCs produce in situ? The third maps the molecular state of individual HSPCs in particular tissues at snapshots in time. Recent single-cell analyses of hematopoiesis at both the mRNA and protein levels have revealed a continuum of states in hematopoiesis, suggesting that the molecular changes that underlie lineage commitment occur on a time scale comparable to the lifetime of mRNA molecules—hours or days. However, little is known about how rapidly individual cells move between these molecular states during steady-state hematopoiesis, and it is not yet possible to accurately predict the lineage potential of a single cell from its transcriptome. To better understand the regulation of lineage commitment, links between the three types of map must be discovered, associating the molecular profile of individual cells to their potential and to their most likely fate in the bone marrow. Further characterization of transcription factor protein levels and genomic binding in single cells across different stages of differentiation and new methods combining cell fate tracking and single-cell transcriptomics will provide new information to help address this and allow the development of more accurate models of the molecular regulation of hematopoietic lineage commitment. These findings have important implications for how we interpret the perturbations in hematopoiesis that underlie hematological diseases, as well as our efforts to develop new therapies for these diseases.
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