New paradigms on hematopoietic stem cell differentiation
Ever since hematopoietic stem cells (HSCs) were first identified half a century ago, their differentiation roadmap has been extensively studied. The classical model of hematopoiesis has long held as a dogma that HSCs reside at the top of a hierarchy in which HSCs possess self-renewal capacity and can progressively give rise to all blood lineage cells. However, over the past several years, with advances in single cell technologies, this developmental scheme has been challenged. In this review, we discuss the evidence supporting heterogeneity within HSC and progenitor populations as well as the hierarchical models revised by novel approaches mainly in mouse system. These evolving views provide further understanding of hematopoiesis and highlight the complexity of hematopoietic differentiation.
Keywordshematopoietic stem cell hierarchy heterogeneity differentiation
common lymphoid progenitors
common myeloid progenitors
common myeloid repopulating progenitors
fluorescence-activated cell sorting
hematopoietic stem cells
hematopoietic stem and progenitor cells
intermediate-term hematopoietic stem cells
lymphoid-primed multipotent progenitors
long-term hematopoietic stem cells
megakaryocyte-erythrocyte repopulating progenitors
megakaryocyte repopulating progenitors
single cell assay for transposase-accessible chromatin using sequencing
single-cell RNA sequencing
signaling lymphocyte activation molecule
stem-like megakaryocyte committed progenitors
short-term hematopoietic stem cells
von Willebrand factor
THE CLASSICAL AND BALANCED HEMATOPOIETIC HIERARCHICAL MODEL
The cellular potential of hematopoietic stem cells (HSCs) has been traditionally defined by transplanting donor cells (or a single cell) into recipients that are preconditioned by lethal irradiation and therefore devoid of a functional endogenous hematopoietic system. This assay has long been the gold-standard for functional HSCs.
The first in vivo evidence for the existence of HSCs, in 1961, was based on the rescue of lethally irradiated recipient mice by bone marrow transplantation, followed by observing hematopoietic colonies in the spleens of recipients (Till and Mc, 1961). Thereafter, scientists were interested in developing methods to purify HSCs from bone marrow to better understand their function and molecular regulatory networks. Separation of HSCs became possible with the utilization of antibodies and fluorescence-activated cell sorting (FACS). Weissman and colleagues first described HSC-enriched cells using the combination of several surface markers in 1988 (Spangrude et al., 1988). Since then, different groups have put great effort into identifying more surface markers to further purify HSCs. To date, CD34, Sca-1, c-Kit, the signaling lymphocyte activation molecule (SLAM) markers, etc. are still commonly used to isolate HSCs in different labs (Ikuta and Weissman, 1992; Okada et al., 1992; Osawa et al., 1996; Kiel et al., 2005; Oguro et al., 2013). Since similar approaches can be used to identify multi- and unipotent progenitors, different progenitor populations were also isolated based on surface markers (Kondo et al., 1997; Akashi et al., 2000; Adolfsson et al., 2005; Wilson et al., 2008; Pietras et al., 2015).
ADVANCES IN THE HEMATOPOIETIC HIERARCHY
Although the classical model has been very useful for understanding the differentiation process of HSCs, it is worth noting that this model has some shortcomings in that it oversimplifies the complexity of hematopoietic stem and progenitor cells (HSPCs), and it is only based on the surface markers and transplantation using bulk cells. Bulk cell analysis assumes that each cell, which has the same phenotype, possesses an identical function. With advances in single cell technology and genetic mouse models, this classical model has been challenged over the past several years, especially in the elucidation of megakaryopoiesis. Moreover, new types of HSPCs have been identified and extensively studied due to their lineage biases.
Heterogeneity in HSCs lineage output and debates on megakaryocyte differentiation
The origin of megakaryocytes has been under debate for several years. The Jacobsen group identified lymphoid-primed MPPs (LMPPs) that give rise to granulocyte/macrophage and lymphoid lineages but not a megakaryocyte/ erythrocyte lineage (Adolfsson et al., 2005) (Fig. 2C). However, lineage tracing studies challenge this view and suggest that LMPPs also differentiate into a megakaryocyte/erythrocyte lineage (Forsberg et al., 2006; Boyer et al., 2011). The different outcomes may be attributed to the cell dose used for transplantation and the methods, including mouse models, used in different labs. More recently, mRNA expression of the megakaryocyte marker von Willebrand factor (vWF) and the surface receptor c-Kit were suggested to be indicative of a platelet-biased, but multipotent HSC sub-population (Sanjuan-Pla et al., 2013; Shin et al., 2014). Evidence for a platelet-biased population of HSCs was provided by the Jacobsen group (Sanjuan-Pla et al., 2013). They found that 25% of LT-HSCs express vWF and vWF+ HSCs are primed for platelet-specific gene expression, with enhanced propensity for long-term reconstitution of platelets. The vWF+ platelet-primed HSCs also have a long-term myeloid lineage bias, can self-renew, and can give rise to vWF− lymphoid-biased HSCs (Fig. 2C). Therefore, the platelet-primed HSCs sit at the top of the hematopoietic hierarchy. Moreover, based on single-cell transplantation experiments, the existence of megakaryocyte-lineage restricted cells in the phenotypic HSC compartment was proposed (Yamamoto et al., 2013). Pair-daughter cell transplantation assay indicates that megakaryocyte precursors are directly derived from HSCs (Yamamoto et al., 2013) (Fig. 2D). Later, a study reported that the HSC compartment contains stem-like megakaryocyte committed progenitors (SL-MkPs), a cell population that shares many features with HSCs (Haas et al., 2015). This population becomes activated upon inflammatory stress to efficiently replenish platelets, thus a potential shortcut from HSCs to megakaryocytes has been suggested under inflammatory conditions (Haas et al., 2015). Furthermore, by tracking progenitors and mature lineage cells produced from single transplanted HSCs, a recent report from Jacobsen’s lab showed that a distinct class of HSCs adopts a fate towards long-term and effective replenishment of megakaryocytes/platelets without replenishment of any other blood cell lineages, whereas no HSCs contribute exclusively to any other single blood cell lineage (Carrelha et al., 2018).
Collectively, HSCs and megakaryocytes share several features, for example, expression of thrombopoietin receptor (MPL), CD150, CXCR4 and vWF, etc. (Wang et al., 1998; Sugiyama et al., 2006; Pronk et al., 2007; Yoshihara et al., 2007; Huang and Cantor, 2009). More importantly, megakaryocytes also serve as an HSC niche component and tightly regulate the maintenance of HSC function (Bruns et al., 2014; Zhao et al., 2014). Platelet- and myeloid-biased vWF+ HSCs, but not lymphoid-biased vWF− HSCs, associate with megakaryocytes and are regulated by megakaryocytes (Pinho et al., 2018). All the evidence suggests that HSCs and the megakaryocyte (or its progenitors) are closer to one another in the hematopoietic developmental hierarchy than previously appreciated. In consideration of the heterogeneity observed in the HSC population, our view is that lineage (cell fate) predetermination occurs in HSCs, prior to their differentiation towards progenitors. Moreover, the megakaryocyte can arise independent of other lineages, and the megakaryocyte differentiation route is first separated from other blood cell lineages in the hierarchy.
Heterogeneity in MPPs
The Trumpp (Wilson et al., 2008) and Passegue (Pietras et al., 2015) groups further divided the MPP population into MPP1, MPP2, MPP3 and MPP4 according to their immuno-phenotype, cell cycle status, lineage bias, resistance to drug treatment and bone marrow abundance. MPP1 is more similar to the previously defined IT-HSC or ST-HSC, which have multiple-lineage reconstitution ability up to 4 months in the first transplantation, whereas MPP2/3/4 are devoid of self-renewal potential and only exhibit short-term myeloid reconstitution ability (<1 month). More importantly, MPP2 and MPP3 produce low levels of T and B cells and MPP4 generates low levels of myeloid cells in vivo. In addition, compared with MPP3 and MPP4, MPP2 produces higher levels of platelets. Taken together, MPP2 is a megakaryocyte-biased MPP subset and MPP3 is a myeloid-biased MPP subset. Both MPP2 and MPP3 are functionally distinct from the lymphoid-primed MPP4. HSCs independently generate all three types of lineage-biased MPPs (MPP2-4), but among them, no MPPs are able to generate other MPPs in vivo (Fig. 2E). After transplantation, HSCs first produce myeloid-biased MPPs (MPP1/2) to quickly establish myeloid output, followed by the lymphoid-primed MPP4 subpopulation to rebuild the lymphoid compartment. Therefore, MPPs are a heterogeneous population with different lineage-biased potential both at the cellular and molecular levels.
Heterogeneity and hierarchy within myeloid progenitors
In the classical model, CMPs and MEPs are separated according to CD34 expression. In the Lin-cKit+Sca1- (LKS-) population, CMPs are CD34+CD16/32-, whereas MEPs are CD34-CD16/32-. CMPs are thought to possess oligo-potency, including granulocyte, macrophage, megakaryocyte and erythrocyte differentiation potential. However, CMPs have a low clonal frequency of mixed myeloid colonies, and MEPs also possess a low level of megakaryocyte potential (Nakorn et al., 2003). Therefore, it prompts us to know whether each CMP or MEP has a different lineage potential at the single cell level, i.e., whether every CMP is indeed oligo-potent, and whether MEP is bipotent.
To understand the heterogeneity and lineage commitment in the LKS- myeloid progenitor population, especially in CMPs, Pronk et al. (Pronk et al., 2007) used CD150, CD105 (Endoglin), CD41 and CD16/32 to re-segregate the LKS- myeloid progenitors. In the LKS- population, CD41+CD150+ cells are defined as megakaryocyte progenitors (MkPs), which are exclusively associated with megakaryocyte generation. CD41-CD150- CD16/32+ cells are GMPs. In the CD41-CD150-CD16/32- population (classical CMPs and MEPs mixture), there are four newly defined sub-populations, including pre MegEs, pre GMs, Pre CFU-Es and CFU-Es (Pronk et al., 2007). Single Pre MegE cells can effectively produce megakaryocytic, erythroid as well as mixed megakaryocyte/erythroid colonies. In contrast, Pre CFU-E cells give rise almost exclusively to erythroid colonies of various sizes. Pre GMs lie developmentally upstream of GMPs, and have a remarkably similar clonal lineage output to the GMPs. Therefore, the Pronk et al. study explores the processes of myeloid cell differentiation, reveals a number of novel intermediate progenitors, and orchestrates a new hierarchy model, including unipotent proliferative neutrophil precursors (Kim et al., 2017; Evrard et al., 2018; Zhu et al., 2018). Classical CMPs consist of pre GMs, and the majority of pre MegEs and MEPs are separated into CFU-E, Pre CFU-Es, and part of pre MegEs. Moreover, MkPs are located mainly in CMPs (Pronk et al., 2007) (Fig. 2F).
In line with the work discussed above, a landmark paper from Amit’s lab reporting the transcriptomes for more than 2,600 mouse single LKS- myelo-erythroid progenitor cells (Paul et al., 2015) and a subsequent work from Gottgens’ lab reporting the transcriptomes of 1,600 HSPCs (Nestorowa et al., 2016) both revealed heterogeneity in LKS- progenitors. The single cells from classical MEPs do not show any expression of megakaryocyte markers or prominent megakaryocyte TFs. However, both megakaryocyte markers (Pf4 and CD41) and TFs (Pbx1, Fli1, Mef2c) are expressed in the cells from classical CMPs (Paul et al., 2015).
Taken together, all the studies explained why megakaryocytes mainly differentiate from CMPs, but not MEPs, and MEPs mostly give rise to erythrocytes. Therefore, this suggests that classical MEPs may not be the true precursor for megakaryocytes.
Hematopoietic differentiation is a continuous process
By using scRNA-seq combined with computational analysis, a recent study on comprehensively sampled human bone marrow HSPCs suggested a model in which acquisition of lineage-specific fates is a continuous process, and unilineage restricted cells emerge directly from a continuum of low-primed undifferentiated HSPCs, without any major transition through the multi- and bi-potent stages (Velten et al., 2017). This view is supported by a zebrafish study that suggested that the continuum of HSPC differentiation is characterized by a highly coordinated transcriptional program, displaying simultaneous suppression of cell proliferation-related genes and upregulation of lineage specific genes (Macaulay et al., 2016). In addition, another study using human cord blood lympho-myeloid progenitor cells, including LMPPs, GMPs and multi-lymphoid progenitors (MLPs), further suggested a model in which a continuum of progenitors execute lymphoid and myeloid differentiation, rather than only unilineage progenitors being present downstream of stem cells (Karamitros et al., 2018). Although most progenitors have uni-lineage potential, bi- and oligo-lineage progenitors are present among LMPPs, GMPs and MLPs. These aforementioned studies change our view that hematopoietic differentiation is a continuous process, rather than a discrete hierarchy, which suggests that there is no obvious boundary among stem cells and progenitors (Laurenti and Gottgens, 2018) (Fig. 3B).
The roadmap of human hematopoiesis
In human hematopoiesis, the Dick group sorted MPPs, CMPs and MEPs from fetal liver and adult bone marrow, and compared their lineage potential from different developmental stages (Notta et al., 2016). They showed that previously defined MPPs, CMPs and MEPs are heterogeneous. Importantly, MEPs, from both fetal liver and bone marrow, uniformly produce erythroid-only clones. Therefore, the classically defined MEPs are principally erythroid precursors when analyzed at single cell resolution and are not megakaryocyte/erythroid progenitors as previously thought, which is consistent with the observation in a mouse model (Pronk et al., 2007). Interestingly, fetal liver contains large numbers of distinct oligopotent progenitors. However, few oligopotent progenitors were present in the adult bone marrow. Instead only two progenitor classes predominate, multipotent and unipotent, with megakaryocyte/erythroid lineages emerging from multipotent cells. The Dick group’s study provides a revised model to understand normal hematopoiesis, which is indeed flexible in developmental time.
HEMATOPOIESIS UNDER PHYSIOLOGICAL CONDITION
Hematopoiesis is regulated by microenvironment or niche (Morrison and Scadden, 2014; Crane et al., 2017), therefore, an unperturbed niche and a pretreated niche have different effects on hematopoiesis (Mendelson and Frenette, 2014). LT-HSC is considered to sit at the apex of the hierarchy, and reconstitutes or maintains the whole hematopoiesis. However, it is important to remember that almost all the supporting evidence was obtained from in vitro colony assay and in vivo transplantation. Irradiation or drug treatment can disrupt the niche, ablate the hematopoietic cells in the recipient, and create the space for donor HSPC engraftment, expansion and differentiation. Moreover, HSPCs are retained in the niche under hypoxic regulation (Suda et al., 2011; Nombela-Arrieta et al., 2013; Spencer et al., 2014; Itkin et al., 2016), and it is reported that transient exposure of HSPCs to normal oxygen impairs their function (Mantel et al., 2015). Therefore, transplantation of HSPCs into pretreated recipients cannot truly reflect the behaviors of hematopoiesis under physiological conditions (steady state).
To understand the dynamics of blood formation in steady state, various lineage-tracing approaches have been used to assess the lineage contribution of individual HSPCs in unperturbed hematopoiesis. By using a doxycycline-induced Sleeping Beauty transposon tagging approach in HSPCs, Sun et al. (Sun et al., 2014) reported that MPPs, rather than HSCs, are the main drivers of steady-state hematopoiesis during adulthood. In addition, Rodewald’s lab devised a mouse model allowing inducible genetic labeling of the most primitive Tie2+ HSCs in bone marrow, and quantified label progression along hematopoietic development by limiting dilution analysis and mathematical modelling (Busch et al., 2015). They found that adult hematopoiesis is largely sustained by ST-HSC, and in contrast, LT-HSCs are rapidly used to establish the immune and blood system in fetal and early postnatal life. Another study from the same group used an alternative genetic fate-mapping system, called polylox barcoding (Pei et al., 2017), and demonstrated that when HSCs are labeled at the fetal liver stage, their descendants in the adult will mostly contribute to multiple lineages. However, when the analysis was repeated in adult stage, few barcodes were detected in HSCs as well as mature progenies. The above studies support the view that MPPs or ST-HSCs contribute predominantly to mature progenies, whereas HSCs do not have a notable role in steady-state hematopoiesis. However, Sawai et al. (2016) reported a paradoxical result. Pdzk1ip1 (Map17) is specifically expressed in the murine HSC population, therefore, they developed Pdzk1ip1-GFP and Pdzk1ip1-CreER; R26-TdTomato mice and showed that LT-HSCs provide a major contribution to all lineage-committed progenitors and mature blood cells. These controversial observations might be owing to the differences of tracing methods and labeling efficiency in HSCs. Therefore, new approaches should be developed and further investigation will be necessary to reconcile this paradox.
Interestingly, in Rodewald’s polylox tracking study, they revealed a basic split between common myeloid–erythroid development and common lymphocyte development, supporting the bifurcating tree model of hematopoiesis that has not been proved in steady state (Pei et al., 2017). However, megakaryocytic fate was not analyzed in this study. Another study from the Camargo group addressed this issue more comprehensively (Rodriguez-Fraticelli et al., 2018). They performed a long-term (30-week) pulse-chase experiment in adult mice with the sleeping beauty barcode system, and found that during unperturbed hematopoiesis, the megakaryocyte lineage arises largely independently of other hematopoietic fates, and the LT-HSCs predominantly contribute to megakaryocyte output.
The kinetics of adult HSC differentiation under different stress conditions have been studied recently in great detail (Lu et al., 2019). However, whether expansion of HSCs in recipients after transplantation occurs is still poorly understood. Furthermore, future studies can focus more on the changes of heterogeneity and hierarchy of HSPCs in disease. For example, one study reported that a c-Kithi progenitor subset positive for IL-7Rα emerged after infection of mice with Plasmodium chabaudi (Belyaev et al., 2010). These cells have both lymphoid and myeloid potential. Therefore, studying HSPCs under different diseases is interesting and also important.
We appreciate insightful suggestions from Dr. Hideo Ema. This work was supported by grants from the National Key Research and Development Program of China (2016YFA0100600, 2017YFA0103400); the National Natural Science Foundation of China (81421002, 81730006, 81430004, 81670106, 81870086, 8181101081); CAMS Initiative for Innovative Medicine (2017-I2M-3-009, 2016-I2M-1-017); and the CAMS Fundamental Research Funds for Central Research Institutes (2016GH3100001, 2018PT31005).
COMPLIANCE WITH ETHICS GUIDELINES
Hui Cheng, Zhaofeng Zheng and Tao Cheng declare that they have no conflict of interest
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