Christina T. Jensen

Identification of Flt3 + Lympho-Myeloid Stem Cells Lacking Erythro-Megakaryocytic Potential: A Revised Road Map for Adult Blood Lineage Commitment

All blood cell lineages derive from a common hematopoietic stem cell (HSC). The current model implicates that the first lineage commitment step of adult pluripotent HSCs results in a strict separation into common lymphoid and common myeloid precursors. We present evidence for a population of cells which, although sustaining a high proliferative and combined lympho-myeloid differentiation potential, have lost the ability to adopt erythroid and megakaryocyte lineage fates. Cells in the Lin-Sca-1+c-kit+ HSC compartment coexpressing high levels of the tyrosine kinase receptor Flt3 sustain granulocyte, monocyte, and B and T cell potentials but in contrast to Lin-Sca-1+c-kit+Flt3- HSCs fail to produce significant erythroid and megakaryocytic progeny. This distinct lineage restriction site is accompanied by downregulation of genes for regulators of erythroid and megakaryocyte development. In agreement with representing a lymphoid primed progenitor, Lin-Sca-1+c-kit+CD34+Flt3+ cells display upregulated IL-7 receptor gene expression. Based on these observations, we propose a revised road map for adult blood lineage development.

Critical Role of Thrombopoietin in Maintaining Adult Quiescent Hematopoietic Stem Cells

The role of cytokines in regulation of hematopoietic stem cells (HSCs) remains poorly understood. Herein we demonstrate that thrombopoietin (THPO) and its receptor, MPL, are critically involved in postnatal steady-state HSC maintenance, reflected in a 150-fold reduction of HSCs in adult Thpo(-/-) mice. Further, whereas THPO and MPL proved not required for fetal HSC expansion, HSC expansion posttransplantation was highly MPL and THPO dependent. The distinct role of THPO in postnatal HSC maintenance is accompanied by accelerated HSC cell-cycle kinetics in Thpo(-/-) mice and reduced expression of the cyclin-dependent kinase inhibitors p57(Kip2) and p19(INK4D) as well as multiple Hox transcription factors. Although also predicted to be an HSC viability factor, BCL2 failed to rescue the HSC deficiency of Thpo(-/-) mice. Thus, THPO regulates posttransplantation HSC expansion as well as the maintenance of adult quiescent HSCs, of critical importance to avoid postnatal HSC exhaustion.

Platelet-biased stem cells reside at the apex of the haematopoietic stem-cell hierarchy

The blood system is maintained by a small pool of haematopoietic stem cells (HSCs), which are required and sufficient for replenishing all human blood cell lineages at millions of cells per second throughout life. Megakaryocytes in the bone marrow are responsible for the continuous production of platelets in the blood, crucial for preventing bleeding—a common and life-threatening side effect of many cancer therapies—and major efforts are focused at identifying the most suitable cellular and molecular targets to enhance platelet production after bone marrow transplantation or chemotherapy. Although it has become clear that distinct HSC subsets exist that are stably biased towards the generation of lymphoid or myeloid blood cells, we are yet to learn whether other types of lineage-biased HSC exist or understand their inter-relationships and how differently lineage-biased HSCs are generated and maintained. The functional relevance of notable phenotypic and molecular similarities between megakaryocytes and bone marrow cells with an HSC cell-surface phenotype remains unclear. Here we identify and prospectively isolate a molecularly and functionally distinct mouse HSC subset primed for platelet-specific gene expression, with enhanced propensity for short- and long-term reconstitution of platelets. Maintenance of platelet-biased HSCs crucially depends on thrombopoietin, the primary extrinsic regulator of platelet development. Platelet-primed HSCs also frequently have a long-term myeloid lineage bias, can self-renew and give rise to lymphoid-biased HSCs. These findings show that HSC subtypes can be organized into a cellular hierarchy, with platelet-primed HSCs at the apex. They also demonstrate that molecular and functional priming for platelet development initiates already in a distinct HSC population. The identification of a platelet-primed HSC population should enable the rational design of therapies enhancing platelet output.

Kit Regulates Maintenance of Quiescent Hematopoietic Stem Cells

Hematopoietic stem cell (HSC) numbers are tightly regulated and maintained in postnatal hematopoiesis. Extensive studies have supported a role of the cytokine tyrosine kinase receptor Kit in sustaining cycling HSCs when competing with wild-type HSCs posttransplantation, but not in maintenance of quiescent HSCs in steady state adult bone marrow. In this study, we investigated HSC regulation in White Spotting 41 (KitW41/W41) mice, with a partial loss of function of Kit. Although the extensive fetal HSC expansion was Kit-independent, adult KitW41/W41 mice had an almost 2-fold reduction in long-term HSCs, reflecting a loss of roughly 10,000 Lin−Sca-1+Kithigh (LSK)CD34−Flt3− long-term HSCs by 12 wk of age, whereas LSKCD34+Flt3− short-term HSCs and LSKCD34+Flt3+ multipotent progenitors were less affected. Whereas homing and initial reconstitution of KitW41/W41 bone marrow cells in myeloablated recipients were close to normal, self-renewing KitW41/W41 HSCs were progressively depleted in not only competitive but also noncompetitive transplantation assays. Overexpression of the anti-apoptotic regulator BCL-2 partially rescued the posttransplantation KitW41/W41 HSC deficiency, suggesting that Kit might at least in the posttransplantation setting in part sustain HSC numbers by promoting HSC survival. Most notably, accelerated in vivo BrdU incorporation and cell cycle kinetics implicated a previously unrecognized role of Kit in maintaining quiescent HSCs in steady state adult hematopoiesis.

Lymphomyeloid Contribution of an Immune-Restricted Progenitor Emerging Prior to Definitive Hematopoietic Stem Cells.

In jawed vertebrates, development of an adaptive immune-system is essential for protection of the born organism against otherwise life-threatening pathogens. Myeloid cells of the innate immune system are formed early in development, whereas lymphopoiesis has been suggested to initiate much later, following emergence of definitive hematopoietic stem cells (HSCs). Herein, we demonstrate that the embryonic lymphoid commitment process initiates earlier than previously appreciated, prior to emergence of definitive HSCs, through establishment of a previously unrecognized entirely immune-restricted and lymphoid-primed progenitor. Notably, this immune-restricted progenitor appears to first emerge in the yolk sac and contributes physiologically to the establishment of lymphoid and some myeloid components of the immune-system, establishing the lymphomyeloid lineage restriction process as an early and physiologically important lineage-commitment step in mammalian hematopoiesis.

FLT3 ligand and not TSLP is the key regulator of IL-7–independent B-1 and B-2 B lymphopoiesis

Phenotypically and functionally distinct progenitors and developmental pathways have been proposed to exist for fetally derived B-1 and conventional B-2 cells. Although IL-7 appears to be the primary cytokine regulator of fetal and adult B lymphopoiesis in mice, considerable fetal B lymphopoiesis and postnatal B cells are sustained in the absence of IL-7; in humans, B-cell generation is suggested to be largely IL-7-independent, as severe combined immune-deficient patients with IL-7 deficiency appear to have normal B-cell numbers. However, the role of other cytokines in IL-7-independent B lymphopoiesis remains to be established. Although thymic stromal lymphopoietin (TSLP) has been proposed to be the main factor driving IL-7-independent B lymphopoiesis and to distinguish fetal from adult B-cell progenitor development in mice, recent studies failed to support a primary role of TSLP in IL-7-independent fetal B-cell development. However, the role of TSLP in IL-7-independent adult B lymphopoiesis and in particular in regulation of B-1 cells remains to be established. Here we demonstrate that, rather than TSLP, IL-7 and FLT3 ligand are combined responsible for all B-cell generation in mice, including recently identified B-1-specified cell progenitors. Thus, the same IL-7- and FLT3 ligand-mediated signal-ing regulates alternative pathways of fetal and adult B-1 and B-2 lymphopoiesis.

Downregulation of Mpl marks the transition to lymphoid-primed multipotent progenitors with gradual loss of granulocyte-monocyte potential.

Evidence for a novel route of adult hematopoietic stem-cell lineage commitment through Lin-Sca-1+Kit+Flt3hi (LSKFlt3hi) lymphoid-primed multipotent progenitors (LMPPs) with granulocyte/monocyte (GM) and lymphoid but little or no megakaryocyte/erythroid (MkE) potential was recently challenged, as LSKFlt3hi cells were reported to possess MkE potential. Herein, residual (1%-2%) MkE potential segregated almost entirely with LSKFlt3hi cells expressing the thrombopoietin receptor (Mpl), whereas LSKFlt3hiMpl- LMPPs lacked significant MkE potential in vitro and in vivo, but sustained combined GM and lymphoid potentials, and coexpressed GM and lymphoid but not MkE transcriptional lineage programs. Gradually increased transcriptional lymphoid priming in single LMPPs from Rag1GFP mice was shown to occur in the presence of maintained GM lineage priming, but gradually reduced GM lineage potential. These functional and molecular findings reinforce the existence of GM/lymphoid-restricted progenitors with dramatically down-regulated probability for committing toward MkE fates, and support that lineage restriction occurs through gradual rather than abrupt changes in specific lineage potentials.

Expression and role of FLT3 in regulation of the earliest stage of normal granulocyte-monocyte progenitor development

Mice deficient in c-fms-like tyrosine kinase 3 (FLT3) signaling have reductions in early multipotent and lymphoid progenitors, whereas no evident myeloid phenotype has been reported. However, activating mutations of Flt3 are among the most common genetic events in acute myeloid leukemia and mice harboring internal tandem duplications within Flt3 (Flt3-ITD) develop myeloproliferative disease, with characteristic expansion of granulocyte-monocyte (GM) progenitors (GMP), possibly compatible with FLT3-ITD promoting a myeloid fate of multipotent progenitors. Alternatively, FLT3 might be expressed at the earliest stages of GM development. Herein, we investigated the expression, function, and role of FLT3 in recently identified early GMPs. Flt3-cre fate-mapping established that most progenitors and mature progeny of the GM lineage are derived from Flt3-expressing progenitors. A higher expression of FLT3 was found in preGMP compared with GMP, and preGMPs were more responsive to stimulation with FLT3 ligand (FL). Whereas preGMPs and GMPs were reduced in Fl(-/-) mice, megakaryocyte-erythroid progenitors were unaffected and lacked FLT3 expression. Notably, mice deficient in both thrombopoietin (THPO) and FL had a more pronounced GMP phenotype than Thpo(-/-) mice, establishing a role of FL in THPO-dependent and -independent regulation of GMPs, of likely significance for myeloid malignancies with Flt3-ITD mutations.

Quiescent leukaemic cells account for minimal residual disease in childhood lymphoblastic leukaemia.

Quiescent leukaemic cells account for minimal residual disease in childhood lymphoblastic leukaemia

TSLP-mediated fetal B lymphopoiesis?

To the editor: In the August 2003 issue of Nature Immunology1, as well as in later studies2, Voβhenrich et al. presented experimental data on the cytokine requirements of developing B cells, which led them to conclude that “TSLP [thymic stromal lymphopoietin] is the factor responsible for most of the fetal and perinatal B cell production that takes place when the IL-7–γc [interleukin 7–common γ-chain] signaling pathway is disrupted.”1 Although the data reported were technically sound and compatible with such a conclusion, the authors did not provide direct evidence to support (or exclude) the idea of a critical function for TSLP in IL-7-independent fetal B lymphopoiesis. The conclusions of Voβhenrich et al. were based on the demonstration that B lymphopoiesis was much more affected (tenfold more) in mice deficient in IL-7 receptor α-chain, essential for IL-7 as well as TSLP signaling, than in mice deficient in the common γ-chain (γc), required for IL-7 but not TSLP-mediated signaling3,4. However, these data could at best be considered strong indirect support for the idea of TSLP as the main cytokine driving IL-7-independent fetal B lymphopoiesis, as there could be other reasons for a difference in the phenotypes of γcdeficient mice and those deficient in the IL-7 receptor α-chain. Furthermore, Voβhenrich et al. used bone marrow of mice 4–12 weeks of age, not fetal liver, for their comparative in vivo analysis of B lymphopoiesis in these mice1,2. Instead, the extrapolation to the idea that TSLP is key to the fetal stages of B lymphopoiesis was based on the finding that fetal but not adult pro–B cells were responsive to TSLP in vitro1,2. In contrast, a lack of an important function for TSLP in adult B lymphopoiesis has been indicated by studies of TSLP receptor–deficient (Tpte2–/–) mice5,6. As fetal lymphopoiesis had not been examined in singly deficient Tslp–/– or Tpte2–/– mice, we investigated B lymphopoiesis in the livers of Tpte2–/– mice at embryonic day 17.5 but found no deficiency in Tpte2–/– fetuses at any stage of B cell development (Fig. 1a and Supplementary Fig. 1 online). Furthermore, when comparing B lymphopoiesis in the fetal livers of Il7–/– and Il7–/–Tpte2–/– mice, we obtained no evidence for substantial involvement of TSLP in IL-7independent regulation of fetal pro–B cells or pre–B cells, whereas we noted a slight additional reduction in the number of immature B cells in Il7–/–Tpte2–/– fetuses relative to that in Il7–/– fetuses (Fig. 1b and Supplementary Fig. 1). Thus, although Voβhenrich et al. provided compelling evidence that fetal pro–B cells are highly responsive to TSLP1, our studies of Tpte2–/– and Il7–/–Tpte2–/– fetuses fail to support their claim that TSLP is the most important cytokine promoting IL-7independent fetal B lymphopoiesis. Instead, although Voβhenrich et al. also concluded that “Flk-2 is involved, but TSLP is the main factor driving IL-7-independent fetal and perinatal lymphopoiesis,”1 we have done additional studies of mice deficient in the cytokine Flt3L (also called Flk-2 ligand) and IL-7 (Flt3l–/–Il7–/– mice) and of Flt3l–/–Tpte2–/– mice and have found that the reported complete loss of B-1 as well as B-2 B lymphopoiesis in Flt3l–/–Il7r–/– mice7 and Flk2–/–Il7r–/– mice1 is entirely due to the simultaneous loss of function of IL-7 and Flt3L (C.T.J. and S.E.W.J., unpublished observations). Collectively, our findings suggest that Flt3L rather than TSLP is the key regulator of IL-7-independent B lymphopoiesis and that intact TSLP function is insufficient to restore any detectable B lymphopoiesis in the absence of these two critical regulators of B cell progenitors.