Natalija Buza-Vidas

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.

Key Role of flt3 Ligand in Regulation of the Common Lymphoid Progenitor but Not in Maintenance of the Hematopoietic Stem Cell Pool

The first lineage commitment step of hematopoietic stem cells (HSC) results in separation into distinct lymphoid and myeloid differentiation pathways, reflected in the generation of common lymphoid and myeloid progenitors (CLP and CMP, respectively). In this report we present the first evidence for a nonredundant regulator of this process, in that adult mice deficient in expression of the flt3 ligand (FL) have severely (10-fold) reduced levels of the CLP, accompanied by reductions in the earliest identifiable B and T cell progenitors. In contrast, CMP and HSC are unaffected in FL-deficient mice. Noteworthy, CLP express high levels of both the flt3 receptor and ligand, indicating a potential autocrine role of FL in regulation of the earliest lymphoid commitment step from HSC.

Persistent malignant stem cells in del(5q) myelodysplasia in remission.

BACKGROUND The in vivo clinical significance of malignant stem cells remains unclear. METHODS Patients who have the 5q deletion (del[5q]) myelodysplastic syndrome (interstitial deletions involving the long arm of chromosome 5) have complete clinical and cytogenetic remissions in response to lenalidomide treatment, but they often have relapse. To determine whether the persistence of rare but distinct malignant stem cells accounts for such relapses, we examined bone marrow specimens obtained from seven patients with the del(5q) myelodysplastic syndrome who became transfusion-independent while receiving lenalidomide treatment and entered cytogenetic remission. RESULTS Virtually all CD34+, CD38+ progenitor cells and stem cells that were positive for CD34 and CD90, with undetectable or low CD38 (CD38−/low), had the 5q deletion before treatment. Although lenalidomide efficiently reduced these progenitors in patients in complete remission, a larger fraction of the minor, quiescent, CD34+,CD38-/low, CD90+ del(5q) stem cells as well as functionally defined del(5q) stem cells remained distinctly resistant to lenalidomide. Over time, lenalidomide resistance developed in most of the patients in partial and complete remission, with recurrence or expansion of the del(5q) clone and clinical and cytogenetic progression. CONCLUSIONS In these patients with the del(5q) myelodysplastic syndrome, we identified rare and phenotypically distinct del(5q) myelodysplastic syndrome stem cells that were also selectively resistant to therapeutic targeting at the time of complete clinical and cytogenetic remission. (Funded by the EuroCancerStemCell Consortium and others.)

Complementary signaling through flt3 and interleukin-7 receptor alpha is indispensable for fetal and adult B cell genesis

Extensive studies of mice deficient in one or several cytokine receptors have failed to support an indispensable role of cytokines in development of multiple blood cell lineages. Whereas B1 B cells and Igs are sustained at normal levels throughout life of mice deficient in IL-7, IL-7Rα, common cytokine receptor gamma chain, or flt3 ligand (FL), we report here that adult mice double deficient in IL-7Rα and FL completely lack visible LNs, conventional IgM+ B cells, IgA+ plasma cells, and B1 cells, and consequently produce no Igs. All stages of committed B cell progenitors are undetectable in FL−/− × IL-7Rα−/− BM that also lacks expression of the B cell commitment factor Pax5 and its direct target genes. Furthermore, in contrast to IL-7Rα−/− mice, FL−/− × IL-7Rα−/− mice also lack mature B cells and detectable committed B cell progenitors during fetal development. Thus, signaling through the cytokine tyrosine kinase receptor flt3 and IL-7Rα are indispensable for fetal and adult B cell development.

The earliest thymic T cell progenitors sustain B cell and myeloid lineage potential

The stepwise commitment from hematopoietic stem cells in the bone marrow to T lymphocyte–restricted progenitors in the thymus represents a paradigm for understanding the requirement for distinct extrinsic cues during different stages of lineage restriction from multipotent to lineage-restricted progenitors. However, the commitment stage at which progenitors migrate from the bone marrow to the thymus remains unclear. Here we provide functional and molecular evidence at the single-cell level that the earliest progenitors in the neonatal thymus had combined granulocyte-monocyte, T lymphocyte and B lymphocyte lineage potential but not megakaryocyte-erythroid lineage potential. These potentials were identical to those of candidate thymus-seeding progenitors in the bone marrow, which were closely related at the molecular level. Our findings establish the distinct lineage-restriction stage at which the T cell lineage–commitment process transits from the bone marrow to the remote thymus.

Distinct myeloid progenitor-differentiation pathways identified through single-cell RNA sequencing

According to current models of hematopoiesis, lymphoid-primed multi-potent progenitors (LMPPs) (Lin−Sca-1+c-Kit+CD34+Flt3hi) and common myeloid progenitors (CMPs) (Lin−Sca-1+c-Kit+CD34+CD41hi) establish an early branch point for separate lineage-commitment pathways from hematopoietic stem cells, with the notable exception that both pathways are proposed to generate all myeloid innate immune cell types through the same myeloid-restricted pre–granulocyte-macrophage progenitor (pre-GM) (Lin−Sca-1−c-Kit+CD41−FcγRII/III−CD150−CD105−). By single-cell transcriptome profiling of pre-GMs, we identified distinct myeloid differentiation pathways: a pathway expressing the gene encoding the transcription factor GATA-1 generated mast cells, eosinophils, megakaryocytes and erythroid cells, and a pathway lacking expression of that gene generated monocytes, neutrophils and lymphocytes. These results identify an early hematopoietic-lineage bifurcation that separates the myeloid lineages before their segregation from other hematopoietic-lineage potential.

FLT3 expression initiates in fully multipotent mouse hematopoietic progenitor cells

Lymphoid-primed multipotent progenitors with down-regulated megakaryocyte-erythroid (MkE) potential are restricted to cells with high levels of cell-surface FLT3 expression, whereas HSCs and MkE progenitors lack detectable cell-surface FLT3. These findings are compatible with FLT3 cell-surface expression not being detectable in the fully multipotent stem/progenitor cell compartment in mice. If so, this process could be distinct from human hematopoiesis, in which FLT3 already is expressed in multipotent stem/progenitor cells. The expression pattern of Flt3 (mRNA) and FLT3 (protein) in multipotent progenitors is of considerable relevance for mouse models in which prognostically important Flt3 mutations are expressed under control of the endogenous mouse Flt3 promoter. Herein, we demonstrate that mouse Flt3 expression initiates in fully multipotent progenitors because in addition to lymphoid and granulocyte-monocyte progenitors, FLT3(-) Mk- and E-restricted downstream progenitors are also highly labeled when Flt3-Cre fate mapping is applied.

Delineation of the earliest lineage commitment steps of haematopoietic stem cells: new developments, controversies and major challenges

Purpose of reviewThis review addresses recently reported evidence for alternative cellular pathways for haematopoietic stem cell lineage commitment. Recent findingsUsing various approaches, several laboratories suggested the existence of adult as well as foetal multipotent progenitor cells with combined B cell, T cell and granulocyte/macrophage potential, but little or no megakaryocyte/erythroid potential. Compared with haematopoietic stem cells, these multipotent progenitor cells exhibited downregulated transcriptional expression of genes of the megakaryocyte/erythroid lineages and upregulated expression of lymphoid lineage genes. The existence of these lineage-restricted multipotent progenitor cells suggests that the first lineage commitment step of haematopoietic stem cells does not result in strict separation into myelopoiesis and lymphopoiesis, and that there might be alternative pathways for commitment toward different lineage fates. These findings have been questioned by other studies, however. To resolve this controversy and establish the complete road map for haematopoietic lineage commitment, improved tools and more stringent standards for how to identify and characterize lineage fate options of distinct stem and progenitor cells are needed. SummaryCurrent and future progress in establishing the complete cellular roadmap for haematopoietic lineage commitment will permit identification and characterization of key regulators of lineage fate decisions in haematopoietic stem cells.

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.