Cornelis J.H. Pronk

Elucidation of the phenotypic, functional, and molecular topography of a myeloerythroid progenitor cell hierarchy

The major myeloid blood cell lineages are generated from hematopoietic stem cells by differentiation through a series of increasingly committed progenitor cells. Precise characterization of intermediate progenitors is important for understanding fundamental differentiation processes and a variety of disease states, including leukemia. Here, we evaluated the functional in vitro and in vivo potentials of a range of prospectively isolated myeloid precursors with differential expression of CD150, Endoglin, and CD41. Our studies revealed a hierarchy of myeloerythroid progenitors with distinct lineage potentials. The global gene expression signatures of these subsets were consistent with their functional capacities, and hierarchical clustering analysis suggested likely lineage relationships. These studies provide valuable tools for understanding myeloid lineage commitment, including isolation of an early erythroid-restricted precursor, and add to existing models of hematopoietic differentiation by suggesting that progenitors of the innate and adaptive immune system can separate late, following the divergence of megakaryocytic/erythroid potential.

Tumor Necrosis Factor Receptor 1 Is a Negative Regulator of Progenitor Proliferation in Adult Hippocampal Neurogenesis

Tumor necrosis factor-α (TNF-α) is a proinflammatory cytokine, acting through the TNF-R1 and TNF-R2 receptors. The two receptors have been proposed to mediate distinct TNF-α effects in the CNS, TNF-R1 contributing to neuronal damage and TNF-R2 being neuroprotective. Whether TNF-α and its receptors play any role for neurogenesis in the adult brain is unclear. Here we used mouse models with loss of TNF-R1 and TNF-R2 function to establish whether signaling through these receptors could influence hippocampal neurogenesis in vivo under basal conditions, as well as after status epilepticus (SE), which is associated with inflammation and elevated TNF-α levels. Notably, in the intact brain, the number of new, mature hippocampal neurons was elevated in TNF-R1−/− and TNF-R1/R2−/− mice, whereas no significant changes were detected in TNF-R2−/− mice. Also after SE, the TNF-R1−/− and TNF-R1/R2−/− mice produced more new neurons. In contrast, the TNF-R2−/− mice showed reduced SE-induced neurogenesis. Cell proliferation in the dentate subgranular zone was elevated in TNF-R1−/− and TNF-R1/R2−/− mice both under basal conditions and after SE. The TNF-R2−/− mice either showed no change or minor decrease of cell proliferation. TNF-R1 and TNF-R2 receptors were expressed by hippocampal progenitors, as assessed with reverse transcription-PCR on sorted or cultured cells and immunocytochemistry on cultures. Our data reveal differential actions of TNF-R1 and TNF-R2 signaling in adult hippocampal neurogenesis and identify for the first time TNF-R1 as a negative regulator of neural progenitor proliferation in both the intact and pathological brain.

Accumulating mitochondrial DNA mutations drive premature hematopoietic aging phenotypes distinct from physiological stem cell aging.

Somatic stem cells mediate tissue maintenance for the lifetime of an organism. Despite the well-established longevity that is a prerequisite for such function, accumulating data argue for compromised stem cell function with age. Identifying the mechanisms underlying age-dependent stem cell dysfunction is therefore key to understanding the aging process. Here, using a model carrying a proofreading-defective mitochondrial DNA polymerase, we demonstrate hematopoietic defects reminiscent of premature HSC aging, including anemia, lymphopenia, and myeloid lineage skewing. However, in contrast to physiological stem cell aging, rapidly accumulating mitochondrial DNA mutations had little functional effect on the hematopoietic stem cell pool, and instead caused distinct differentiation blocks and/or disappearance of downstream progenitors. These results show that intact mitochondrial function is required for appropriate multilineage stem cell differentiation, but argue against mitochondrial DNA mutations per se being a primary driver of somatic stem cell aging.

Hematopoietic stem cell ageing is uncoupled from p16(INK4A)-mediated senescence.

Somatic stem cells are ultimately responsible for mediating appropriate organ homeostasis and have therefore been proposed to represent a cellular origin of the ageing process—a state often characterized by inappropriate homeostasis. Specifically, it has been suggested that ageing stem cells might succumb to replicative senescence by a mechanism involving the cyclin-dependent kinase inhibitor p16INK4A. Here, we tested multiple functional and molecular parameters indicative of p16INK4A activity in primary aged murine hematopoietic stem cells (HSCs). We found no evidence that replicative senescence accompanies stem cell ageing in vivo, and in line with p16INK4A being a critical determinant of such processes, most aged HSCs (>99%) failed to express p16INK4A at the mRNA level. Moreover, whereas loss of epigenetically guided repression of the INK4A/ARF locus accompanied replicative senescent murine embryonic fibroblasts, such repression was maintained in aged stem cells. Taken together, these studies indicate that increased senescence as mediated by the p16INK4A tumor suppressor has only a minor function as an intrinsic regulator of steady-state HSC ageing in vivo.

Phosphatidylinositol 3-kinase is essential for kit ligand-mediated survival, whereas interleukin-3 and flt3 ligand induce expression of antiapoptotic Bcl-2 family genes.

Cytokines such as interleukin 3 (IL‐3), kit ligand (KL), and flt3 ligand (FL) promote survival of hematopoietic stem cells and myeloid progenitor cells. In many cell types, members of the Bcl‐2 gene family are major regulators of survival, but the mediating mechanisms are not fully understood. Using two myeloid progenitor cell lines, FDCP‐mix and FDC‐P1, as well as primary mouse bone marrow progenitors, we demonstrate that KL‐mediated survival is dependent on the activation of phosphatidylinositol‐3 (PI‐3) kinase. The inhibitor LY294002 was able to completely abolish survival mediated by KL, whereas IL‐3 and FL were only partially affected. Although all three cytokines induced phosphorylation of protein kinase B (PKB), only KL required PI‐3 kinase activity to elicit survival in hematopoietic progenitors. In contrast, pretreatment of cells with inhibitors to the MAP kinase pathway did not affect the survival. We next established if IL‐3 and FL activated antiapoptotic Bcl‐2 and the related genes Bcl‐XL and Mcl‐1. By RNA protection assay and Western blot analysis, we show that all three genes are induced by IL‐3, whereas FL induces Bcl‐2 and to some extent Bcl‐XL. Importantly, KL could not sustain their expression. Moreover, use of inhibitors implied that IL‐3 was mainly exerting its effect on Bcl‐2 at the level of transcription. The addition of LY294002 did not affect the expression of Bcl‐2 and Bcl‐XL, and thus, we conclude that expression of antiapoptotic Bcl‐2 family member genes is not dependent on PI‐3 kinase activity. Our results indicate that cytokines exert distinct survival effects and that FL and IL‐3 are capable of sustaining progenitor survival by up‐regulating the expression of Bcl‐2 and related genes.

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.

Long-term persistence of human donor alveolar macrophages in lung transplant recipients

Background Alveolar macrophages (AMFs) are critical regulators of lung function, and may participate in graft rejection following lung transplantation. Recent studies in experimental animals suggest that most AMFs are self-maintaining cells of embryonic origin, but knowledge about the ontogeny and life span of human AMFs is scarce. Methods To follow the origin and longevity of AMFs in patients with lung transplantation for more than 100 weeks, we obtained transbronchial biopsies from 10 gender-mismatched patients with lung transplantation. These were subjected to combined in situ hybridisation for X/Y chromosomes and immunofluorescence staining for macrophage markers. Moreover, development of AMFs in humanised mice reconstituted with CD34+ umbilical cord-derived cells was assessed. Results The number of donor-derived AMFs was unchanged during the 2 year post-transplantation period. A fraction of the AMFs proliferated locally, demonstrating that at least a subset of human AMFs have the capacity to self-renew. Lungs of humanised mice were found to abundantly contain populations of human AMFs expressing markers compatible with a monocyte origin. Moreover, in patients with lung transplantation we found that recipient monocytes seeded the alveoli early after transplantation, and showed subsequent phenotypical changes consistent with differentiation into proliferating mature AMFs. This resulted in a stable mixed chimerism between donor and recipient AMFs throughout the 2-year period. Conclusions The finding that human AMFs are maintained in the lung parenchyma for several years indicates that pulmonary macrophage transplantation can be a feasible therapeutic option for patients with diseases caused by dysfunctional AMFs. Moreover, in a lung transplantation setting, long-term persistence of donor AMFs may be important for the development of chronic graft rejection.

Tumor necrosis factor receptor-1 is essential for LPS-induced sensitization and tolerance to oxygen-glucose deprivation in murine neonatal organotypic hippocampal slices.

Inflammation and ischemia have a synergistic damaging effect in the immature brain. The role of tumor necrosis factor (TNF) receptors 1 and 2 in lipopolysaccharide (LPS)-induced sensitization and tolerance to oxygen—glucose deprivation (OGD) was evaluated in neonatal murine hippocampal organotypic slices. Hippocampal slices from balb/c, C57BL/6 TNFR1−/-, TNFR2−/-, and wild-type (WT) mice obtained at P6 were grown in vitro for 9 days. Preexposure to LPS immediately before OGD increased propidium iodide-determined cell death in regions CA1, CA3, and dentate gyrus from 4 up to 48 h after OGD (P < 0.001). Extending the time interval between LPS exposure and OGD to 72 h resulted in tolerance, that is reduced neuronal cell death after OGD (P < 0.05). Slices from TNFR1−/- mice showed neither LPS-induced sensitization nor LPS-induced tolerance to OGD, whereas both effects were present in slices from TNFR2−/- and WT mice. Cytokine secretion (TNFα and interleukin-6) during LPS exposure was decreased in TNFR1−/- slices and increased in TNFR2−/- as compared with WT slices. We conclude that LPS induces sensitization or tolerance to OGD depending on the time interval between exposure to LPS and OGD in murine hippocampal slice cultures. Both paradigms are dependent on signaling through TNFR1.

Hematopoietic Stem Cells Are Intrinsically Protected against MLL-ENL-Mediated Transformation.

Studies of developmental pathways of hematopoietic stem cells (HSCs) have defined lineage relationships throughout the blood system. This is relevant to acute myeloid leukemia (AML), where aggressiveness and therapeutic responsiveness can be influenced by the initial stage of transformation. To address this, we generated a mouse model in which the mixed-lineage leukemia/eleven-nineteen-leukemia (MLL-ENL) transcription factor can be conditionally activated in any cell type. We show that AML can originate from multiple hematopoietic progenitor subsets with granulocytic and monocytic potential, and that the normal developmental position of leukemia-initiating cells influences leukemic development. However, disease failed to arise from HSCs. Although it maintained or upregulated the expression of target genes associated with leukemic development, MLL-ENL dysregulated the proliferative and repopulating capacity of HSCs. Therefore, the permissiveness for development of AML may be associated with a narrower window of differentiation than was previously appreciated, and hijacking the self-renewal capacity of HSCs by a potent oncogene is insufficient for leukemic development.

Flow cytometry-based identification of immature myeloerythroid development.

Precursor cells of the myeloerythroid cell lineages give rise to mature cells of the granulocyte, monocyte, erythroid, and/or thrombocytic lineages. High-resolution profiling of the developmental stages, from hematopoietic stem cells to mature progeny, is important to study and understand the underlying mechanisms that guide various cell fate decisions. In addition, this approach provides greater insights into pathogenic events such as leukemia, diseases that are most often characterized by halted differentiation at defined immature precursor levels. In this chapter, we provide protocols and discuss approaches concerning the analysis and purification of immature myeloerythroid lineages by multiparameter flow cytometry. Although recent data have demonstrated the feasibility of similar approaches also for the human system, we will focus our chapter on C57BL/6 mice, in which immunophenotypic applications have been most widely developed. This should also allow for its application in genetically modified models on this background. For maximal reproducibility, all protocols described have been established using reagents from commercial vendors to be analyzed on a three-laser flow cytometer with factory standard configuration.