Developmental programming of adult haematopoiesis system.

The Barker hypothesis of 'foetal origin of adult diseases' has led to emphasize the concept of 'developmental programming', based on the crucial role of epigenetic factors. Accordingly, it has been demonstrated that parental adversity (before conception and during pregnancy) and foetal factors (i.e., hypoxia, malnutrition and placental insufficiency) permanently modify the physiological systems of the progeny, predisposing them to premature ageing and chronic disease during adulthood. Thus, an altered functionality of the endocrine, immune, nervous and cardiovascular systems is observed in the progeny. However, it remains to be understood whether the haematopoietic system itself also represents a portrait of foetal programming. Here, we provide evidence, reporting and discussing related theories, and results of studies described in the literature. In addition, we have outlined our opinions and suggest how it is possible to intervene to correct foetal mal-programming. Some pro-health interventions and recommendations are proposed, with the hope of guarantee the health of future generations and trying to combat the continuous increase in age-related diseases in human populations.

Circadian Regulation in Tissue Regeneration.

Circadian rhythms regulate over 40% of protein-coding genes in at least one organ in the body through mechanisms tied to the central circadian clock and to cell-intrinsic auto-regulatory feedback loops. Distinct diurnal differences in regulation of regeneration have been found in several organs, including skin, intestinal, and hematopoietic systems. Each regenerating system contains a complex network of cell types with different circadian mechanisms contributing to regeneration. In this review, we elucidate circadian regeneration mechanisms in the three representative systems. We also suggest circadian regulation of global translational activity as an understudied global regulator of regenerative capacity. A more detailed understanding of the molecular mechanisms underlying circadian regulation of tissue regeneration would accelerate the development of new regenerative therapies.

Human Bone Marrow Mesenchymal Stem Cells Rescue Endothelial Cells Experiencing Chemotherapy Stress by Mitochondrial Transfer Via Tunneling Nanotubes.

Tunneling nanotubes (TNTs) are newly discovered tubular structures between two distant cells that facilitate the intercellular exchange of signals and components. Recent reports show that mesenchymal stem cells (MSCs) can rescue injured target cells and promote recovery from a variety of stresses via TNT-mediated mitochondrial transfer. In this study, we explored how TNTs form between bone marrow MSCs and endothelial cells (ECs) by using a human umbilical cord vein endothelial cell (HUVEC) model. TNT formation between MSCs and HUVECs could be induced by treating HUVECs with cytarabine (Ara-C), and human bone marrow mesenchymal stem cells (hBMMSCs) could transfer mitochondria to injured HUVECs through TNTs. Mitochondrial transfer from hBMMSCs to HUVECs via TNTs rescued the injured HUVECs by reducing apoptosis, promoting proliferation and restoring the transmembrane migration ability as well as the capillary angiogenic capacity of HUVECs. This study provides novel insights into the cell-cell communication between MSCs and ECs and supports the results of prior studies indicating that ECs promote hematopoietic regeneration. An improved understanding of MSC-EC cross-talk will promote the development of MSC-directed strategies for improving EC function and hematopoietic system regeneration following myelosuppressive and myeloablative injuries.

ETS transcription factor ETV2/ER71/Etsrp in hematopoietic and vascular development, injury, and regeneration.

The major goal in regenerative medicine is to repair and restore injured, diseased or aged tissue function, thereby promoting general health. As such, the field of regenerative medicine has great translational potential in undertaking many of the health concerns and needs that we currently face. In particular, hematopoietic and vascular systems supply oxygen and nutrients and thus play critical roles in tissue development and tissue regeneration. Additionally, tissue vasculature serves as a tissue stem cell niche and thus contributes to tissue homeostasis. Notably, hematopoietic and vascular systems are sensitive to injury and subject to regeneration. As such, successful hematopoietic and vascular regeneration is prerequisite for efficient tissue repair and organismal survival and health. Recent studies have established that the interplay among the ETS transcription factor ETV2, vascular endothelial growth factor, and its receptor VEGFR2/FLK1 is essential for hematopoietic and vascular development. Emerging studies also support the role of these three factors and possible interplay in hematopoietic and vascular regeneration. Comprehensive understanding of the molecular mechanisms involved in the regulation and function of these three factors may lead to more effective approaches in promoting tissue repair and regeneration. Developmental Dynamics 246:318-327, 2017. © 2016 Wiley Periodicals, Inc.

The emerging role of GATA transcription factors in development and disease.

The GATA family of transcription factors consists of six proteins (GATA1-6) which are involved in a variety of physiological and pathological processes. GATA1/2/3 are required for differentiation of mesoderm and ectoderm-derived tissues, including the haematopoietic and central nervous system. GATA4/5/6 are implicated in development and differentiation of endoderm- and mesoderm-derived tissues such as induction of differentiation of embryonic stem cells, cardiovascular embryogenesis and guidance of epithelial cell differentiation in the adult.

klf2ash317 Mutant Zebrafish Do Not Recapitulate Morpholino-Induced Vascular and Haematopoietic Phenotypes.

INTRODUCTION AND OBJECTIVES: The zinc-finger transcription factor Krϋppel-like factor 2 (KLF2) transduces blood flow into molecular signals responsible for a wide range of responses within the vasculature. KLF2 maintains a healthy, quiescent endothelial phenotype. Previous studies report a range of phenotypes following morpholino antisense oligonucleotide-induced klf2a knockdown in zebrafish. Targeted genome editing is an increasingly applied method for functional assessment of candidate genes. We therefore generated a stable klf2a mutant zebrafish and characterised its cardiovascular and haematopoietic development. METHODS AND RESULTS: Using Transcription Activator-Like Effector Nucleases (TALEN) we generated a klf2a mutant (klf2ash317) with a 14bp deletion leading to a premature stop codon in exon 2. Western blotting confirmed loss of wild type Klf2a protein and the presence of a truncated protein in klf2ash317 mutants. Homozygous klf2ash317 mutants exhibit no defects in vascular patterning, survive to adulthood and are fertile, without displaying previously described morphant phenotypes such as high-output cardiac failure, reduced haematopoetic stem cell (HSC) development or impaired formation of the 5th accessory aortic arch. Homozygous klf2ash317 mutation did not reduce angiogenesis in zebrafish with homozygous mutations in von Hippel Lindau (vhl), a form of angiogenesis that is dependent on blood flow. We examined expression of three klf family members in wildtype and klf2ash317 zebrafish. We detected vascular expression of klf2b (but not klf4a or biklf/klf4b/klf17) in wildtypes but found no differences in expression that might account for the lack of phenotype in klf2ash317 mutants. klf2b morpholino knockdown did not affect heart rate or impair formation of the 5th accessory aortic arch in either wildtypes or klf2ash317 mutants. CONCLUSIONS: The klf2ash317 mutation produces a truncated Klf2a protein but, unlike morpholino induced klf2a knockdown, does not affect cardiovascular development.

Development of hematological and immunological characteristics in neonatal rats.

As major immunological and hematological parameters evolve during the early period of life, laboratory data must be interpreted in relation to developmental changes. Wistar (WU) rats were sacrificed on PND2, 4, 7, 10, 14, 17 and 21. Peripheral blood, bone marrow, thymus samples and spleen cells were collected and a bronchoalveolar lavage (BAL) performed. Parameters of blood counts changed considerably between time points. IgM and IgG levels steadily increased. Spontaneous spleen cell proliferation was low before PND21, although mitogens had stimulatory effects above baseline. In the spleen, T-lymphocyte counts tripled by PND17 (mainly attributed to CD8(+) cytotoxic T-cells and CD4(+) T-helper cells). In peripheral blood an increase in B-lymphocytes to about 60% of the cell number was observed. In BAL fluid, macrophages represented 95-98% of the cells. In thymus architecture, lymphoblast migration was seen and epithelial structures appeared. The data presented will help to distinguish between maturational changes and treatment-related effects.

RUNX1: A microRNA hub in normal and malignant hematopoiesis.

Hematopoietic development is orchestrated by gene regulatory networks that progressively induce lineage-specific transcriptional programs. To guarantee the appropriate level of complexity, flexibility, and robustness, these networks rely on transcriptional and post-transcriptional circuits involving both transcription factors (TFs) and microRNAs (miRNAs). The focus of this review is on RUNX1 (AML1), a master hematopoietic transcription factor which is at the center of miRNA circuits necessary for both embryonic and post-natal hematopoiesis. Interference with components of these circuits can perturb RUNX1-controlled coding and non-coding transcriptional programs in leukemia.

Ontogeny of erythroid gene expression.

Erythroid ontogeny is characterized by overlapping waves of primitive and definitive erythroid lineages that share many morphologic features during terminal maturation but have marked differences in cell size and globin expression. In the present study, we compared global gene expression in primitive, fetal definitive, and adult definitive erythroid cells at morphologically equivalent stages of maturation purified from embryonic, fetal, and adult mice. Surprisingly, most transcriptional complexity in erythroid precursors is already present by the proerythroblast stage. Transcript levels are markedly modulated during terminal erythroid maturation, but housekeeping genes are not preferentially lost. Although primitive and definitive erythroid lineages share a large set of nonhousekeeping genes, annotation of lineage-restricted genes shows that alternate gene usage occurs within shared functional categories, as exemplified by the selective expression of aquaporins 3 and 8 in primitive erythroblasts and aquaporins 1 and 9 in adult definitive erythroblasts. Consistent with the known functions of Aqp3 and Aqp8 as H2O2 transporters, primitive, but not definitive, erythroblasts preferentially accumulate reactive oxygen species after exogenous H2O2 exposure. We have created a user-friendly Web site (http://www.cbil.upenn.edu/ErythronDB) to make these global expression data readily accessible and amenable to complex search strategies by the scientific community.

The CCN family acting throughout the body: recent research developments.

The animal body is composed of a variety of cells and extracellular matrices that are organized and orchestrated in a harmonized manner to support life. Therefore, the critical importance of a comprehensive understanding of the molecular network surrounding and integrating the cells is now emphasized. The CCN family is a novel group of matricellular proteins that interact with and orchestrate a number of extracellular signaling and matrix molecules to construct and maintain living tissues. This family comprises six distinct members in mammals, which are characterized by a unique and conserved modular structure. These proteins are not targeted to limited and specific receptors to execute specific missions, but manipulate a vast number of biomolecules in the network by serving as a molecular hub at the center. The unified nomenclature, CCN, originates from a simple acronym of the three classical members, which helps us to avoid having any preconception about their pleiotropic and anonymous functional nature. In this review, after a brief summary of the general molecular concepts regarding the CCN family, new aspects of each member uncovered by recent research are introduced, which represent, nevertheless, only the tip of the iceberg of the profound functionality of these molecules.

The BAF53a subunit of SWI/SNF-like BAF complexes is essential for hemopoietic stem cell function.

ATP-dependent SWI/SNF-like BAF chromatin remodeling complexes are emerging as key regulators of embryonic and adult stem cell function. Particularly intriguing are the findings that specialized assemblies of BAF complexes are required for establishing and maintaining pluripotent and multipotent states in cells. However, little is known on the importance of these complexes in normal and leukemic hemopoiesis. Here we provide the first evidence that the actin-related protein BAF53a, a subunit of BAF complexes preferentially expressed in long-term repopulating stem cells, is essential for adult hemopoiesis. Conditional deletion of BAF53a resulted in multilineage BM failure, aplastic anemia, and rapid lethality. These severe hemopoietic defects originate from a proliferative impairment of BM HSCs and progenitors and decreased progenitor survival. Using hemopoietic chimeras, we show that the impaired function of BAF53a-deficient HSCs is cell-autonomous and independent of the BM microenvironment. Altogether, our studies highlight an unsuspected role for BAF chromatin remodeling complexes in the maintenance of HSC and progenitor cell properties.

The transcription factor Mxd4 controls the proliferation of the first blood precursors at the onset of hematopoietic development in vitro.

OBJECTIVE: The balance between proliferation and differentiation during hematopoietic development in the embryo is a complex process, the detailed molecular mechanisms of which remain to be fully characterized. The transcription factor Mxd4, a member of the Myc-Max-Mad network, was identified in a global gene expression profiling screen as being tightly regulated at the onset of hematopoietic lineage specification upon in vitro differentiation of mouse embryonic stem cells. Our study investigated the Mxd4 expression pattern at the onset of hematopoiesis and the biological relevance of its sharp and transient downregulation. MATERIALS AND METHODS: To study the expression pattern and role of Mxd4 at the onset of hematopoiesis, the in vitro differentiation of embryonic stem cells was used as a model system. Gain of function assays were performed using a doxycycline-inducible embryonic stem cell system. RESULTS: We show here that Mxd4 expression is transiently downregulated at an early stage of commitment to the hematopoietic lineage. Enforced expression of Mxd4 at this period of differentiation results in a defect in hematopoietic progenitor development, with impaired development of both primitive and definitive blood lineages. This effect is due to a severe decrease in cell proliferation, with an increased frequency of cells in the G(0)/G(1) phase of the cell cycle, alongside a reduced frequency of cells in the S phase. CONCLUSIONS: Together our results indicate that during embryonic hematopoietic differentiation Mxd4 is an important player in the regulation of blood progenitor proliferation, and suggest that downregulation of its expression might be required for a proliferative burst preceding lineage specification.

Aging of hematopoietic stem cells: Intrinsic changes or micro-environmental effects?

During development hematopoietic stem cells (HSCs) expand in number and persist throughout life by undergoing self-renewing divisions. Nevertheless, the hematopoietic system does not escape the negative effects of aging, suggesting that self-renewal is not complete. A fundamental issue in stem cell biology relates to such age-dependent loss of stem cell activity. Both stem cell intrinsic factors and extrinsic factors associated with an aging micro-environment could contribute to aging of the hematopoietic system. Recently, changes in the clonal composition of the HSC compartment during aging have been put forward as a key factor. Here, we discuss these recent developments and speculate how they may be of clinical relevance.

Runx1 isoforms show differential expression patterns during hematopoietic development but have similar functional effects in adult hematopoietic stem cells.

OBJECTIVE: RUNX1 (also known as acute myeloid leukemia 1) is an essential regulator of hematopoiesis and has multiple isoforms arising from differential splicing and utilization of two promoters. We hypothesized that the rare Runx1c isoform has a distinct role in hematopoietic stem cells (HSCs). MATERIALS AND METHODS: We have characterized the expression pattern of Runx1c in mouse embryos and human embryonic stem cell (hESC)-derived embryoid bodies using in situ hybridization and expression levels in mouse and human HSCs by real-time polymerase chain reaction. We then determined the functional effects of Runx1c using enforced retroviral overexpression in mouse HSCs. RESULTS: We observed differential expression profiles of RUNX1 isoforms during hematopoietic differentiation of hESCs. The RUNX1a and RUNX1b isoforms were expressed consistently throughout hematopoietic differentiation, whereas the RUNX1c isoform was only expressed at the time of emergence of definitive HSCs. RUNX1c was also expressed in the AGM region of E10.5 to E11.5 mouse embryos, the region where definitive HSCs arise. These observations suggested that the RUNX1c isoform may be important for the specification or function of definitive HSCs. However, using retroviral overexpression to study the effect of RUNX1 isoforms on HSCs in a gain-of-function system, no discernable functional difference could be identified between RUNX1 isoforms in mouse HSCs. Overexpression of both RUNX1b and RUNX1c induced quiescence in mouse HSCs in vitro and in vivo. CONCLUSIONS: Although the divergent expression profiles of Runx1 isoforms during development suggest specific roles for these proteins at different stages of HSC maturation, we could not detect an important functional distinction in adult mouse HSCs using our assay systems.

Ontogeny and nutritional control of adipogenesis in zebrafish (Danio rerio).

The global obesity epidemic demands an improved understanding of the developmental and environmental factors regulating fat storage. Adipocytes serve as major sites of fat storage and as regulators of energy balance and inflammation. The optical transparency of developing zebrafish provides new opportunities to investigate mechanisms governing adipocyte biology, however zebrafish adipocytes remain uncharacterized. We have developed methods for visualizing zebrafish adipocytes in vivo by labeling neutral lipid droplets with Nile Red. Our results establish that neutral lipid droplets first accumulate in visceral adipocytes during larval stages and increase in number and distribution as zebrafish grow. We show that the cellular anatomy of zebrafish adipocytes is similar to mammalian white adipocytes and identify peroxisome-proliferator activated receptor gamma and fatty acid binding protein 11a as markers of the zebrafish adipocyte lineage. By monitoring adipocyte development prior to neutral lipid deposition, we find that the first visceral preadipocytes appear in association with the pancreas shortly after initiation of exogenous nutrition. Zebrafish reared in the absence of food fail to form visceral preadipocytes, indicating that exogenous nutrition is required for adipocyte development. These results reveal homologies between zebrafish and mammalian adipocytes and establish the zebrafish as a new model for adipocyte research.

The cholinergic system is involved in regulation of the development of the hematopoietic system.

Gene expression profiling demonstrated that components of the cholinergic system, including choline acetyltransferase, acetylcholinesterase and nicotinic acetylcholine receptors (nAChRs), are expressed in embryonic stem cells and differentiating embryoid bodies (EBs). Triggering of nAChRs expressed in EBs by nicotine resulted in activation of MAPK and shifts of spontaneous differentiation toward hemangioblast. In vivo, non-neural nAChRs are detected early during development in fetal sites of hematopoiesis. Similarly, in vivo exposure of the developing embryo to nicotine resulted in higher numbers of hematopoietic progenitors in fetal liver. However postpartum, the number of hematopoietic stem/progenitor cells (HSPC) was decreased, suggesting an impaired colonization of the fetal bone marrow with HSPCs. This correlated with increased number of circulating HSPC and decreased expression of CXCR4 that mediates migration of circulating cells into the bone marrow regulatory niche. In addition, protein microarrays demonstrated that nicotine changed the profile of cytokines produced in the niche. While the levels of IL1alpha, IL1beta, IL2, IL9 and IL10 were not changed, the production of hematopoiesis-supportive cytokines including G-CSF, GM-CSF, IL3, IL6 and IGFBP-3 was decreased. This correlated with the decreased repopulating ability of HSPC in vivo and diminished hematopoietic activity in bone marrow cultures treated with nicotine. Interestingly, nicotine stimulated the production of IL4 and IL5, implying a possible role of the cholinergic system in pathogenesis of allergic diseases. Our data provide evidence that the nicotine-induced imbalance of the cholinergic system during gestation interferes with normal development and provides the basis for negative health outcomes postpartum in active and passive smokers.

[Relationship between hematopoietic development and endothelial cells (hemangioblast or hemogenic endothelium)--review].

Developmental programs of blood and endothelium are closely correlated and remarkably conserved among species. To categorize the ontogeny relationship between hematopoietic and endothelial lineages, two putative models are presented here. In the yolk sac, hematopoietic and endothelial cells are more likely derived from a common precursor--the hemangioblast. By comparison, the hemogenic endothelium is proposed to characterize the generation of hematopoietic stem cells from mature endothelium in the P-Sp/AGM region. Furthermore, couples of molecules, including Scl, Flk-1 and Runx-1, are involved in formation and subsequent differentiation of the hemangioblast or hemogenic endothelium during embryonic hematopoiesis. In this article, the development of primitive and definitive hematopoiesis, two models associated with development of hematopoietic and vascular endothelial cells as well as molecules associated with development of hematopoietic and endotheliate cells were summaried.

Children are not little adults: just ask their hematopoietic stem cells.

HSCs differ during ontogeny in some important parameters, including anatomic site of residence and cell cycling characteristics. In this issue of the JCI, Bowie et al. show that postnatal HSCs as well as fetal liver HSCs in mice are active in the cell cycle at much higher rates than that of adult HSCs; however, this increased frequency of cycling abruptly ceases 4 weeks after birth (see the related article beginning on page 2808). The cycling postnatal HSCs expressed high levels of CXC chemokine ligand 12 (CXCL12, also known as stromal cell-derived factor 1 [SDF-1]), a chemokine previously implicated in stem cell trafficking to the marrow cavity and shown to be expressed by cells within the hematopoietic microenvironment. These cells also possessed an engraftment defect impeding reconstitution in irradiated recipient mice, which was reversible by pretransplant administration of antagonists of the CXCL12 receptor, CXCR4. Such agents are currently clinically available, suggesting that this approach could be used to improve stem cell transplantation and engraftment.

Hematopoietic stem cells proliferate until after birth and show a reversible phase-specific engraftment defect.

The regulation of HSC proliferation and engraftment of the BM is an important but poorly understood process, particularly during ontogeny. Here we show that in mice, all HSCs are cycling until 3 weeks after birth. Then, within 1 week, most became quiescent. Prior to 4 weeks of age, the proliferating HSCs with long-term multilineage repopulating activity displayed an engraftment defect when transiting S/G2/M. During these cell cycle phases, their expression of CXC chemokine ligand 12 (CXCL12; also referred to as stromal cell-derived factor 1 [SDF-1]) transiently increased. The defective engrafting activity of HSCs in S/G2/M was reversed when cells were allowed to progress into G1 prior to injection or when the hosts (but not the cells) were pretreated with a CXCL12 antagonist. Interestingly, the enhancing effect of CXCL12 antagonist pretreatment was exclusive to transplants of long-term multilineage repopulating HSCs in S/G2/M. These results demonstrate what we believe to be a new HSC regulatory checkpoint during development. They also suggest an ability of HSCs to express CXCL12 in a fashion that changes with cell cycle progression and is associated with a defective engraftment that can be overcome by in vivo administration of a CXCL12 antagonist.