Inflammatory signaling regulates embryonic hematopoietic stem and progenitor cell production

Identifying signaling pathways that regulate hematopoietic stem and progenitor cell (HSPC) formation in the embryo will guide efforts to produce and expand HSPCs ex vivo. Here we show that sterile tonic inflammatory signaling regulates embryonic HSPC formation. Expression profiling of progenitors with lymphoid potential and hematopoietic stem cells (HSCs) from aorta/gonad/mesonephros (AGM) regions of midgestation mouse embryos revealed a robust innate immune/inflammatory signature. Mouse embryos lacking interferon γ (IFN-γ) or IFN-α signaling and zebrafish morphants lacking IFN-γ and IFN-ϕ activity had significantly fewer AGM HSPCs. Conversely, knockdown of IFN regulatory factor 2 (IRF2), a negative regulator of IFN signaling, increased expression of IFN target genes and HSPC production in zebrafish. Chromatin immunoprecipitation (ChIP) combined with sequencing (ChIP-seq) and expression analyses demonstrated that IRF2-occupied genes identified in human fetal liver CD34⁺ HSPCs are actively transcribed in human and mouse HSPCs. Furthermore, we demonstrate that the primitive myeloid population contributes to the local inflammatory response to impact the scale of HSPC production in the AGM region. Thus, sterile inflammatory signaling is an evolutionarily conserved pathway regulating the production of HSPCs during embryonic development.     

Control of human hematopoietic stem/progenitor cell migration by the extracellular matrix protein Slit3

Patients whose hematopoietic system is compromised by chemo- and/or radiotherapy require transplantation of hematopoietic stem and progenitor cells (HSPCs) to restore hematopoiesis. Successful homing of transplanted HSPCs to the bone marrow (BM) largely depends on their migratory potential, which is critically regulated by the chemokine CXCL12. In this study, we have investigated the expression and function of Slit proteins and their corresponding Roundabout (Robo) receptors in human HSPC migration. Slit proteins are extracellular matrix proteins that can modulate the (chemoattractant-induced) migration of mature leukocytes. We show that mRNAs for all Slits (Slit1-3) are expressed in primary BM stroma and BM-derived endothelial and stromal cell lines, but not in CD34? HSPCs. Human CD34? HSPCs expressed mRNAs for all Robos (Robo1-4), but only the Robo1 protein was detected on their cell surface. Functionally, Slit3 treatment increased the in vivo homing efficiency of CD34? HSPCs to the BM in NOD/SCID mice, whereas Slit3-exposed HSPC migration in vitro was inhibited. These effects do not appear to result from modulated CXCL12 responsiveness as CXCR4 expression, CXCL12-induced actin polymerization or the basal and CXCL12-induced adhesion to fibronectin or BM-derived endothelial cells of CD34? HSPC were not altered by Slit3 exposure. However, we show that Slit3 rapidly reduced the levels of active RhoA in HL60 cells and primary CD34? HSPC, directly affecting a pathway involved in actin cytoskeleton remodeling and HSPC migration. Together, our results support a role for Slit3 in human HSPC migration in vitro and homing in vivo and might contribute to the design of future approaches aimed at improving transplantation efficiency of human CD34? HSPCs.     

Pluripotent hematopoietic stem cell development during embryogenesis

Significant progress has been made towards a better understanding of the establishment of hematopoiesis in the embryo. Hematopoietic precursors have been shown to arise independently in the yolk sac and in the intra-embryonic mesoderm. From the combined analysis of differentiation potentials, expression patterns and mutant phenotypes, a picture has emerged: definitive hematopoietic precursors are transiently generated in a specific environment close to the endothelium of the main arteries.     

TLRs control hematopoiesis during infection

Recent research has shown that (i) Toll‐like receptor (TLR) agonists drive hematopoietic stem and progenitor cells (HSPCs) to proliferate and differentiate along the myeloid lineage in vitro, and (ii) direct TLR‐mediated stimulation of HSPCs also promotes macrophage differentiation in vivo following infection. These new insights demonstrate that TLR signaling in HSPCs, in addition to other TLR‐dependent mechanisms, can contribute to HSPC expansion and myeloid differentiation after infection. Evidence is, therefore, mounting that direct TLR‐induced programming of hematopoiesis plays a key role in host defense by rapidly replenishing the innate immune system with the cells needed to deal with pathogens.     

Noncanonical NF-κB signaling regulates hematopoietic stem cell self-renewal and microenvironment interactions

RelB and nuclear factor κB (NF-κB2) are the main effectors of NF-κB noncanonical signaling and play critical roles in many physiological processes. However, their role in hematopoietic stem/progenitor cell (HSPC) maintenance has not been characterized. To investigate this, we generated RelB/NF-κB2 double-knockout (dKO) mice and found that dKO HSPCs have profoundly impaired engraftment and self-renewal activity after transplantation into wild-type recipients. Transplantation of wild-type bone marrow cells into dKO mice to assess the role of the dKO microenvironment showed that wild-type HSPCs cycled more rapidly, were more abundant, and had developmental aberrancies: increased myeloid and decreased lymphoid lineages, similar to dKO HSPCs. Notably, when these wild-type cells were returned to normal hosts, these phenotypic changes were reversed, indicating a potent but transient phenotype conferred by the dKO microenvironment. However, dKO bone marrow stromal cell numbers were reduced, and bone-lining niche cells supported less HSPC expansion than controls. Furthermore, increased dKO HSPC proliferation was associated with impaired expression of niche adhesion molecules by bone-lining cells and increased inflammatory cytokine expression by bone marrow cells. Thus, RelB/NF-κB2 signaling positively and intrinsically regulates HSPC self-renewal and maintains stromal/osteoblastic niches and negatively and extrinsically regulates HSPC expansion and lineage commitment through the marrow microenvironment.     

Expansion of engrafting human hematopoietic stem/progenitor cells in three-dimensional scaffolds with surface-immobilized fibronectin

An efficient and practical ex vivo expansion methodology for human hematopoietic stem/progenitor cells (HSPCs) is critical in realizing the potential of HSPC transplantation in treating a variety of hematologic disorders and as a supportive therapy for malignant diseases. We report here an expansion strategy using a three-dimensional (3D) scaffold conjugated with an extracellular matrix molecule, fibronectin (FN), to partially mimic the hematopoietic stem cell niche. FN-immobilized 3D polyethylene terephthalate (PET) scaffold was synthesized and evaluated for HSPC expansion efficiency, in comparison with a FN-immobilized 2D PET substrate and a 3D scaffold with FN supplemented in the medium. Covalent conjugation of FN produced substrate and scaffold with higher cell expansion efficiency than that on their unmodified counterparts. After 10 days of culture in serum-free medium, human umbilical cord blood CD34+ cells cultured in FN-conjugated scaffold yielded the highest expansion of CD34+ cells (approximately 100 fold) and long-term culture initiating cells (approximately 47-fold). The expanded human CD34+ cells successfully reconstituted hematopoiesis in NOD/SCID mice. This study demonstrated the synergistic effect between the three-dimensionality of the scaffold and surface-conjugated FN, and the potential of this FN-conjugated 3D scaffold for ex vivo expansion of HSPCs.     

Long-Acting IL-33 Mobilizes High-Quality Hematopoietic Stem and Progenitor Cells More Efficiently Than Granulocyte Colony-Stimulating Factor or AMD3100

Mobilization of hematopoietic stem and progenitor cells (HSPCs) has become increasingly important for hematopoietic cell transplantation. Current mobilization approaches are insufficient because they fail to mobilize sufficient numbers of cells in a significant fraction of patients and are biased toward myeloid immune reconstitution. A novel, single drug mobilization agent that allows a more balanced (myeloid and lymphoid) reconstitution would therefore be highly favorable to improve transplantation outcome. In this present study, we tested commercially available IL-33 molecules and engineered novel variants of IL-33. These molecules were tested in cell-based assays in vitro and in mobilization models in vivo. We observed for the first time that IL-33 treatment in mice mobilized HSPCs and common myeloid progenitors more efficiently than clinical mobilizing agents granulocyte colony-stimulating factor (G-CSF) or AMD3100. We engineered several oxidation-resistant IL-33 variants with equal or better in vitro activity. In vivo, these variants mobilized HSPCs and, interestingly, also hematopoietic stem cells, common lymphoid progenitor cells, and endothelial progenitor cells more efficiently than wild-type IL-33 or G-CSF. We then engineered an IL-33-Fc fusion molecule, a single dose of which was sufficient to significantly increase the mobilization of HSPCs after 4 days. In conclusion, our findings suggest that long-acting, oxidation-resistant IL-33 may be a novel approach for HSPC transplantation. IL-33-mobilized HSPCs differ from cells mobilized with G-CSF and AMD3100, and it is possible that these differences may result in better transplantation outcomes.     

The ontogenesis of the hematopoietic system revisited

The ontogenesis of hematopoiesis is classically described as a series of successive steps: the first takes in the yolk sac where blood islands differentiate. Then, cells deriving from these structures migrate and populate the transient hematopoietic organs such as the liver and the spleen. At last, the eventual migration allows the establishment of bone marrow hematopoiesis. This theory described in almost all the textbooks of Human Embryology does not fit with recent experimental data. Indeed, the construction of quail-chick chimeras shows that the yolk sac does not contribute to the adult hematopoiesis in birds. Adult hematopoietic cells arise from a population located on the ventral side of the aorta both in birds and mammals. The aortic population derives from the para-aortic splanchnopleura and its derivative, the so-called AGM (Aorta-Gonad-Mesonephros). These new data provide new concepts to understand the process of ontogenesis of the hematopoietic system in vertebrates.     

The human embryo, but not its yolk sac, generates lympho-myeloid stem cells: mapping multipotent hematopoietic cell fate in intraembryonic mesoderm

We have traced emerging hematopoietic cells along human early ontogeny by culturing embryonic tissue rudiments in the presence of stromal cells that promote myeloid and B cell differentiation, and by assaying T cell potential in the NOD-SCID mouse thymus. Hematogenous potential was present inside the embryo as early as day 19 of development in the absence of detectable CD34+ hematopoietic cells, and spanned both lymphoid and myeloid lineages from day 24 in the splanchnopleural mesoderm and derived aorta where CD34+ progenitors appear at day 27. By contrast, hematopoietic cells arising in the third week yolk sac, as well as their progeny at later stages, were restricted to myelopoiesis and therefore are unlikely to contribute to definitive hematopoiesis in man.     

Intraembryonic, but not yolk sac hematopoietic precursors, isolated before circulation, provide long-term multilineage reconstitution

The relative contribution of yolk sac and intraembryonic precursors to hematopoiesis has been a matter of long-standing controversy. As reconstitution activity has so far only been found in embryonic tissues after the onset of circulation, the origin of reconstituting cells could not be formally established. Here, we separated yolk sac and intraembryonic splanchnopleura prior to circulation and maintained the explants in organ culture before transfer. Precursors derived from the intraembryonic site generated multilineage hematopoietic progeny in adult mice for more than 6 months. Yolk sac cells only provided myeloid short-term reconstitution. The results reveal a differential hematopoietic capacity of precirculation embryonic tissues in vivo, and indicate that the only cells capable of adult long-term hematopoiesis are of intraembryonic origin.     

The effect of the humoral action of the cells from chick embryo hematopoietic organs on the colony-forming capacity of normal mouse bone-marrow cells and in activated erythropoiesis

The cells of chick embryo hematopoietic organs were cultivated together with mouse bone marrow cells in bicameral diffusion chambers under normal conditions and in activated erythropoiesis. The yolk sac cells inhibited colony formation on the 2nd, 4th days of development, the hepatic cells--on the 8th, 12th days, the splenic cells--on the 13th, 17th days, and the bone marrow cells--on the 15th day. The cells of the yolk sac sharply intensify the formation of colonies by the marrow mononuclears, in particular, on the 6th developmental day in activated erythropoiesis. The yolk sac (on the 4th, 6th days of development), the liver, and the spleen produce humoral factors which facilitate the formation of erythroid colonies by the marrow cells of mice. The cells of a 4-day-old yolk sac inhibit the formation of granulocytic colonies, and the synthesis of inhibitors ceases in a 6-day-old yolk sac. It is suggested that the synthesis of local humoral inhibitors of the proliferation of colony-forming units controls the intensity of hematopoiesis in the organ and, by reducing proliferation, facilitates the emergency of cells into differentiation independently, according to the law or under the effect of local humoral poetins.     

Patterns of Wnt/Fzd/LRP gene expression during embryonic hematopoiesis

Wnt signaling plays several roles in hematopoiesis, promoting hemopoietic stem cell (HSC) self-renewal, providing proliferative signals for immature progenitors and regulating lineage commitment. To ascertain which Wnt proteins and receptors are important during hematopoietic development, we used two systems; in vitro hematopoietic differentiation of embryonic stem (ES) cells and tissues isolated from sites specific for hematopoiesis during mouse embryogenesis. Initially genes involved in hematopoiesis were profiled and indicate differentiating ES cells undergo a wave of primitive hematopoiesis (Day 3.75) similar to the mouse yolk sac, followed by a wave of more definitive hematopoiesis (Day 7.75) comparable to the aorta-gonad-mesonephros (AGM) and E15.5 liver with lineage commitment by Day 15. A similar biphasic expression pattern occurred for Wnt/Fzd/LRP genes with Wnt 3, 5a, 8a, Fzd4, and LRP5 becoming upregulated during primitive hematopoiesis, followed by Wnt3a, 6, 7b, 10b, and 16 during more definitive hematopoiesis. High expression of Wnt5a, Fzd4, and LRP5 during the first phase of hematopoiesis suggests these genes are involved in early hematopoietic regulation. Wnt3a and 16 were also expressed at specific stages, with Wnt16 detected when the earliest lymphoid progenitors are formed (AGM and 2 degrees BC of ES differentiation). Wnt3a expression corresponded with the induction of definitive hematopoiesis a period, which involves rapid expansion of HSC (Day 7.75 of ES differentiation, AGM and E15.5 liver). Supplementation with Wnt3a during ES hematopoietic differentiation increased proliferation and appeared to promote stem cell expansion. Overall this study provides valuable information on the Wnt/Fzd/LRP involved in supporting embryonic hematopoiesis.     

Commentary: the role of cell migration in the ontogeny of the lymphoid system

The foundations of experimental hematology were laid by histologists, and while their contributions were enormous, they were limited in their interpretation of very dynamic processes by the static nature of the methodology. The middle of the twentieth century saw the introduction of techniques for hematopoietic cell marking and development of in vitro and in vivo assays for primitive hematopoietic cells, allowing dynamic studies of hematopoiesis. Paralleling this was an understanding of cellular immunology with the discovery of the role of the thymus and the identification of T and B lymphocyte lineages. In the 1960s a series of ontogenetic studies in birds and subsequently in mice revealed that hematopoietic and lymphoid development involved migration streams of primitive cells that colonized developing primary lymphoid organs as well as spleen, marrow, and liver. The yolk sac was proposed as the ultimate origin of these lympho-hematopoietic precursors. Subsequent studies identified a region associated with the dorsal aorta as the primary site of "definitive" stem cells. These opposing views are currently achieving a compromise that recognizes that both sites contribute stem cells involved in seeding the developing tissues. The clear distinction between the local origin of the inducing microenvironment provided by the endoderm or by stroma derived from mesenchymal stem cells of mesodermal origin, and the immigrant origin of the hematopoietic stem cells and progenitors, raises intriguing questions in the current climate of stem cell plasticity, cell fusion, and discovery of stem cells in adult marrow with the capacity to generate hematopoiesis as well as other mesodermal, ectodermal, and endodermal lineages.     

小鼠胚胎早期血细胞发生的研究进展

根据时空上的不同,小鼠胚胎发育时期的血细胞发生目前被分为原始造血、红系/髓系祖细胞（EMPs）的产生和造血干细胞（HSCs）成熟并分化为各种血细胞3个阶段。最新的观点也把原始造血和EMPs的产生归结为非HSCs依赖性的血细胞谱系分化阶段,将第3阶段称为HSCs依赖性的血细胞谱系分化阶段。尽管胚胎时期的血细胞发生涉及多个造血器官,但本文中我们主要对近年在细胞和分子水平进行的造血细胞和HSCs在卵黄囊和腹主动脉-性腺-中肾（AGM）区发育的研究进行归纳总结,以此展示新近发现的胚胎发育早期血细胞发生的特点及其潜在机制。     

Stromal-derived factor-1/CXCR4 signaling: indispensable role in homing and engraftment of hematopoietic stem cells in bone marrow

Homing and engraftment of hematopoietic stem/progenitor cells (HSPCs) in bone marrow is the major determining factor in success of hematopoietic stem cell transplantation. This is a complex, multistep process orchestrated by the coordinated interplay between adhesion molecules, cytokines, growth factors, and regulatory cofactors, many of which remain to be defined. Recent studies have highlighted the pivotal role of unique stromal-derived factor-1 (SDF-1)/CXCR4 signaling in the regulation of HSPC homing and subsequent engraftment. In addition, studies suggest that SDF-1/CXCR4 signaling acts as an essential survival-promoting factor of transplanted HSPCs as well as maintenance of quiescent HSCs in bone marrow niche. These pleiotropic effects exerted by SDF-1/CXCR4 axis make this unique signaling initiator very promising, not only for optimal hematopoietic reconstitution but also for the development of innovative approaches to achieve restoration, regeneration, or repair of other damaged tissues potentially amendable to reversal by stem cell transplantation. This goal can only be achieved when the role of SDF-1/CXCR4 axis in hematopoietic transplantation is clearly defined. Hence, this review presents current knowledge of the mechanisms through which SDF-1/CXCR4 signaling promotes restoration of hematopoiesis by regulating the homing and engraftment of HSPCs.     

Distinctive contact between CD34+ hematopoietic progenitors and CXCL12+ CD271+ mesenchymal stromal cells in benign and myelodysplastic bone marrow

Mesenchymal stromal cells (MSCs) support hematopoiesis and are cytogenetically and functionally abnormal in myelodysplastic syndrome (MDS), implying a possible pathophysiologic role in MDS and potential utility as a diagnostic or risk-stratifying tool. We have analyzed putative MSC markers and their relationship to CD34+ hematopoietic stem/progenitor cells (HSPCs) within intact human bone marrow in paraffin-embedded bone marrow core biopsies of benign, MDS and leukemic (AML) marrows using tissue microarrays to facilitate scanning, image analysis and quantitation. We found that CD271+, ALP+ MSCs formed an extensive branching perivascular, periosteal and parenchymal network. Nestin was brightly positive in capillary/arteriolar endothelium and occasional subendothelial cells, whereas CD146 was most brightly expressed in SMA+ vascular smooth muscle/pericytes. CD271+ MSCs were distinct by double immunofluorescence from CD163+ macrophages and were in close contact with but distinct from brightly nestin+ and from brightly CD146+ vascular elements. Double immunofluorescence revealed an intimate spatial relationship between CD34+ HSPCs and CD271+ MSCs; remarkably, 86% of CD34+ HSPCs were in direct contact with CD271+ MSCs across benign, MDS and AML marrows, predominantly in a perivascular distribution. Expression of the intercrine chemokine CXCL12 was strong in the vasculature in both benign and neoplastic marrow, but was also present in extravascular parenchymal cells, particularly in MDS specimens. We identified these parenchymal cells as MSCs by ALP/CXCL12 and CD271/CXCL12 double immunofluorescence. The area covered by CXCL12+ ALP+ MSCs was significantly greater in MDS compared with benign and AML marrow (P=0.021, Kruskal-Wallis test). The preservation of direct CD271+ MSC/CD34+ HSPC contact across benign and neoplastic marrow suggests a physiologically important role for the CD271+ MSC/CD34+ HSPC relationship and possible abnormal exposure of CD34+ HSPCs to increased MSC CXCL12 expression in MDS.     

MT1-MMP and RECK: opposite and essential roles in hematopoietic stem and progenitor cell retention and migration

Migratory capacity is a fundamental property of hematopoietic stem and progenitor cells (HSPCs). This feature is employed in clinical mobilization of HSPCs to the circulation and constitutes the basis for modern bone marrow (BM) transplantation procedures which are routinely used to treat hematological malignancies. Therefore, characterization of new players in the complex process of HSPC motility in steady-state conditions as well as during stress situations is a major challenge. We report that while the metalloproteinase membrane type 1-metalloprotease (MT1-MMP) has an essential role in human HSPC trafficking during granulocyte colony-stimulating factor (G-CSF)-induced mobilization, its inhibitor reversion-inducing cysteine-rich protein with Kazal motifs (RECK) and the adhesion molecule CD44 are required for HSPC retention to the BM in steady-state conditions. The nervous system via Wnt signaling along with HGF/c-Met signaling and the complement cascade play a major role in regulating MT1-MMP increased activity, CD44 cleavage, and RECK-reduced expression during G-CSF-induced mobilization. This review will elaborate on the opposite roles of MT1-MMP and RECK in HSPC migration and retention and suggest targeting them in order to facilitate HSPC mobilization and engraftment upon BM transplantation in patients.