Bone Marrow Hematopoietic Niche Research Articles

Inducible CXCL12/CXCR4–dependent extramedullary hematopoietic niches in the adrenal gland

AbstractAdult hematopoietic stem and progenitor cells (HSPCs) reside in the bone marrow (BM) hematopoietic niche, which regulates HSPC quiescence, self-renewal, and commitment in a demand-adapted manner. Although the complex BM niche is responsible for adult hematopoiesis, evidence exists for simpler, albeit functional and more accessible, extramedullary hematopoietic niches. Inspired by the anecdotal description of retroperitoneal hematopoietic masses occurring at higher frequency upon hormonal dysregulation within the adrenal gland, we hypothesized that the adult adrenal gland could be induced into a hematopoietic-supportive environment in a systematic manner, thus revealing mechanisms underlying de novo niche formation in the adult. Here, we show that upon splenectomy and hormonal stimulation, the adult adrenal gland of mice can be induced to recruit and host functional HSPCs, capable of serial transplantation, and that this phenomenon is associated with de novo formation of platelet-derived growth factor receptor α/leptin receptor (PDGFRα+/LEPR+/–)–expressing stromal nodules. We further show in CXCL12–green fluorescent protein reporter mice that adrenal glands contain a stromal population reminiscent of the CXCL12-abundant reticular cells, which compose the BM HSPC niche. Mechanistically, HSPC homing to hormonally induced adrenal glands was found dependent on the CXCR4–CXCL12 axis. Mirroring our findings in mice, we found reticular CXCL12+ cells coexpressing master niche regulator FOXC1 in primary samples from human adrenal myelolipomas, a benign tumor composed of adipose and hematopoietic tissue. Our findings reignite long-standing questions regarding hormonal regulation of hematopoiesis and provide a novel model to facilitate the study of adult-specific inducible hematopoietic niches, which may pave the way to therapeutic applications.

Bone Marrow Stroma Co-cultivation Model of Breast Cancer Dormancy.

Breast cancer cells metastasize to the bone marrow before a primary tumor is detected. Most micrometastases die in this hostile microenvironment, but some survive and enter a state of dormancy and chemoresistance due to their close interaction with cells in the bone marrow hematopoietic niche. Over many years, some of the cells reawaken and result in metastatic disease that cannot be cured. Analyzing the cellular and molecular interactions between cancer and bone marrow niche cells requires relevant models that can be manipulated and studied. Generation of bone marrow stromacultures in vitro permits the formation of cellular monolayers upon which breast cancer cells can be co-cultivated and their behavior interrogated using a variety of techniques. This manuscript describes the basic techniques for generating bone marrow stromal monolayers, co-incubating cancer cells and determining the effects on cancer cell proliferation and molecular signaling.

The effect of platelet infusion on the repair of bone marrow hematopoietic niche damage in mice induced by (60)Co radiation and hematopoietic reconstruction after bone marrow transplantation

Objective: To observe the effect of platelets on hematopoietic stem cell (HSCs) implantation in mice with radiation-induced bone marrow injury and bone marrow transplantation models. Methods: ①Male C57BL/6 mice were divided into a single irradiation group and a radiation infusion group after receiving (60)Co semimyeloablative irradiation for 18-10 weeks. The irradiation infusion group received 1×10(8) platelets expressing GFP fluorescent protein. ② The allogeneic bone marrow transplantation model was established. The experimental groups included the simple transplantation group (BMT) and the transplantation infusion group (BMT+PLT). The BMT group was infused through the tail vein only 5 × 10(6) bone marrow cells, the BMT+PLT group needs to be infused with bone marrow cells at the same time 1× 10(8) platelets. ③ Test indicators included peripheral blood cell and bone marrow cell counts, flow cytometry to detect the proportion of hematopoietic stem cell (HSC) and hematopoietic progenitor cells, bone marrow cell proliferation and apoptosis, and pathological observation of vascular niche damage and repair. Results: ①On the 3rd, 7th, 14(th), and 21st days after irradiation, the bone marrow cell count of the infusion group was higher than that in the single irradiation group (P<0.05), and the peripheral blood cell count was also higher. A statistically significant difference was found between the white blood cell count on the 21st day and the platelet count on the 7th day (P<0.05). In the observation cycle, the percentage of bone marrow cell proliferation in the infusion group was higher, while the percentage of apoptosis was lower. ② The results of bone tissue immunofluorescence after irradiation showed that the continuity of hematopoietic niche with red fluorescence was better in the irradiation infusion group. ③The chimerism percentage in the BMT+PLT group was always higher than that in the BMT group after transplantation.④ The BMT+PLT group had higher bone marrow cell count and percentage of bone marrow cell proliferation on the 7th and 28th day after transplantation than that in the BMT group, and the percentage of bone marrow cell apoptosis on the 14th day was lower than that in the BMT group (P<0.05). After the 14th day, the percentage of stem progenitor cells in the bone marrow cells of mice was higher than that in the BMT group (P<0.05). ⑤The immunohistochemical results of bone marrow tissue showed that the continuity of vascular endothelium in the BMT+PLT group was better than that in the BMT group. Conclusion: Platelet transfusion can alleviate the injury of vascular niche, promotes HSC homing, and is beneficial to hematopoietic reconstruction.

Young donor hematopoietic stem cells revitalize aged or damaged bone marrow niche by transdifferentiating into functional niche cells.

The bone marrow niche maintains hematopoietic stem cell (HSC) homeostasis and declines in function in the physiologically aging population and in patients with hematological malignancies. A fundamental question is now whether and how HSCs are able to renew or repair their niche. Here, we show that disabling HSCs based on disrupting autophagy accelerated niche aging in mice, whereas transplantation of young, but not aged or impaired, donor HSCs normalized niche cell populations and restored niche factors in host mice carrying an artificially harassed niche and in physiologically aged host mice, as well as in leukemia patients. Mechanistically, HSCs, identified using a donor lineage fluorescence-tracing system, transdifferentiate in an autophagy-dependent manner into functional niche cells in the host that include mesenchymal stromal cells and endothelial cells, previously regarded as "nonhematopoietic" sources. Our findings thus identify young donor HSCs as a primary parental source of the niche, thereby suggesting a clinical solution to revitalizing aged or damaged bone marrow hematopoietic niche.

Anoxia Rapidly Induces Changes in Expression of a Large and Diverse Set of Genes in Endothelial Cells

Sinusoidal endothelial cells are the predominant vascular surface of the bone marrow and constitute the functional hematopoietic niche where hematopoietic stem and progenitor cells receive cues for self-renewal, survival, and differentiation. In the bone marrow hematopoietic niche, the oxygen tension is usually very low, and this condition affects stem and progenitor cell proliferation and differentiation and other important functions of this region. Here, we have investigated in vitro the response of endothelial cells to a marked decrease in O2 partial pressure to understand how the basal gene expression of some relevant biological factors (i.e., chemokines and interleukins) that are fundamental for the intercellular communication could change in anoxic conditions. Interestingly, mRNA levels of CXCL3, CXCL5, and IL-34 genes are upregulated after anoxia exposure but become downmodulated by sirtuin 6 (SIRT6) overexpression. Indeed, the expression levels of some other genes (such as Leukemia Inhibitory Factor (LIF)) that were not significantly affected by 8 h anoxia exposure become upregulated in the presence of SIRT6. Therefore, SIRT6 mediates also the endothelial cellular response through the modulation of selected genes in an extreme hypoxic condition.

Экспрессия генов семейства WNT у больных множественной миеломой с различным ответом на противоопухолевую терапию

Aim. To compare the expression levels of the WNT family genes in mesenchymal stromal cells (MSC) of the bone marrow (BM) hematopoietic niche in multiple myeloma (MM) patients vs. healthy donors.&#x0D; Materials &amp; Methods. The study enrolled 12 MM patients aged 49–71 years (the median age 61 years) after standard induction bortezomib therapy. The treatment efficacy was assessed in accordance with the criteria of International Myeloma Working Group (IMWG). Patients were stratified in groups with complete and partial response (CPR; group 1, n = 9) and no response (group 2, n = 3). Besides, a group of primary untreated patients was formed (n = 2). The control group included healthy donors of BM (n = 3). The levels of the WNT and CTNNB1 gene expression were assessed by real-time PCR on cDNA isolated from MSC.&#x0D; Results. In the group of 2 primary patients, two genes (WNT2B and WNT9B) considerably differed in the degree of expression. In non-responders (n = 3), the WNT2B expression could not be determined, whereas the WNT15 expression appeared to be increased. In group CPR (n = 9), mRNA level of the WNT5A gene increased after therapy, whereas the WNT3A gene expression returned to the normal level. The WNT7B gene transcription level did not differ in the control and comparison groups. In group CPR, a significant expression increase in the β-catenin-coding CTNNB1 gene was detected.&#x0D; Conclusion. The differences identified in the expression of the WNT2B, WNT9B, and CTNNB1 genes suggest the possibility of their use as prognostic molecular markers in MM.

Frizzled-6 promotes hematopoietic stem/progenitor cell mobilization and survival during LPS-induced emergency myelopoiesis

SummaryEmergency hematopoiesis involves the activation of bone marrow hematopoietic stem/progenitor cells (HSPCs) in response to systemic inflammation by a combination of cell-autonomous and stroma-dependent signals and leads to their release from bone marrow and migration to periphery. We have previously shown that FZD6 plays a pivotal role in regulating HSPC expansion and long-term maintenance. Now we sought to better understand the underlying mechanisms. Using lipopolysaccharide (LPS)-induced emergency granulopoiesis as a model, we show that failed expansion was intrinsic to FZD6-deficient HSPCs but also required a FZD6-deficient environment. FZD6-deficient HSPCs became more strongly activated, but their mobilization to peripheral blood was impaired and they were more susceptible to inflammatory cell death, leading to enhanced release of pro-inflammatory cytokines in the marrow. These studies indicate that FZD6 has a protective effect in the bone marrow to prevent an overactive inflammatory response and further suggest that mobilization improves HSPC survival during bone marrow inflammation.

Mouse dendritic cells in the steady state: Hypoxia, autophagy, and stem cell factor.

Dendritic cells (DCs) are innate immune cells with a central role in immunity and tolerance. Under steady-state, DCs are scattered in tissues as resting cells. Upon infection or injury, DCs get activated and acquire the full capacity to prime antigen-specific CD4+ and CD8+ T cells, thus bridging innate and adaptive immunity. By secreting different sets of cytokines and chemokines, DCs orchestrate diverse types of immune responses, from a classical proinflammatory to an alternative pro-repair one. DCs are highly heterogeneous, and physiological differences in tissue microenvironments greatly contribute to variations in DC phenotype. Oxygen tension is normally low in some lymphoid areas, including bone marrow (BM) hematopoietic niches; nevertheless, the possible impact of tissue hypoxia on DC physiology has been poorly investigated. We assessed whether DCs are hypoxic in BM and spleen, by staining for hypoxia-inducible-factor-1α subunit (HIF-1α), the master regulator of hypoxia-induced response, and pimonidazole (PIM), a hypoxic marker, and by flow cytometric analysis. Indeed, we observed that mouse DCs have a hypoxic phenotype in spleen and BM, and showed some remarkable differences between DC subsets. Notably, DCs expressing membrane c-kit, the receptor for stem cell factor (SCF), had a higher PIM median fluorescence intensity (MFI) than c-kit- DCs, both in the spleen and in the BM. To determine whether SCF (a.k.a. kit ligand) has a role in DC hypoxia, we evaluated molecular pathways activated by SCF in c-kit+ BM-derived DCscultured in hypoxic conditions. Gene expression microarrays and gene set enrichment analysissupported the hypothesis that SCF had an impact on hypoxia response and inhibited autophagy-related gene sets. Our results suggest that hypoxic response and autophagy, and their modulation by SCF, can play a role in DC homeostasis at the steady state, in agreement with our previous findings on SCF's role in DC survival.

Mesenchymal Stromal Cells (MSCs): An Ally of B-Cell Acute Lymphoblastic Leukemia (B-ALL) Cells in Disease Maintenance and Progression within the Bone Marrow Hematopoietic Niche.

Simple SummaryB-cell acute lymphoblastic leukemia (B-ALL) is the most common pediatric cancer. Even though the cure rate actually exceeds 85%, the prognosis of relapsed/refractory patients is dismal. Recent literature data indicate that the bone marrow (BM) microenvironment could play a crucial role in the onset, maintenance and progression of the disease. In particular, mesenchymal stromal cells (MSCs), which are key components of the BM niche, actively crosstalk with leukemic cells providing crucial signals for their survival and resistance to therapy. We hereby review the main mechanisms exploited by MSCs to nurture and protect B-ALL cells that could become appealing targets for innovative microenvironment remodeling therapies to be coupled with classical leukemia-directed strategies.Mesenchymal stromal cells (MSCs) are structural components of the bone marrow (BM) niche, where they functionally interact with hematopoietic stem cells and more differentiated progenitors, contributing to hematopoiesis regulation. A growing body of evidence is nowadays pointing to a further crucial contribution of MSCs to malignant hematopoiesis. In the context of B-cell acute lymphoblastic leukemia (B-ALL), MSCs can play a pivotal role in the definition of a leukemia-supportive microenvironment, impacting on disease pathogenesis at different steps including onset, maintenance and progression. B-ALL cells hijack the BM microenvironment, including MSCs residing in the BM niche, which in turn shelter leukemic cells and protect them from chemotherapeutic agents through different mechanisms. Evidence is now arising that altered MSCs can become precious allies to leukemic cells by providing nutrients, cytokines, pro-survivals signals and exchanging organelles, as hereafter reviewed. The study of the mechanisms exploited by MSCs to nurture and protect B-ALL blasts can be instrumental in finding new druggable candidates to target the leukemic BM microenvironment. Some of these microenvironment-targeting strategies are already in preclinical or clinical experimentation, and if coupled with leukemia-directed therapies, could represent a valuable option to improve the prognosis of relapsed/refractory patients, whose management represents an unmet medical need.

P1399: EXTRAMEDULLARY HEMATOPOIESIS IN THE ADULT ADRENAL GLAND AS A MODEL OF THE MINIMAL HEMATOPOIETIC NICHE

Background: Hematopoietic Stem and Progenitor Cells (HSPCs) reside in the hematopoietic niche, a structure that regulates the balance of cellular quiescence, self-renewal and commitment towards progenitor cells in a demand-adapted manner. The bone marrow (BM) hematopoietic niche is formed by several cellular players, mainly endothelial cells, osteoblasts, adipocytes, and a variety of stromal cells. While the BM niche contains a complex stromal population, evidence exists for simpler, albeit functional, extramedullary hematopoietic (EMH) niches. EMH can be found associated to adipose tissue. An example of adipose-associated EMH is myelolipoma, an adrenal tumor composed of myeloid and adipose tissue. Myelolipoma is frequent in patients with congenital adrenal hyperplasia, an endocrine disorder that associates an increase in ACTH. Aims: We aimed to produce an EMH animal model that would recapitulate myelolipoma to study a minimalistic, boneless hematopoietic niche. Methods: We designed a strategy to induce EMH in the adrenal gland. For this, we injected hematopoietic cytokines (Granulocyte colony-stimulating factor (G-CSF)) and pituitary axis hormones (ACTH) as well as androgens (testosterone) in splenectomized C57BL/6J mice and studied the hematopoietic-supportive capacity of the induced adrenal glands. Results: Here, we show that the adult adrenal gland can be transformed into a hematopoietic supportive environment. Upon hormonal stimulation, the adult adrenal gland can be induced to recruit and host hematopoietic cells, including serially transplantable HSCPs. The adrenal stroma supports HSPC proliferation and function; furthermore, the adrenal stroma contains a CXCL12+ population, reminiscent of BM CXCL12-Abundant Reticular (CAR). These findings are mirrored by the presence of CXCL12+ cells in human myelolipoma samples, a benign adrenal tumor composed of adipose and hematopoietic tissue. Summary/Conclusion: We present our model as a novel tool to increase our understanding of the physiology of hematopoietic support and to facilitate the development of a boneless niche model.

TGF-β Signaling in Stromal Cells Contributes to Myelofibrosis, but Not Niche Disruption, in Myeloproliferative Neoplasms

Myeloproliferative neoplasms are associated with significant alterations in the bone marrow microenvironment that contribute to disease pathogenesis. The most striking alteration is the development of myelofibrosis, which is characterized by extensive collagen deposition in the bone marrow and is associated with a poor prognosis. Recent evidence suggests that expression of key niche factors, including CXCL12 (stromal derived factor-1, SDF-1) and Kit ligand are reduced in MPNs. This is relevant, since studies by our group and others have shown that deleting these niche factors from stromal cells results in a shift in hematopoiesis from the bone marrow to spleen. Indeed, a prominent feature of MPN is the development of splenomegaly and extramedullary hematopoiesis. There is evidence implicating inflammatory mediators in the development of myelofibrosis. In particular, increased production of TGF-β produced by megakaryocytes and monocytes is found in most patients with MPNs. To assess the role of TGF-β signaling in mesenchymal stromal cells in the bone marrow in the development of myelofibrosis, we generated Osx-Cre; Tgfbr2 f/- mice, in which TGF-β signaling is abrogated in all bone marrow mesenchymal stromal cells (including Lepr + stromal cells), but not endothelial cells or hematopoietic cells. We transplanted MPL W515L transduced hematopoietic stem and progenitor cells (HSPCs) or JAK2 V617F bone marrow into these mice and quantified myelofibrosis using reticulin staining and Collagen 1 and 3 immunostaining. We previously reported that deletion of TGF-β signaling in mesenchymal stromal cells in these mice abrogated the development of myelofibrosis, and we presented evidence that this was mediated by non-canonical JNK-dependent TGF-β signaling. Here, we describe the impact of stromal TGF-β signaling on the bone marrow hematopoietic niche in MPN.MPL W515L transduced HSPCs were transplanted into Osx-Cre; Tgfbr2 f/- mice, and the impact on hematopoietic niche disruption and development of extramedullary hematopoiesis was assessed. In control recipients, transplantation of MPL W515L HSPCs resulted in marked decreases in bone marrow Cxcl12 and Kit ligand expression (Figure 1A-B). Surprisingly, a similar decrease was observed in Osx-Cre; Tgfbr2 f/- recipients. The loss of these key niche factors is predicted to impair hematopoietic niche function in the bone marrow. Consistent with this prediction, total bone marrow cellularity and HSC number were significantly reduced in both control and Osx-Cre; Tgfbr2 f/- recipients (Figure 1C-D). Finally, disruption of the bone marrow niche is often associated with extramedullary hematopoiesis. Indeed, a significant increase in spleen size and spleen HSCs and erythroid progenitors was observed in control recipients (Figure 1E-G). Again, a similar phenotype was observed in Osx-Cre; Tgfbr2 f/- recipients.Collectively, these data show that TGF-β signaling in bone marrow mesenchymal stromal cells is required for the development of myelofibrosis but not hematopoietic niche disruption in MPNs. Thus, these data show for the first time that the signals that induce a fibrogenic program in bone marrow mesenchymal stromal cells are distinct from those that suppress Cxcl12 and Kit ligand expression. Our data show that the fibrogenic program is dependent on non-canonical JNK-dependent TGF-β signaling, while the signals that regulate niche factor expression remain unknown. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Direct Intravital Imaging of the Bone Marrow and Splenic Hematopoietic Niches in Individual Mice to Define the Early Engraftment Kinetics Following HSC-Transplant

Hematopoietic stem cells (HSCs) give rise to and maintain the entire hematopoietic system for the life of an organism. This remarkable feat has established HSC transplant as an indispensable tool for treating a variety of hematological disorders. Yet the initial steps of homing, engraftment, and clonal expansion, which lead to eventual long-term hematopoietic recovery after HSC transplant, remain incompletely characterized. Given the determinative effect that early HSC activity has on transplant outcomes, a more complete understanding of initial engraftment dynamics is imperative for bettering HSC therapies.Preliminary studies aimed at functional characterization of classic HSC and hematopoietic stem and progenitor cell (HSPC) populations-namely the CD150 +CD48 -Sca-1 +c-Kit +Lin - (SLAM SKL) and Sca-1 +c-Kit +Lin - (SKL) populations, respectively-revealed that these populations exhibit disparate early engraftment dynamics. Using previously developed intravital imaging techniques, we were able to partially characterize early HSC and HSPC engraftment dynamics in mice competitively transplanted with GFP + SLAM SKL and DsRed + SKL cells. The SKL population was found to primarily engraft in the bone marrow, completely recapitulating engraftment behavior of transplanted whole bone marrow. In contrast, the more purified SLAM SKL population engrafted poorly in the marrow space and instead preferentially engrafted in the spleen, where it produced the majority of donor-derived blood at early stages (7 days) after competitive transplant into lethally irradiated mice. However, by 14 days post-transplant, SLAM SKL-derived cells migrated from the spleen to repopulate the majority of bone marrow space. These results reflect the dynamic nature of hematopoietic recovery in a myeloablative model and highlight the need for in vivo imaging techniques to fully understand hematopoietic reconstitution by the SLAM SKL population.In order to further dissect the interactive processes of bone marrow hematopoiesis and splenic extramedullary hematopoiesis, we have developed a novel, multi-organ intravital imaging technique that allows for simultaneous analysis of defined hematopoietic compartments in a single animal. Our multimodal imaging approach combines direct visualization of fluorescently labeled hematopoietic cells in the spleen via our recently developed spleen window, with concomitant observation of hematopoietic cells in tibia marrow environment. Our spleen window is a specially engineered biocompatible ring with an affixed coverslip to allow for direct, non-invasive microscopic visualization of labeled hematopoietic cells in the spleen. The spleen window can be installed with the tibia window in an individual mouse. Multimodal mice can be visualized repeatedly over a minimum of 7 days post-HSC transplant to follow individual cell behaviors within the living recipient. Preliminary results from competitive repopulation assays utilizing our multimodal imaging approach suggest that the SLAM SKL population is an active one that confers rapid hematopoietic recovery in lethally irradiated recipients primarily from extramedullary hematopoiesis in the spleen (CFU-S). The results of ongoing work characterizing the active use of the splenic and marrow niches will be presented. DisclosuresCogle: Celgene: Membership on an entity's Board of Directors or advisory committees; Aptevo therapeutics: Research Funding.

The Therapeutic Potential of Hematopoietic Stem Cells in Bone Regeneration.

The repair process of bone fractures is a complex biological mechanism requiring the recruitment and in situ functionality of stem/stromal cells from the bone marrow (BM). BM mesenchymal stem/stromal cells have been widely explored in multiple bone tissue engineering applications, whereas the use of hematopoietic stem cells (HSCs) has been poorly investigated in this context. A reasonable explanation is the fact that the role of HSCs and their combined effect with other elements of the hematopoietic niches in the bone-healing process is still elusive. Therefore, in this review we intend to highlight the influence of HSCs in the bone repair process, mainly through the promotion of osteogenesis and angiogenesis at the bone injury site. For that, we briefly describe the main biological characteristics of HSCs, as well as their hematopoietic niches, while reviewing the biomimetic engineered BM niche models. Moreover, we also highlighted the role of HSCs in translational in vivo transplantation or implantation as promoters of bone tissue repair. Impact statement The ability of bone to natural self-heal depends on the size and stabilization level of the tissue fracture, and it is impaired in several pathophysiological conditions. Considering that the available treatment options have demonstrated limited regenerative performance, the hematopoietic stem cells (HSCs) co-cultured in different tissue engineering strategies have emerged as a powerful tool to promote effective bone regeneration and healing. Here, we reviewed the most important biomimetic bone-marrow hematopoietic niches and showed the regenerative potential of these cells, both in vitro and in translational in vivo transplantation/implantation approaches. This knowledge encourages the development of new HSC-related bone regenerative therapies.

Human Red Blood Cells Modulate Cytokine Expression in Monocytes/Macrophages Under Anoxic Conditions.

In the bone marrow (BM) hematopoietic niche, the oxygen tension is usually very low. Such condition affects stem and progenitor cell proliferation and differentiation and, at cellular level regulates hematopoietic growth factors, chemokines and adhesion molecules expression. In turn, these molecules affect the proliferation and maturation of other cellular components of the niche. Due to the complexity of the system we started the in vitro investigations of the IL-6, IL-8, TNFα cytokines expression and the vascular endothelial growth factor (VEGF), considered key mediators of the hematopoietic niche, in human macrophages and macrophage cell line. Since in the niche the oxygen availability is mediated by red blood cells (RBCs), we have influenced the anoxic cell cultures by the administration of oxygenated or deoxygenated RBCs (deoxy RBCs). The results reported in this brief paper show that the presence of RBCs up-regulates IL-8 mRNA while IL-6 and VEGF mRNA expression appears down-regulated. This does not occur when deoxy RBCs are used. Moreover, it appears that the administration of RBCs leads to an increase of TNFα expression levels in MonoMac 6 (MM6). Interestingly, the modulation of these factors likely occurs in a hypoxia-inducible factor-1α (HIF-1α) independent manner. Considering the role of oxygen in the hematopoietic niche further studies should explore these preliminary observations in more details.

Methodological considerations for the enrichment of bone marrow endothelial and mesenchymal stromal cells

Stromal cells are critical regulators of bone marrow hematopoietic niches, but assessment of their regulatory roles has been impeded by difficult and ineffective dissociation methods. Here, we methodically address bone marrow stromal cell dissociation. Yield of bone marrow CD45−/Ter119−/CD31+/CD202b+ endothelial cells (ECs) and CD45−/Ter119−/CD44−/PDGFR+ mesenchymal stromal cells (MSCs) were determined by flow cytometry. Liberase DL, Collagenase D, and Dispase II (all supplemented with DNase) enhanced EC and MSC yields, with Dispase II + DNase proving most effective. Combinations of these enzymes did not exhibit additive benefits, nor did the addition of Elastase, TrypLE, Hyaluronidase, or Accutase. Similarly, common mechanical dissociation approaches also proved ineffective. However, the combination of gentle Dispase II + DNase dissociation with magnetic sorting dramatically enriched both ECs and MSCs. This work methodically addressed common approaches for bone marrow stromal dissociation and established an effective approach for enrichment.

G-CSF treatment of healthy pediatric donors affects their hematopoietic microenvironment through changes in bone marrow plasma cytokines and stromal cells

Although G-CSF mobilized peripheral blood stem cell (PBSC) transplantation is commonly used in adults, bone marrow (BM) is still the preferred stem cell source in pediatric stem cell transplantation. Despite the fact that G-CSF is increasingly being used to enhance the hematopoietic stem/progenitor cell (HSPC) yield in BM transplantation (G-BM), the direct effects of G-CSF on the pediatric BM microenvironment have never been investigated. The BM hematopoietic niche provides the physical space where the HSPCs reside. This BM niche regulates HSPC quiescence and proliferation through direct interactions with other niche cells, including Mesenchymal Stromal Cells (MSCs). These cells have been shown to secrete a wide range of hematopoietic cytokines (CKs) and growth factors (GFs) involved in differentiation, retention and homing of hematopoietic cells. Here, we assessed changes in the BM microenvironment by measuring levels of 48 different CKs and GFs in G-BM and control BM (C-BM) plasma from pediatric donors. In addition, the effect of G-CSF on cell numbers and characteristics of HSPCs and MSCs was assessed. IL-16, SCGF-b, MIP-1b (all >1000 pg/mL) and RANTES (>10.000 pg/mL) were highly expressed in healthy donor pediatric BM plasma. Levels of IL-3, IL-18, GROa, MCP-3 (p<0.05) were increased in G-BM, whereas levels of RANTES (p<0.001) decreased after G-CSF treatment. We found a negative correlation with increasing age for IL2-Ra and LIF (p<0.05). In addition, a concomitant increase in the number of both hematopoietic and fibroblast colony forming units was observed, indicating that G-CSF affects both HSPC and MSC numbers. In conclusion, G-CSF treatment of healthy pediatric donors affects the hematopoietic BM microenvironment by expansion of HSPC and MSC numbers and modifying local CK and GF levels.

Macrophage Regulation of Granulopoiesis and Neutrophil Functions.

Significance: Neutrophils are potent effector cells of innate immunity requiring precise regulation of their numbers and functions in blood and tissues. Recent Advances: Macrophages have emerged as modulators of neutrophil properties. In inflammatory conditions, tissue macrophages modulate neutrophil trafficking and activation. Further, macrophages govern granulopoiesis in the bone marrow hematopoietic niche. Interactions of macrophages and neutrophils can be induced by cytokines and damage-associated molecular patterns, and they are also regulated by oxidative signaling. Critical Issues: We review the impact of macrophages on neutrophil development and function, and its consequences in health and disease. Future Directions: Targeting the liaison between macrophages and neutrophils might provide an interesting therapeutic strategy to reduce tissue inflammation and promote immune tolerance. Antioxid. Redox Signal. 35, 182-191.

Inferring Gene Networks in Bone Marrow Hematopoietic Stem Cell-Supporting Stromal Niche Populations.

SummaryThe cardinal property of bone marrow (BM) stromal cells is their capacity to contribute to hematopoietic stem cell (HSC) niches by providing mediators assisting HSC functions. In this study we first contrasted transcriptomes of stromal cells at different developmental stages and then included large number of HSC-supportive and non-supportive samples. Application of a combination of algorithms, comprising one identifying reliable paths and potential causative relationships in complex systems, revealed gene networks characteristic of the BM stromal HSC-supportive capacity and of defined niche populations of perivascular cells, osteoblasts, and mesenchymal stromal cells. Inclusion of single-cell transcriptomes enabled establishing for the perivascular cell subset a partially oriented graph of direct gene-to-gene interactions. As proof of concept we showed that R-spondin-2, expressed by the perivascular subset, synergized with Kit ligand to amplify ex vivo hematopoietic precursors. This study by identifying classifiers and hubs constitutes a resource to unravel candidate BM stromal mediators.

Canonical signaling by TGF family members in mesenchymal stromal cells is dispensable for hematopoietic niche maintenance under basal and stress conditions.

Mesenchymal stromal cells are an important component of the bone marrow hematopoietic niche. Prior studies showed that signaling from members of the transforming growth factor (TGF) superfamily in mesenchymal stromal cells is required for normal niche development. Here, we assessed the impact of TGF family signaling on niche maintenance and stress responses by deleting Smad4 in mesenchymal stromal cells at birth, thereby abrogating canonical TGF signaling. No alteration in the number or spatial organization of CXCL12-abundant reticular (CAR) cells, osteoblasts, or adipocytes was observed in Osx-Cre, Smad4fl/fl mice, and expression of key niche factors was normal. Basal hematopoiesis and stress erythropoiesis responses to acute hemolytic anemia were normal. TGF-β potently inhibits stromal CXCL12 expression in vitro; however, G-CSF induced decreases in bone marrow CXCL12 expression and subsequent hematopoietic stem/progenitor cell mobilization were normal in Osx-Cre, Tgfbr2fl/fl mice, in which all TGF-β signaling in mesenchymal stromal is lost. Finally, although a prior study showed that TGF-β enhances recovery from myeloablative therapy, hematopoietic recovery following single or multiple doses of 5-flurauracil were normal in Osx-Cre, Tgfbr2fl/fl mice. Collectively, these data suggest that TGF family member signaling in mesenchymal stromal cells is dispensable for hematopoietic niche maintenance under basal and stress conditions.

The Posterior Signaling Center Is an Important Microenvironment for Homeostasis of the Drosophila Lymph Gland.

Hematopoiesis is a necessary process for development and immune defense in Drosophila from the embryonic period to adulthood. There are two main stages in this process: the first stage occurs in the head mesoderm during the embryonic stage, and the second occurs in a specialized hematopoietic organ along the dorsal vessel, the lymph gland, during the larval stage. The lymph gland consists of paired lobes, each of which has distinct regions: the cortical zone (CZ), which contains mature hemocytes; the medullary zone (MZ), which contains hematopoietic progenitors; and the posterior signaling center (PSC), which specifically expresses the early B-cell factor (EBF) transcription factor Collier (Col) and the HOX factor Antennapedia (Antp) to form a microenvironment similar to that of the mammalian bone marrow hematopoietic stem cell niche. The PSC plays a key role in regulating hematopoietic progenitor differentiation. Moreover, the PSC contributes to the cellular immune response to wasp parasitism triggered by elevated ROS levels. Two recent studies have revealed that hematopoietic progenitor maintenance is directly regulated by Col expressed in the MZ and is independent of the PSC, challenging the traditional model. In this review, we summarize the regulatory networks of PSC cell proliferation, the controversy regarding PSC-mediated regulation of hematopoietic progenitor differentiation, and the wasp egg infection response. In addition, we discuss why the PSC is an ideal model for investigating mammalian hematopoietic stem cell niches and leukemia.