Endothelial JAK3 Expression Enhances Pro-Hematopoietic Angiocrine Function of Sinusoidal Endothelial Cells

Unlike Jak1, Jak2, and Tyk2, Jak3 is the only member of the Jak family of secondary messengers that signals exclusively by binding the common gamma chain of interleukin receptors IL2, IL4, IL7, IL9, IL15, and IL21. Jak3-null mice display defective T and NK cell development, which results in a mild SCID phenotype. Still, functional Jak3 expression outside the hematopoietic system remains unreported. Our data show that Jak3 is expressed in endothelial cells across hematopoietic and non-hematopoietic organs, with heightened expression in the bone marrow and spleen. Increased arterial zonation in the bone marrow of Jak3-null mice further suggests that Jak3 is a marker of sinusoidal endothelium, which is confirmed by fluorescent microscopy staining and single-cell RNA-sequencing. We also show that the Jak3-null niche is deleterious for the maintenance of long-term repopulating hematopoietic stem and progenitor cells (LT-HSCs) and that Jak3-overexpressing endothelial cells have increased potential to expand LT-HSCs in vitro. In addition, we identify the soluble factors downstream of Jak3 that provide endothelial cells with this functional advantage and show their localization to the bone marrow sinusoids in vivo. Our work serves to identify a novel function for a non-promiscuous tyrosine kinase in the bone marrow vascular niche and further characterize the hematopoietic stem cell niche of sinusoidal endothelium. Disclosures No relevant conflicts of interest to declare.

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An In Vivo Functional Screen Identifies miRNA-150 As a Regulator of Hematopoietic Regeneration Post Chemotherapeutic Injury

Blood ◽

10.1182/blood.v118.21.2333.2333 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2333-2333

Author(s):

Brian D. Adams ◽

Shangqin Guo ◽

Haitao Bai ◽

Changchun Xiao ◽

E. Premkumar Reddy ◽

...

Keyword(s):

Bone Marrow ◽

Conflicts Of Interest ◽

Negative Regulator ◽

Post Transplantation ◽

Hematopoietic Stem ◽

Hematopoietic Recovery ◽

Expression Construct

Abstract Abstract 2333 . MicroRNAs are important regulators of many hematopoietic processes, yet little is known with regard to the role of microRNAs in controlling normal hematopoietic regeneration. The most common methodology for in vivo microRNA studies follows a hypothesis-driven candidate approach. Here, we report the establishment of an unbiased, in vivo, microRNA gain-of-function screen, and the identification of miR-150 as a negative regulator of hematopoietic recovery post chemotherapeutic challenge. Specifically, a retroviral-library consisting of 135 hematopoietic-expressed microRNAs was generated, with each expression construct containing a barcode sequence that can be specifically recognized using a novel bead-based platform. Hematopoietic-stem-and-progenitor-cell (HSPC)-enriched wild-type bone marrow was transduced with this library and transplanted into lethally-irradiated recipients. Analysis of peripheral blood samples from each recipient up to 11 weeks post transplantation revealed that 87% of the library barcodes are reliably detected. To identify microRNAs that regulate hematopoietic regeneration after chemotherapy-induced injury, we measured the change in barcode abundance for specific microRNA constructs after 5-fluorouracil (5-FU) challenge. Notably, a small number of barcodes were consistently depleted in multiple recipient mice after treatment. Among the top hits was the miR-150-associated barcode, which was selected for further experimentation. Indeed, overexpression of miR-150 in a competitive environment resulted in significantly lower recovery rates for peripheral myeloid and platelet populations after 5-FU treatment, whereas the effects on B- and T-cells were milder. Furthermore, full recovery of these cell populations did not occur until ∼12 weeks after treatment, suggesting the involvement of HSPCs and/or common lineage progenitors. Conversely, knocking out miR-150 led to an opposite phenotype, with platelets and myeloid cells displaying faster recovery in both competitive and non-competitive settings. Interestingly, we could not observe the described effects of miR-150 in bone marrow primary cell cultures, suggesting that such effects cannot be recapitulated in vitro. Overall, these data indicate that miR-150 is a novel regulator of hematopoietic recovery after chemotherapeutic-induced injury, and highlight the important role of microRNAs in the intrinsic wiring of the hematopoietic regeneration program. Our experiments also demonstrate the feasibility and power of functional in vivo screens for studying normal hematopoietic functions, which can become an important tool in the hematology field. Disclosures: No relevant conflicts of interest to declare.

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Primary Bone Marrow-Derived MSC Subsets Have Distinct Wnt-Signatures Compared to Conventionally Cultured MSC and Differ in Their Capacity to Support Hematopoiesis in Vitro,

Blood ◽

10.1182/blood.v118.21.3414.3414 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3414-3414 ◽

Cited By ~ 1

Author(s):

Marijke W Maijenburg ◽

Marion Kleijer ◽

Kim Vermeul ◽

Erik P.J. Mul ◽

Floris P.J. van Alphen ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Wnt Signaling ◽

Mononuclear Cells ◽

Conflicts Of Interest ◽

Cell Types ◽

Hematopoietic Stem ◽

Wnt Proteins

Abstract Abstract 3414 Mesenchymal stromal cells (MSC) are of promising therapeutic use to suppress immunogenic responses following transplantation, and to support expansion of hematopoietic stem- and progenitors cells (HSPC) from small transplants derived for instance from cord blood. Culture-expanded MSC produce a wide variety and quantity of Wnt-proteins and the crucial role of Wnt-signaling in the hematopoietic stem cell niche is well established. However, studies addressing Wnt-signaling in MSC have (i) only focused on culture-expanded MSC and (ii) did not discriminate between phenotypically distinct subpopulations which are present in bulk cultures of expanded MSC. Recently we identified three new subpopulations of MSC in human bone marrow (BM) based on expression of CD271 and CD146: CD271brightCD146−, CD271brightCD146+, CD271−CD146+. These fractions co-express the “classical” MSC markers CD90 and CD105 and lack expression of CD45 and CD34 (Maijenburg et al, Blood 2010, 116, 2590). We and others demonstrated that the adult BM-derived CD271brightCD146− and CD271brightCD146+ cells contain all colony forming units-fibroblasts (Maijenburg et al, Blood 2010, 116, 2590; Tormin et al, Blood 2010, 116, 2594). To investigate how these primary subsets functionally compare to conventional, culture-expanded MSC, we investigated their Wnt-signature and hematopoietic support capacity. To this end, we sorted CD271brightCD146− and CD271brightCD146+ cells from human adult BM (n=3) and compared their Wnt-signatures obtained by Wnt-PCR array to the profiles from cultured MSC from the same donors. Fifteen genes were consistently differentially expressed in the two sorted uncultured subsets compared to their conventionally cultured counterparts. Expression of CCND1, WISP1 and WNT5B was strongly increased, and WNT5A was only detected in the conventionally cultured MSC. In contrast, WNT3A was exclusively expressed by sorted primary CD271brightCD146− and CD271brightCD146+ cells, that also expressed higher levels of JUN, LEF1 and WIF1. The differences in Wnt (target)-gene expression between CD271brightCD146− and CD271brightCD146+ cells were more subtle. The Wnt-receptors LRP6 and FZD7 were significantly higher expressed in CD271brightCD146+ cells, and a trend towards increased expression in the same subset was observed for CTNNB1, WNT11 and MYC. When the sorted subsets were cultured for 14 days (one passage), the differences in Wnt-related gene expression between the subsets was lost and the expanded sorted cells acquired an almost similar Wnt-signature as the MSC cultured from BM mononuclear cells from the same donors. The cultured subsets lost the expression of Wnt3a and gained the expression of Wnt5a, similar to the unsorted MSC cultured from the same donors in parallel. Despite the loss of a distinct Wnt-signature, co-culture experiments combining the sorted MSC subsets with human HSPC revealed that CD271brightCD146+ cells have a significantly increased capacity to support HSPC in short-term co-cultures (2 weeks) compared to CD271brightCD146− cells (p<0.021, n=3), which was analyzed in hematopoietic colony assays following co-culture. In contrast, a trend towards better long-term hematopoietic support (co-culture for 6 weeks) was observed on CD271brightCD146− cells. In conclusion, we demonstrate for the first time that primary sorted uncultured MSC subsets have a distinct Wnt-signature compared to cultured unsorted MSC and display differences in hematopoietic support. As it was recently shown that CD271brightCD146− and CD271brightCD146+ MSC localize to separate niches in vivo (Tormin et al, Blood 2011), our data indicate that the two MSC subsets are not necessarily distinct cell types and that the different Wnt-signature may be a reflection of these distinct microenvironments. Cell culturing for only one passage dramatically changed the Wnt-signature of the sorted MSC subsets, indicating that Wnt-signaling in in vitro expanded MSC does not resemble the Wnt-signature in their tissue resident counterparts in vivo. Disclosures: No relevant conflicts of interest to declare.

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Human Cytomegalovirus Selectively Suppresses the Megakaryo/Thrombopoiesis of PDGFR+ and αvβ3+ Megakaryocytes Via the TPO/c-Mpl Pathway after Allo-HSCT

Blood ◽

10.1182/blood-2020-137115 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 25-25

Author(s):

Feng-qi Liu ◽

Fei-er Feng ◽

Gao-chao Zhang ◽

Yan Su ◽

Xue-yan Sun ◽

...

Keyword(s):

Bone Marrow ◽

Human Cytomegalovirus ◽

Hcmv Infection ◽

Conflicts Of Interest ◽

Antiviral Treatment ◽

Infection Model ◽

Hematopoietic Stem ◽

The Impact

Introduction Virus-induced thrombocytopenia is a severe complication in immunocompromised hosts. Among patients following allogeneic hematopoietic stem cell transplantation (allo-HSCT), human cytomegalovirus (HCMV) infection contributes to a variety of end-organ diseases and hematological complications, leading to increased mortality. Even with antiviral treatment, HCMV remains a potentially lethal infection due to the lack of understanding of the underlying mechanisms of host-virus interactions. The key to solving this problem is to identify the factors that predispose patients to HCMV infection and carry out targeted therapy. Here, we investigated the megakaryo/thrombopoiesis process, including the thrombopoietin (TPO)/c-Mpl pathway, after HCMV infection in vivo and in vitro, screened for susceptible subsets of megakaryocytes (MKs) and explored novel therapeutic targets for HCMV infection. Methods To test whether thrombocytopenia induced by HCMV results from an impaired megakaryo/thrombopoiesis process, we studied the impact of HCMV in an in vivo model of HCMV DNAemia patients following allo-HSCT and an in vitro model of bone marrow CD34+-derived MKs infected with serum from HCMV DNAemia patients. Forty patients who had received allo-HSCT were enrolled in this study, among whom 18 recipients had HCMV DNAemia and 22 were HCMV negative, and bone marrow-derived mononuclear cells (MNCs) from patients were tested for CD41, vWF, pp65, c-Mpl, PDGFR, αvβ3 and TLR2 using flow cytometry (FCM). Transmission electron microscopy (TEM) was used to detect HCMV capsids inside MKs. Cell apoptosis was measured by Annexin V. MK ploidy was determined by FCM for propidium iodide (PI) staining. Finally, inhibitors of PDGFR (IMC-3G3 and Gleevec), αvβ3 and TLR2 were cocultured with MKs. Results Our data showed that pp65+ cells accounted for 40.59±6.12% of total CD41+vWF+ MKs from HCMV DNAemia patients, and there was a significant increase in the expression of αvβ3, PDGFR and TLR2 in pp65+ MKs compared with that in control patients. Furthermore, the percentage of PDGFR+αvβ3+ MKs emerged as an independent factor associated with HCMV infection in multivariate analysis (p = 0.008). MKs in HCMV-infected patients showed increased apoptosis and necrosis and different patterns of MK ploidy distribution compared with those in HCMV-negative patients, with a decreased proportion from 16N to 64N and a peak at 8N. Meanwhile, the expression of TPO receptor c-Mpl was lower in pp65+ MKs from HCMV DNAemia patients (0.77±0.38% in pp65+ MKs from HCMV DNAemia patients, 1.75±0.40% in pp65- MKs from HCMV DNAemia patients, 1.97±0.67% in MKs from HCMV-negative patients, and 2.06±0.29% in MKs from healthy controls, p<0.01) while the TPO level in serum was increased compared with that in controls. Next, we established an in vitro HCMV infection model of CD34+-derived MKs with serum from HCMV DNAemia patients, and the laboratory HCMV strain Towne was used as a positive control. After 9 days of coculturing, the viral capsids of HCMV were observed in the nuclei of MKs (Figure 1A), and HCMV infection increased the apoptosis of MKs and shifted them to low ploidy, with a significant decrease in platelet release. As with the in vivo results, c-Mpl was downregulated in HCMV-infected MKs. The expression levels of PDGFR, TLR2 and αvβ3 on MKs were increased in coculture with HCMV DNAemia serum, and pp65-positive MKs were decreased compared with the control after treatment with inhibitors of PDGFR and αvβ3 (Figure 1B). However, neither Gleevec nor anti-TLR2 altered the HCMV infection rate. Conclusions Our study showed that HCMV could impair megakaryopoiesis throughout maturation, apoptosis, and platelet generation via the TPO/c-Mpl pathway both in vivo and in vitro. MKs with PDGFR+ and αvβ3+ phenotypes are susceptible to HCMV infection and we proposed PDGFR and αvβ3 inhibitors as potential therapeutic alternatives for allo-HSCT patients with HCMV infection. Disclosures No relevant conflicts of interest to declare.

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SDF-1 Acutely Promotes the Physical Association of Megakaryocytes with Vascular Endothelium in the Bone Marrow and Increases the Number of Circulating Platelets.

Blood ◽

10.1182/blood.v120.21.2306.2306 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2306-2306

Author(s):

Lisa M Niswander ◽

Jennifer L McLaughlin ◽

Anne D Koniski ◽

Kathleen E McGrath ◽

James Palis

Keyword(s):

Bone Marrow ◽

Endothelial Cells ◽

Single Dose ◽

Conflicts Of Interest ◽

Nature Medicine ◽

Platelet Response ◽

Platelet Counts ◽

Vascular Niche ◽

Sinusoidal Endothelium

Abstract Abstract 2306 Thrombocytopenia complicates many diseases and can be a life-threatening consequence of genotoxic treatments including chemotherapy and radiation therapy. It is well established that thrombopoiesis occurs in the bone marrow where mature megakaryocyte (MK) precursor cells associate with sinusoidal endothelial cells and extrude pro-platelets into the vasculature. There has been much interest in elucidating mechanisms that control megakaryopoiesis and in utilizing these pathways to increase platelet output. The leading paradigm of megakaryopoiesis centers on the ability of cytokines, chiefly thrombopoietin (TPO), to promote MK progenitor proliferation and MK precursor maturation. More recently, attention has been focused on the ability of the bone marrow microenvironment to promote MK maturation and platelet formation. The chemokine stromal-derived factor-1 (SDF-1, also known as CXCL12), signaling through its receptor CXCR4, is implicated in the chemotaxis of MKs toward sinusoidal vessels, and in vivo evidence demonstrates that sustained plasma elevation of SDF-1 can increase platelet counts (Avecilla et al. Nature Medicine, 2004). To more specifically determine the short-term effects of SDF-1, we injected mice with a single 400ng intravenous dose of SDF-1 and enumerated the progenitor, precursor, and platelet compartments of the MK lineage. At 24 hours, SDF-1 induced a 30% increase in platelets compared to vehicle control (p<0.05). However, MK progenitors, defined functionally by the formation of acetylcholinesterase-positive colonies in vitro, and MK precursors, enumerated by imaging flow cytometry, were both unchanged (p>0.7 and p>0.5). To quantitatively determine if SDF-1 regulates the physical interactions of MK precursors with sinusoidal endothelium, we developed a double immunohistochemistry assay using Gp1Bβ to distinguish MK precursors and MECA32 to identify vascular endothelial cells. In vehicle-treated mice, 39% of MKs in the marrow localized to the sinusoidal endothelium, and this increased to 53% 24 hours following SDF-1 treatment (p<0.01). Thus, a single dose of SDF-1 acutely increases the number of MKs in the vascular niche as well as peripheral platelet counts. Given these results, we tested whether a single dose of SDF-1 could improve thrombocytopenia in the setting of radiation-induced marrow injury. Mice were treated with SDF-1 4 days after sublethal 4Gy total body irradiation (TBI), when radiosensitive MK progenitors are drastically reduced and radioresistant MK precursors and platelets are just beginning to decline. At 5 days post-TBI (24 hours post-SDF-1), SDF-1 treatment increased the number of circulating platelets by 15% (p<0.01) as well as the percentage of MK precursors in the vascular niche by over 15% (p<0.02) without changing the total number of MK progenitors or precursors in the marrow compared to irradiated vehicle controls (p>0.4 and p>0.7). As the platelet response following SDF-1 was less robust in the setting of TBI injury, we hypothesized that SDF-1-induced thrombopoiesis may improve if the number of MK precursors available to move to the vascular niche is increased. To test this, we administered TPO and SDF-1 at 2 hours and at 4 days, respectively, following TBI. TPO treatment alone resulted in 15% more MK precursors than irradiated vehicle controls at 5 days post-TBI (p<0.02), but did not significantly change the platelet count (p>0.2). In contrast, mice receiving both TPO and SDF-1 had over 20% more platelets than irradiated vehicle controls (p<0.01) and over 12% more platelets than mice receiving TPO alone (p<0.05) with the same increase in MK precursors (p>0.9). Correcting for differences in MK precursor numbers, mice receiving TPO and SDF-1 post-TBI had 1.8-fold more MKs in the vascular niche than irradiated vehicle controls (p<0.03) and 1.4-fold more than mice treated with TPO only (p<0.02). Taken together, we provide quantitative data in support of the concept that SDF-1 acutely promotes increases in the number of MK precursors in the vascular niche as well as peripheral platelet counts, and this effect correlates with the number of MK precursors in the marrow. Therapeutic approaches combining agents that first increase MK number and secondly increase MKs poised for thrombopoiesis by localization within the vascular niche may be a novel strategy to maximally increase peripheral platelet counts. Disclosures: No relevant conflicts of interest to declare.

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Novel Method to Study Mouse Bone Marrow Endothelial Cells in Vivo and in Vitro

Blood ◽

10.1182/blood.v120.21.617.617 ◽

2012 ◽

Vol 120 (21) ◽

pp. 617-617 ◽

Cited By ~ 2

Author(s):

Yuxin Feng ◽

Ming Liu ◽

Fukun Guo ◽

Wei Liu ◽

Leesa Sampson ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Endothelial Cells ◽

Culture System ◽

Assay Method ◽

Mouse Bone Marrow ◽

Hematopoietic Stem ◽

Egfp Reporter

Abstract Abstract 617 Self-renewal, differentiation, and proliferation of hematopoietic stem cells (HSCs) and leukemia stem cells (LSCs) are maintained in a complex microenvironment of the adult bone marrow (BM). BM endothelial cells (ECs) have been proposed to be a key component of HSC and LSC niche. However, in contrast to the well-developed culture system of human ECs, current work of murine BM endothelial cells is hindered by a lack of mouse bone marrow endothelial cell primary culture and suitable assay methods to clearly define murine BMEC functionality in vivo and in vitro, which limits genetic and mechanistic studies by using mouse models. To establish an in vivo approach to study the EC function in adult mice, a strain of Tie2-CreER transgenic mice was generated to allow conditional and inducible manipulation of BMECs by Cre recombinase expression under the Tie2 promoter. In vivo lineage tracing was achieved in a Tie2-CreER/TD-tomato or -EGFP reporter mouse strain. Upon a four day Tamoxifen injection regimen, TD-tomato or EGFP reporter was readily visualized in bone marrow vasculature that colocalizes with CD31+ ECs as determined by immunostaining. FACS analysis of Tie2-CreER/EGFP reporter mice showed that the EGFP+ cells in the BM were exclusively in the CD45- VEGFR2+ and CD31+ cell fraction, with no EGFP+ cells being detectable in the CD45+ hematopoietic lineages or osteoblast/stroma cell fractions, suggesting that the Tie2-driven CreER expression is limited to the endothelial lineage in the adult BM. Next, we developed an in vitro method to culture and assay the mouse BMECs functionally. An in vitro selection process allowed us to establish a primary BM cell culture condition that permitted functional expansion and maintenance of mouse BMECs in long-term tissue culture, yielding homogenous CD45- cells expressing endothelial markers CD31, CD34 and VEGFR2. These cells formed capillary-like structures in 2-demensional and 3-demensional tubes/capillaries, and showed TD-tomato reporter color when derived from the Tamoxifen induced Tie2-CreER/TD-tomato mouse BM. They showed expected adhesion and migration activities and morphology of ECs. Lineage chasing assays using isolated CD45+ and CD45- BM cells from the Tie2-CreER/Td-tomato mice demonstrated that the BMECs in our culture system, bearing the Tie2-promoter driven TD-tomato color and CD31+ marker, were exclusively derived from CD45- non-hematopoietic lineage. Taken together, we have established a faithful assay method for studying murine BM EC functions in vivo and in vitro, allowing the tracking and genetic manipulation of adult BM ECs in mice and in culture. The method can be useful for delineating molecular and cellular mechanisms of BMEC regulation and EC-mediated BM niche function, and may have value in testing anti-angiogenic activities of anticancer drugs in animal models. Disclosures: No relevant conflicts of interest to declare.

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Endothelial Jak3 expression enhances pro-hematopoietic angiocrine function in mice

Communications Biology ◽

10.1038/s42003-021-01846-3 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

José Gabriel Barcia Durán ◽

Tyler Lu ◽

Sean Houghton ◽

Fuqiang Geng ◽

Ryan Schreiner ◽

...

Keyword(s):

Bone Marrow ◽

Endothelial Cells ◽

Cell Types ◽

Bone Marrow Niche ◽

Hematopoietic Stem ◽

Sinusoidal Endothelium ◽

Bone Marrow Vascular Niche ◽

Different Cell Types

AbstractJak3 is the only non-promiscuous member of the Jak family of secondary messengers. Studies to date have focused on understanding and targeting the cell-autonomous role of Jak3 in immunity, while functional Jak3 expression outside the hematopoietic system remains largely unreported. We show that Jak3 is expressed in endothelial cells across hematopoietic and non-hematopoietic organs, with heightened expression in the bone marrow. The bone marrow niche is understood as a network of different cell types that regulate hematopoietic function. We show that the Jak3–/– bone marrow niche is deleterious for the maintenance of long-term repopulating hematopoietic stem cells (LT-HSCs) and that JAK3-overexpressing endothelial cells have increased potential to expand LT-HSCs in vitro. This work may serve to identify a novel function for a highly specific tyrosine kinase in the bone marrow vascular niche and to further characterize the LT-HSC function of sinusoidal endothelium.

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Overexpression of the TGF-Beta Modulator .Bambi Promotes Hematopoetic Stem Cell Proliferation.

Blood ◽

10.1182/blood.v114.22.1446.1446 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1446-1446

Author(s):

Rui Mao ◽

Olga Sirin ◽

Margaret Goodell

Keyword(s):

Cell Cycle ◽

Bone Marrow ◽

Conflicts Of Interest ◽

Receptor Activation ◽

Lymphoid Cells ◽

Quiescent State ◽

Tgf Beta ◽

Hematopoietic Stem

Abstract Abstract 1446 Poster Board I-469 Hematopoietic stem cells (HSC) normally reside in a quiescent state in the bone marrow. During times of stress, HSCs are activated to begin differentiation and self-renewal, replenishing the supply of myeloid and lymphoid cells present in the blood. The mechanisms regulating this rapid activation have not been fully elucidated. We previously identified the TGF-beta modulator Bambi (BMP and activin membrane-bound inhibitor) to be upregulated four-fold in HSCs compared to differentiated cells. Bambi codes for a transmembrane pseudoreceptor that inhibits TGF-beta receptor activation. Since TGF-beta signaling has been established to be important for induction of HSC quiescence as well as cell-cycle inhibition in long-term progenitors, we hypothesize that Bambi may play an important role in the regulation of HSCs. Using a retroviral vector, we overexpressed Bambi in bone marrow cells. Overexpression of Bambi resulted in increased colony-formation in vitro when compared to control cells. Furthermore, transduced cells expressed higher levels of the cell-cycle marker Ki-67, indicating a greater proportion of cells in active stages of the cell cycle. To verify the results of these assays in vivo, bone marrow overexpressing Bambi was transplanted into lethally irradiated recipient mice. Bambi-overexpressing cells demonstrated a higher level of engraftment in all lineages than control cells at several time points, which confirms the previous in vitro data suggesting greater cell cycle activity. Moreover, we identified the pathway through which Bambi acts by monitoring the levels of phosphorylated Smad2 (pSmad2), a downstream target of TGF-beta. Overexpression of Bambi resulted in a distinctly lower level of pSmad2, which explains the cell-cycle effects seen in vivo and in vitro. These studies show that Bambi functions to promote HSC proliferation and a probable mode of action in HSCs is through decreased pSmad2 levels from inhibition of the TGF-beta pathway. Bambi has been shown to be upregulated in certain leukemias, and a more complete understanding of the mechanism through which Bambi acts will provide better opportunities for therapeutic innovation. This research was graciously funded by an NIH grant and the ASH Trainee Research Award. Disclosures No relevant conflicts of interest to declare.

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A Stable and Reproducible Xenotransplant Model for AML.

Blood ◽

10.1182/blood.v114.22.1005.1005 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1005-1005

Author(s):

Muriel Malaise ◽

Konstanze Doehner ◽

Dirk Reinhardt ◽

Klaus-Michael Debatin ◽

Selim Corbacioglu

Keyword(s):

Bone Marrow ◽

Peripheral Blood ◽

Conflicts Of Interest ◽

Chromosome 17 ◽

Organ Distribution ◽

Positive Staining ◽

Hematopoietic Stem ◽

Median Delay

Abstract Abstract 1005 Poster Board I-27 Background: Xenotransplant models are invaluable tools to generate an unlimited source for in vivo propagation and extensive in vitro studies through consecutive passages of reproducibly stable supply. In vivo analyses of the pathogenetic relevance of these and other unidentified targets is of importance for the development of molecular targeted drug regimens. Whereas in ALL NOD/SCID based xenotransplant models are well established in AML only in rare subsets and animals with additional immunogenic deficiencies the diseases could be established and propagated because of age-dependant leakiness of functional immunity, residual innate immunity and short life span of the immunodeficient animals despite several strategies to enhance engraftment were applied. Over the years several mouse models with a variety if immunodeficient phenotypes were generated to alleviate this problem. Recently the NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mouse model with an IL-2R common gamma-chain deficiency was established and demonstrated stable engraftment rates with mobilized human hematopoietic stem cells. Methods: In this study 6 to 10 weeks old NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) animals were used for xenotransplant experiments. Fresh and frozen samples from adult and pediatric patients with newly diagnosed AML were transplanted via intramedullary injection. Animals were neither irradiated nor were accessory strategies used to enhance engraftment. Primary AML samples were adjusted to 2×107 cells per animal. Animals were anesthetized and samples were equally distributed between both femurs. All procedures were carried out in accordance with national laws and policies. Blood samples were collected weekly. A complete blood count (CBC) was performed and the samples were analyzed for human cells via FACS staining with fluorescence-labeled human anti-CD45 monoclonal antibodies (hCD45). PCR of the alpha-satellite region of human chromosome 17 was performed for confirmation. Animals were sacrificed when hCD45 was >5% or earliest 18 weeks post-injection. Organ distribution of hCD45 positive cells was assessed via FACS analysis of samples from liver, spleen, bone marrow and peripheral blood. Re-transplantion was performed either directly with fresh or from frozen samples. Results: 20 human samples (16 adult and 4 pediatric) were transplanted. The engraftment rate was 80% (16/20) with a median delay of 43.5 days. All pediatric samples engrafted between 30 to 38 days (median 31 days) post-transplant. hCD45 staining in the blood was positive from 13% to 64%, in the liver 0.1% to 54.6%, in the spleen 0.6% to 60.8% and in the bone marrow 0.6% to 71.4%. Adult samples engrafted from 30 to 142 days (median 45 days) post-transplanted with a human CD45 positive staining between 1.5% to 55.7% in the blood, 0.1% to 54.6% in the liver, 0.6% to 60.8% in the spleen and 0.6% to 71.4% in the bone marrow. The percentage of hCD45 in the peripheral blood did not reflect organ infiltration. Second transplants engrafted with a rate of 57.2%, (8/14) with a median delay of 27 days and with human CD45 positive staining between 0.9 to 81.4%. Thrombocytopenia was observed with a median platelet count of 94.500 PLT/μl in engrafted animals compared to control animals with 484.000 PLT/μl (p<0.05). Conclusion: The NSG xenotransplant model demonstrates to be a stable and reproducible tool for the establishment of primary human AML and it is therefore feasible for in vitro and in vivo studies. Engraftment can be predicted via hCD45 analysis and decreasing PLT counts. Engraftment rates of over 80% and a median time to engraftment of 43 days open the possibility to establish individual xenotransplant models in order to assess aberrant mechanisms and molecular rescue strategies for patients who relapsed after treatment. Disclosures: No relevant conflicts of interest to declare.

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Human Cytokine-Induced Memory-like (CIML) NK Cells Are Active Against Myeloid Leukemia in Vitro and in Vivo

Blood ◽

10.1182/blood.v124.21.1117.1117 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1117-1117 ◽

Cited By ~ 4

Author(s):

Maximillian Rosario ◽

Rizwan Romee ◽

Stephanie E Schneider ◽

Jeffrey W Leong ◽

Ryan P Sullivan ◽

...

Keyword(s):

Bone Marrow ◽

Nk Cells ◽

Low Dose ◽

Nk Cell ◽

Conflicts Of Interest ◽

K562 Cells ◽

Lymphoid Cells ◽

Ifn Gamma

Abstract NK cells are innate lymphoid cells that mediate anti-leukemia responses. The ability of MHC-haploidentical NK cells to recognize and eliminate AML blasts have been established in the setting of stem cell transplantation and early phase adoptive NK cell immunotherapy trials. However, the optimal approach to prepare human NK cells for maximal anti-leukemia capacity is unclear. As one form of innate NK cell memory, cytokine-induced memory-like (CIML) NK cells are induced by a brief (16 hour) pre-activation of human NK cells with the combination of IL-12, IL-15, and IL-18, while control NK cells from the same donor are activated by IL-15 only. In published work, this combined IL-12, IL-15, and IL-18 pre-activation results in enhanced proliferation and augmented IFN-gamma responses to cytokine or activating receptor-based re-stimulation following a rest period of 1 – 6 weeks. We hypothesized that CIML NK cells exhibit improved anti-leukemia properties compared to control NK cells from the same individual. Purified primary human CIML NK cells [both CD56bright and CD56dim subsets] produce more IFN-gamma, compared to control NK cells, upon re-stimulation with K562 cells or primary AML blasts after 7 days of rest (p<0.05 and p<0.001, N=5). CIML NK cells also exhibit higher granzyme B protein expression (p<0.01; N=8), and increased cytotoxicity against K562 leukemia targets in vitro (p<0.001, 2.5:1 and 5:1 E:T ratios). We next established a NOD-SCID-gamma-c-/- (NSG) xenograft model to investigate primary human CIML NK cell responses in vivo, with survival supported by low dose IL-2 administered every other day. Seven days following injection of 4 million NK cells / mouse, human CIML NK cells traffic to the bone marrow, spleen, liver and blood, and exhibited better in vivo expansion and persistence, compared to control NK cells (p=0.05 in the blood and bone marrow). Further, the characteristic enhanced functionality of CIML compared to control NK cells when restimulated with K562 targets was retained when assessed ex vivo 7 days post-transfer (p<0.05). Next, we investigated the ability of CIML versus control NK cells from the same donor to clear K562 AML cells in vivo. First, luciferase expressing K562 cells (1 million / mouse) were engrafted into sub-lethally irradiated (250 cGy) NSG mice. On day 3 after K562 challenge, primary human CIML or control NK cells from the same donor (4 million / mouse) were injected, which were supported in vivo using low dose IL-2. CIML NK cells exhibited significantly improved in vivo leukemia clearance as evidenced by whole mouse bioluminescence imaging (see Figure, P=0.03, N=7 mice per group). Thus, human CIML NK cells exhibit enhanced in vitro and in vivo anti-leukemia effects, compared to control NK cells. Based on these findings, a first-in-human phase 1 study of CIML NK cells in relapsed/refractory AML is currently underway. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Alteration of HSC Functions in Thalassemia

Blood ◽

10.1182/blood.v126.23.4752.4752 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4752-4752 ◽

Cited By ~ 2

Author(s):

Annamaria Aprile ◽

Maria Rosa Lidonnici ◽

Alessandro Gulino ◽

Claudio Tripodo ◽

Giacomo Mandelli ◽

...

Keyword(s):

Bone Marrow ◽

Conflicts Of Interest ◽

Pathological Condition ◽

Autologous Transplantation ◽

Beta Thalassemia ◽

Allogeneic Bone ◽

Erythroid Progenitors ◽

Hematopoietic Stem

Abstract Beta-thalassemia represents one of the most globally widespread monogenic disorders and is characterized by significantly reduced or absent synthesis of hemoglobin beta-chains. In its severe form the insufficient production of adult hemoglobin results in altered erythropoiesis, hemolytic anemia, bone marrow (BM) hematopoietic hyperplasia and splenomegaly often associated with extramedullary hematopoiesis, requiring regular blood transfusions and iron chelation treatment. Over the last two decades many progresses were made in the field of allogeneic bone marrow (BM) transplantation to definitively cure beta-thalassemia. In parallel, experimental autologous transplantation protocols were developed to correct the disease by gene therapy also in patients lacking a compatible donor. Both in the allogeneic and autologous setting, thalassemic hematopoietic stem cells (HSCs) and the BM niche represent central elements. Although many aspects of the pathophysiology of thalassemia have been extensively investigated, the HSC and its niche have never been explored. In thalassemia, the BM is a stressed environment, characterized by the compensatory expansion of erythroid progenitors secondary to ineffective erythropoiesis. Whether other hematopoietic subpopulations, such as primitive progenitors and/or HSCs, might be affected by such an altered hematopoietic microenvironment is unknown. We investigated the frequency of hematopoietic progenitors in a murine model of severe beta-thalassemia intermedia. Immunophenotypic analyses revealed no differences in MEP, GMP, CMP, LMPP and MPP committed precursor subpopulations, whereas a significantly lower frequency of HSCs (Lin- Sca-1+ c-kit+ CD48- CD150+) was observed in thalassemic mice, as compared to age-matched wild-type controls. Competitive transplantation experiments revealed a disadvantage in the engraftment capacity of thalassemic HSCs, which was substantiated by the preliminary results from in vitro and in vivo cell cycle analyses suggesting an accelerated HSC exhaustion. Analyses of other cellular components, such as BM stroma and differentiated hematopoietic cells, revealed that additional elements are altered in the thalassemic BM microenvironment. The cellular and molecular bases of HSC-niche interaction in this pathological condition are under investigation. Our results uncover a previously ignored defect of HSCs in beta-thalassemia. The investigation of cellular and molecular players that might affect in trans HSC functions in the complexity of this altered microenvironment is ongoing. Disclosures No relevant conflicts of interest to declare.

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