Mir-886-3p Contributes to the Regulation of the hematopoietic microenvironment by down-Regulating SDF-1α (CXCL12).

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 561-561
Author(s):  
Manoj M Pillai ◽  
Xiaodong Yang ◽  
Mineo Iwata ◽  
Lynne Bemis ◽  
Beverly Torok-Storb
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Stromal Cells ◽  
Stem Cell Niche ◽  
Expression Profiles ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Proliferation And Differentiation ◽  
Cell Niche ◽  
Angiopoietin 1

Abstract Abstract 561 Two functionally distinct stromal cell lines were isolated from a primary long term culture (LTC) established from aspirated human marrow. Designated HS5 and HS27a, the lines were immortalized and extensively characterized including expression profiles for both messenger (mRNA) and micro-RNA (miRNA, a recently described class of small non-coding RNAs that regulate gene expression by binding to target mRNAs to prevent their translation). HS5 was found to secrete growth factors that stimulate proliferation and differentiation of hematopoietic progenitors (G-CSF, IL-6, IL-1α and IL1β), whereas HS27a expresses activities associated with the stem cell niche (SDF-1αa, angiopoietin-1 etc). In keeping with this HS5 conditioned media stimulated proliferation and differentiation of isolated CD34+ cells whereas HS27a supported CD34+ cells in an undifferentiated state. When cultured together to better mimic in vivo cell-cell interactions, the gene expression of HS27a and HS5 combined differed from the expected sum of the two parts, exemplified by the 5-fold down regulation of SDF-1α. Comparisons of miRNA expression profiles of HS5 and HS27a determined that mir-886-3p, (previously described by deep sequencing of small RNA libraries) was expressed > 40 fold in HS5 compared to HS27a, this was then confirmed by quantitative RT-PCR. Given the abundance of mir-886-3p and the possibility that it could be secreted by HS5 and taken up by cells in contact with HS5, we tested its effect on gene expression in HS27a. Transcript levels of genes associated with the stem cell niche (Jagged1, BMP4, Angiopoietin-1, SDF-1α, VCAM-1 and N-Cadherin) were determined by quantitative RT-PCR after direct transfection of mir-886-3p precursors into HS27a cells and compared to appropriate controls. Results show SDF-1α mRNA expression was down-regulated by as much as 8 fold 3 days after transfection. Levels of secreted SDF-1α in culture media, as determined by ELISA, were also decreased. Since SDF-1α is a chemokine known to be critical for the homing of hematopoietic stem and progenitor cells to their niche, the functional significance of the SDF-1α down-regulation by mir-886-3p was confirmed by decreased chemotaxis of T-lymphocytic cells (Jurkat) following miRNA transfection of stromal cells. To determine if mir-886-3p directly effects the SDF-1α transcript, the 1.5 kbp 3'untranslated region (UTR) of SDF-1α gene was cloned downstream of the luciferase gene, and co-transfected with mir-886-3p into HS27a cells. Results showed the luciferase activity was down-regulated greater than 50% in the presence of mir-886-3p, suggesting a direct effect on the SDF-1 α transcript. Given the concern over the relevance of immortalized cell lines we investigated Mir-886-3p expression in primary marrow stromal cells at early passage sorted on the basis of +/- expression of CD146. (CD146 or MCAM has been reported to define a population that supports the hematopoietic stem/ precursor cell niche and is expressed by HS27a and not HS5 cells). Results indicated that the CD146+ stromal cells had significantly lower expression of mir-886-3p when compared to CD146- cells. In summary, these data suggest a role for miRNA in modulating the expression of gene products that are associated with the hematopoietic stem cell niche. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The identification of fibrosis-driving myofibroblast precursors reveals new therapeutic avenues in myelofibrosis

Blood ◽  
2018 ◽  
Vol 131 (19) ◽  
pp. 2111-2119 ◽  
Author(s):  
Rafael Kramann ◽  
Rebekka K. Schneider
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Stromal Cells ◽  
Stem Cell Niche ◽  
Solid Organ ◽  
Hematopoietic Stem ◽  
Gli Proteins ◽  
Hematopoietic Stem Cell Niche ◽  
Cell Niche ◽  
Organ Fibrosis

Abstract Myofibroblasts are fibrosis-driving cells and are well characterized in solid organ fibrosis, but their role and cellular origin in bone marrow fibrosis remains obscure. Recent work has demonstrated that Gli1+ and LepR+ mesenchymal stromal cells (MSCs) are progenitors of fibrosis-causing myofibroblasts in the bone marrow. Genetic ablation of Gli1+ MSCs or pharmacologic targeting of hedgehog (Hh)-Gli signaling ameliorated fibrosis in mouse models of myelofibrosis (MF). Moreover, pharmacologic or genetic intervention in platelet-derived growth factor receptor α (Pdgfrα) signaling in Lepr+ stromal cells suppressed their expansion and ameliorated MF. Improved understanding of cellular and molecular mechanisms in the hematopoietic stem cell niche that govern the transition of MSCs to myofibroblasts and myofibroblast expansion in MF has led to new paradigms in the pathogenesis and treatment of MF. Here, we highlight the central role of malignant hematopoietic clone-derived megakaryocytes in reprogramming the hematopoietic stem cell niche in MF with potential detrimental consequences for hematopoietic reconstitution after allogenic stem cell transplantation, so far the only therapeutic approach in MF considered to be curative. We and others have reported that targeting Hh-Gli signaling is a therapeutic strategy in solid organ fibrosis. Data indicate that targeting Gli proteins directly inhibits Gli1+ cell proliferation and myofibroblast differentiation, which results in reduced fibrosis severity and improved organ function. Although canonical Hh inhibition (eg, smoothened [Smo] inhibition) failed to improve pulmonary fibrosis, kidney fibrosis, or MF, the direct inhibition of Gli proteins ameliorated fibrosis. Therefore, targeting Gli proteins directly might be an interesting and novel therapeutic approach in MF.

scholarly journals Inhibition of TGFβ improves hematopoietic stem cell niche and ameliorates cancer-related anemia

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Boyan Wang ◽  
Yi Wang ◽  
Hainan Chen ◽  
Senyu Yao ◽  
Xiaofan Lai ◽  
...  
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Stromal Cells ◽  
Stem Cell Niche ◽  
Cancer Cachexia ◽  
Tgfβ Signaling ◽  
Hematopoietic Stem ◽  
Hematopoietic Stem Cell Niche ◽  
Cell Niche ◽  
Cancer Related Anemia

Abstract Background Cancer cachexia is a wasting syndrome that is quite common in terminal-stage cancer patients. Cancer-related anemia is one of the main features of cancer cachexia and mostly results in a poor prognosis. The disadvantages of the current therapies are obvious, but few new treatments have been developed because the pathological mechanism remains unclear. Methods C57BL/6 mice were subcutaneously injected with Lewis lung carcinoma cells to generate a cancer-related anemia model. The treated group received daily intraperitoneal injections of SB505124. Blood parameters were determined with a routine blood counting analyzer. Erythroid cells and hematopoietic stem/progenitor cells were analyzed by flow cytometry. The microarchitecture changes of the femurs were determined by micro-computed tomography scans. Smad2/3 phosphorylation was analyzed by immunofluorescence and Western blotting. The changes in the hematopoietic stem cell niche were revealed by qPCR analysis of both fibrosis-related genes and hematopoietic genes, fibroblastic colony-forming unit assays, and lineage differentiation of mesenchymal stromal cells. Results The mouse model exhibited hematopoietic suppression, marked by a decrease of erythrocytes in the peripheral blood, as well as an increase of immature erythroblasts and reduced differentiation of multipotent progenitors in the bone marrow. The ratio of bone volume/total volume, trabecular number, and cortical wall thickness all appeared to decrease, and the increased osteoclast number has led to the release of latent TGFβ and TGFβ signaling over-activation. Excessive TGFβ deteriorated the hematopoietic stem cell niche, inducing fibrosis of the bone marrow as well as the transition of mesenchymal stromal cells. Treatment with SB505124, a small-molecule inhibitor of TGFβ signaling, significantly attenuated the symptoms of cancer-related anemia in this model, as evidenced by the increase of erythrocytes in the peripheral blood and the normalized proportion of erythroblast cell clusters. Meanwhile, hindered hematopoiesis and deteriorated hematopoietic stem cell niche were also shown to be restored with SB505124 treatment. Conclusion This study investigated the role of TGFβ released by bone remodeling in the progression of cancer-related anemia and revealed a potential therapeutic approach for relieving defects in hematopoiesis.

A Niche-Based Cell Mutagenesis Assay Identifies ABL-Kinase Mutations Appearing in BCR-ABL T315I-Mutated Leukemic Cells Treated with Ponatinib.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2436-2436
Author(s):  
Djamel Aggoune ◽  
Nathalie Sorel ◽  
Marie Laure Bonnet ◽  
Jean-Claude Chomel ◽  
Ali G Turhan
Keyword(s):  
Stem Cell ◽  
Stromal Cells ◽  
Stem Cell Niche ◽  
Leukemic Cells ◽  
Hematopoietic Stem ◽  
Abl Kinase ◽  
Loop Region ◽  
T315i Mutation ◽  
Cell Niche ◽  
Hematopoietic Niche

Abstract Abstract 2436 The occurrence of ABL-kinase (ABL-K) mutation is a major persisting concern in CML patients treated with tyrosine kinase inhibitor (TKI) therapies. Leukemic stem cell niche can protect the leukemic cells by providing survival and/or quiescence signals but also could favor the occurrence of ABL-kinase mutations. Amongst the ABL-kinase mutations, T315I is one of the most problematic as it induces resistance to all three clinically accepted TKI (Imatinib, Dasatinib, Nilotinib) and has been shown to occur at the level of stem cells (Chomel et al, Leuk lymphoma 2010). Ponatinib (Formerly AP24534) is a multi-targeted TKI which overcomes resistance to T315I mutation as well as to other ABL-kinase mutations. To model the role of the niche in the context of T315I in patients treated with Ponatinib, we designed a niche-based cell mutagenesis assay in the human hematopoietic UT7 cells engineered to express BCR-ABL (UT7-BCR-ABL native cells) and BCR-ABL with T315I (UT7-T315I), via retrovirus mediated gene transfer. Western blot analyses demonstrated that these cells express BCR-ABL and UT7 clones harboring the T315I mutation were resistant to Imatinib, Dasatinib and Nilotinib but sensitive to Ponatinib. We have treated UT7-T315I cells with N-ethyl-N-nitrosourea (ENU, 50microg/ml) for 24 hours and seeded them on previously prepared MS5 stromal cells (plate of 96 wells) in the presence of Ponatinib (30 nM final concentration). As a control, ENU-treated cells were also cultured in the absence of MS5 feeders and Ponatinib-based selection was performed in the same conditions. The same niche-based assay was also performed in the UT7-BCR-ABL native cells and selection process has taken place in the presence of Imatinib (2 microM). Cell medium was changed every week with addition of Ponatinib (UT7-T315I cells) or Imatinib (UT7-BCR-ABL native cells). At week+4, Ponatinib-resistant and IM-resistant wells cultured in the presence (MS5+) or in the absence (MS5-) of the hematopoietic niche were enumerated. In UT7-BCR-ABL native plates, the numbers of IM-resistant outgrowth was identical in MS5+ versus MS5- conditions (51/95 and 54/96, respectively). On the other hand, in UT7-T315I plates, there was a major difference in the numbers of Ponatinib-resistant T315I cells in MS5+ (96/96, 100% survival) as compared to MS5- conditions (65/96, 62 % survival). IM and Ponatinib-resistant clones were amplified and 48 clones resistant to Ponatinib (MS5+ n= 24; MS5- n= 24) and 48 clones resistant to IM (MS5+ n= 24: MS5- n=24) were screened for ABL-K mutations using a denaturing gradient gel electrophoresis assay followed by direct sequencing of the ABL-kinase domain. In the native BCR-ABL mutagenesis assay (with Imatinib), all clones were found mutated, in the presence or absence of MS5 stromal cells. Most of them were mutated in the P-loop region (E255K, G250E, Y253H). In addition, we have found 3 clones harboring the T315I mutation (1 in MS5- and 2 in MS5+ conditions). Concerning T315I clones surviving to Ponatinib, in addition to the T315I mutation, most of the resistant clones harbored an additional P-loop mutation which occrred in the absence or presence of MS5. Moreover, in two clones, two additional mutations were detected in addition to the T315I mutation. Interestingly, when Ponatinib-resistant cells were switched from MS5+ conditions to MS5- conditions, Ponatinib resistance could be abrogated in some but not all cases. In conclusion, our assay shows that the hematopoietic stem cell niche could play a crucial role in conferring resistance to Ponatinib, not only via the occurrence of novel mutations but also by providing survival signals. Preliminary results also suggest that the hematopoietic niche could facilitate the emergence of T315I mutation in cells expressing native BCR-ABL. These results could be important to study the mechanisms of the occurrence and selection of ABL-K mutations in patients treated with TKI including Ponatinib, and to develop niche-targeted therapies to overcome TKI-resistance in CML. Disclosures: Turhan: Novartis, Bristol Myers Squibb: Honoraria, Research Funding.

Microenvironmental Cross-Talk of Wnt/Notch Signal in Hematopoietic Stem Cell

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3569-3569
Author(s):  
Jin-A Kim ◽  
Young-Ju Kang ◽  
Kyung-Sin Park ◽  
Eek-Hoon Jho ◽  
Il-Hoan Oh
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Stromal Cells ◽  
Stem Cell Niche ◽  
Bone Marrow Cells ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Notch Signal ◽  
Cell Niche ◽  
Notch Ligands

Abstract Microenvironment in the stem cell niche plays an important role to regulate self-renewal and differentiation of hematopoietic stem cells (HSCs). We previously showed opposing effects of b-catenin activation on HSC depending on target of activation in the bone marrow microenvironment. To further analyze the microenvironmental regulation of HSC by Wnt/b-catenin signal, we examined b-catenin activation mode in the trabecular bone marrows. In-situ immunohistochemistry of bone marrows revealed a compartmentalized enrichment of b-catenin in the spindle shaped, CD45(−) endosteal stroma of bone marrow (SNO cells) compared to CD45(+) hematopoietic cells. Receptors for canonical Wnt signals (Fz1, 2, 7, 8) or co-receptors (LRP5, 6) were also enriched in the CD45(−) mesenchymal stromal cells of bone marrows than hemapoietic cells. Moreover, accumulation of active form b-catenin was selectively observed in the “stimulated” bone marrows that had been irradiated or injected with Wnt 3a conditioned medium (Wnt 3a-CM), but not in the “steady state” bone marrows. To examine the effect of b-catenin activated stroma on HSCs, 5-FU bone marrow cells were co-cultured in-vitro for 5 days and transplanted into irradiated mice. A 3-fold higher expansion of primitive phenotype (Lin-Sca-1+c-kit+) cells were seen after culture without differences in cell cycle progression. Further, CRU analysis of the transplanted co-cultured cells displayed higher numbers of CRUs regenerated in the recipient bone marrows (65 CRU vs. 1155 CRUs for MIG vs. b-catenin MSC group, respectively). To directly test the effect of b-catenin activated stroma on HSC during normal reconstitution process, we compared HSC self-renewal in the marrows reconstituted with control or b-catenin activated MSCs; MIG or b-catenin transduced MSCs were directly injected into femur with bone marrow cells and each group mice marrows were then mixed (1:1) transplanted into secondary recipient mice for competitive CRU assay. A 3-fold higher CRU frequency was seen for the HSCs derived from marrows reconstituted with b-catenin/MSCs, indicating the physiological significance of b-catenin activation for in-vivo reconstitution. We next investigated the underlying mechanism for stromal b-catenin effects on HSCs. Expression analysis of b-catenin transduced or Wnt3a-stimulated MSCs revealed higher levels of notch ligands (jagged-1, dll-1), which was similarly observed in the trabecular endosteum of mice treated with Wnt3a-CM. A microarray-based expression analysis further supported up-regulation of notch ligands in b-catenin transduced MSCs, as evidenced by induction of dlk-1 and microfibrillar glycoprotein-2, a protein facilitating utilization of jagged-1. Importantly, induction of notch down-stream molecules (Hes-1 and Deltex-1) was demonstrated in the hematopoietic cells (Lin-Sca-1+) cells co-cultured on the b-catenin activated MSCs. Furthermore, enhancing effects of b-catenin/MSC for expanding undifferentiated cells were abrogated by treatment of gamma-secretase 2 inhibitors during the co-culture. These results show that b-catenin activated stromal cells activate notch signal in the contacting HSCs and that activated notch signal underlies the observed stimulatory effects of b-catenin activated stroma on HSCs. Taken together, Wnt/β-catenin activated stroma and the cross-talk with HSCs may function as a physiologically regulated microenvironmental cue for HSC self-renewal in the stem cell niche.

scholarly journals The hematopoietic stem cell niche in homeostasis and disease

Blood ◽  
2015 ◽  
Vol 126 (22) ◽  
pp. 2443-2451 ◽  
Author(s):  
Laura M. Calvi ◽  
Daniel C. Link
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Stromal Cells ◽  
Stem Cell Niche ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Stem ◽  
Hematopoietic Malignancies ◽  
Hematopoietic Stem Cell Niche ◽  
Cell Niche ◽  
Stem Cell Niches

Abstract The bone marrow microenvironment contains a heterogeneous population of stromal cells organized into niches that support hematopoietic stem cells (HSCs) and other lineage-committed hematopoietic progenitors. The stem cell niche generates signals that regulate HSC self-renewal, quiescence, and differentiation. Here, we review recent studies that highlight the heterogeneity of the stromal cells that comprise stem cell niches and the complexity of the signals that they generate. We highlight emerging data that stem cell niches in the bone marrow are not static but instead are responsive to environmental stimuli. Finally, we review recent data showing that hematopoietic niches are altered in certain hematopoietic malignancies, and we discuss how these alterations might contribute to disease pathogenesis.

Disruption of Bis Leads to Deterioration of Mesenchymal Stromal Cells Predominantly Affecting Vascular Compartment of Hematopoietic Stem Cell Niche.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3627-3627
Author(s):  
Il-Hoan Oh ◽  
Kyong-Rim Kwon ◽  
Ji-Yeon Ahn ◽  
Myungshin Kim ◽  
Jeong-Hwa Lee
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Stem Cell Niche ◽  
Cellular Stress ◽  
Hematopoietic Stem ◽  
Stromal Component ◽  
Cell Niche ◽  
Vascular Compartment

Abstract Abstract 3627 Poster Board III-563 The stem cell niche plays an important role in the microenvironmental regulation of hematopoietic stem cells (HSCs), but the integration of niche activity remains poorly understood. In this study explored the hematopoietic defect of mice disrupted with Bis/BAG-3/CAIR-1, a protein related to apoptosis and response to cellular stress and show that functional loss of bis leads to series of hematopoietic derangements due to perturbation of vascular stem cell niche. First, mice with targeted disruption of bis (bis−/−) exhibited severe hypocellularity in the bone marrows and spleen starting from 16 days after birth. Affected mice exhibited loss of primitive neonatal HSCs (CD34+Lin-Sca-1+c-kit+) and defect in early stage B-lymphopoiesis including common lymphoid progenitors (IL-17R+LSK), pre-B and pro-B cell populations, but not for mature recycling B-lymphocytes (IgD+B220+). However, this hematological defect of bis−/− mice was not reproduced when bis−/− bone marrow cells were transplanted into wild-type (WT) recipients, pointing to the microenvironmental origin of the phenotypes. Subsequent analysis of bis−/− mice bone marrow revealed a characteristic defect in the mesenchymal stromal component that included a quantitative loss of stromal cells (CD45-CD31-TER119-CD105+) in the bone marrows and rapid sensescence of stromal cells comprising colony forming unit-fibroblast (CFU-F) when re-plated in the ex-vivo culture. Moreover, mesenchymal stromal cells expressing CXCL-12 or IL-7 was lost in the affected bone marrows with lowered density of vascular development, together indicating a perturbation of peri-vascular stem cell niche in the bone marrow. In contrast, no abnormalities were observed in the growth and hematopoietic supporting activities of osteoblasts obtained from bis−/− mice. Collectively, these results indicate that Bis functions to mediate cellular regulation of the stem cell niche activities selectively on the vascular compartment without affecting osteoblastic niche, and suggest that Bis may serve as a molecule that can bridge the microenvironment niche and cellular stress/apoptotic signals during the in-vivo orchestration of hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

Mechanism of Calcium Sensing Receptor Mediated Hematopoietic Stem Cell Lodgment In the Adult Bone Marrow Niche

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 399-399
Author(s):  
Ben S. Lam ◽  
Cynthia Cunningham ◽  
Gregor B. Adams
Keyword(s):  
Stem Cell ◽  
Stem Cell Niche ◽  
Calcium Sensing Receptor ◽  
Hematopoietic Stem ◽  
Endosteal Surface ◽  
Calcium Sensing ◽  
Cd34 Cells ◽  
Cell Niche

Abstract Abstract 399 The ability of hematopoietic stem cells (HSCs) to maintain an undifferentiated state and undergo self-renewal is partly regulated by external signals originating from the stem cell niche. One receptor expressed on HSCs that is known to be involved in HSC niche biology is the calcium-sensing receptor (CaR). Our previous study using HSCs obtained from the fetal liver of mice deficient in CaR has shown the crucial role of CaR in HSC lodgment and engraftment in the bone marrow (BM), where CaR-/- HSCs lose their ability to lodge in the endosteal surface of the bone, leading to defective engraftment. To further investigate the mechanism of CaR-mediated lodgment of HSCs, we used a pharmacological approach to activate the receptor and assess the in vitro and in vivo effects. Cinacalcet treatment, which acts as a positive allosteric modulator of CaR to increase the sensitivity of the receptor to activation by extracellular Ca2+, leads to a 3-fold increase in primitive hematopoietic cell activity in vitro as assessed by the cobblestone forming cell assay. The increase in activity in vitro does not appear to be an effect of alterations in cell proliferation, cell survival or the expression of cell adhesion molecules such as VLA-4 or L-selectin. Rather, with CaR stimulation, long-term HSCs have an increased adhesion capability to collagen I, a major ECM molecule present predominantly in the BM endosteal region. We also observed that activation of the CaR following Cinacalcet treatment significantly enhances HSC homing to the BM, lodgment at the endosteal surface and in vivo engraftment capabilities as assessed by a competitive repopulation assay. This enhancement of in vivo activity correlates with increased CXCR4 signaling and migration towards an SDF-1alpha stimulus. Signaling through this receptor is known to be important in cell migration, proliferation, survival, and retention of HSCs in the BM following transplantation. Analysis of the CaR on human cells has demonstrated that there is a distinct population of CaR+ cells present in the CD34+ cell population. The frequency of this population varies between approximately 2% on mobilized peripheral blood CD34+ cells and 6% on BM CD34+ cells. Further analysis is being performed to define the identity of this subpopulation of CaR+CD34+ cells. These mechanisms by which the CaR dictates preferential localization of HSCs in the BM endosteal region may provide additional insights for the fundamental interrelationship between the stem cell niche and stem cell fate. These studies also have implications in the area of clinical stem cell transplantation, where ex vivo modulation of the CaR may be envisioned as a strategy to enhance HSC engraftment in the BM. Disclosures: No relevant conflicts of interest to declare.

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