KIT D816V Mutation Positive Bone Marrow Mesenchymal Stem Cells in Indolent Systemic Mastocytosis Are Associated with Disease Progression

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4058-4058
Author(s):  
Andres C Garcia-Montero ◽  
Maria Jara-Acevedo ◽  
Ivan Alvarez-Twose ◽  
Cristina Teodosio ◽  
Laura Sanchez-Muñoz ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Disease Progression ◽  
X Chromosome ◽  
Conflicts Of Interest ◽  
Systemic Mastocytosis ◽  
Kit Mutation ◽  
Indolent Systemic Mastocytosis ◽  
Kit D816v

Abstract PURPOSE: Multilineageinvolvement of bone marrow (BM) hematopoiesis by the somatic KIT D816V mutation is present in a subset of adult indolent systemic mastocytosis (ISM) patients in association with a poorer prognosis. Here we investigated the potential involvement of BM mesenchymal stem cells (MSC) from ISM patients by the KIT D816V mutation and its potential impact on disease progression and outcome. METHODS: The KIT D816V mutation was investigated in highly-purified BM MSC and other BM cell populations from 83 ISM patients followed for a median of 116 months. MC clonality was further evaluated in female patients by the pattern of inactivation of the X chromosome (XCIP). RESULTS: KIT D816V-mutated MSC were detected in 22/83 (27%) ISM patients. All MSC-mutated patients had multilineage KIT mutation (100% vs. 30%, p=0.0001) and they more frequently showed involvement of lymphoid plus myeloid BM cells (59% vs 22%; P =.03) and a polyclonal XCIP of the KIT- mutated BM MC (64% vs 0%; P =0.01) vs other multilineage ISM cases. Moreover, presence of KIT D816V-mutated MSC was associated with more advanced disease features of ISM, a greater rate of disease progression (50% vs 17%; P =.04) and a shorter progression-free survival at 10, 20 and 30 years (P ≤.003). CONCLUSION: Overall, these results support the notion that ISM patients with mutated MSC may have acquired the KIT mutation in a common pluripotent progenitor cell, prior to differentiation into MSC and hematopoietic precursor cells, before the X-chromosome inactivation process occurs. From a clinical point of view, acquisition of the KIT mutation in an earlier BM precursor cell confers a significantly greater risk for disease progression and a poorer outcome. Disclosures No relevant conflicts of interest to declare.

scholarly journals KIT D816V–mutated bone marrow mesenchymal stem cells in indolent systemic mastocytosis are associated with disease progression

Blood ◽  
2016 ◽  
Vol 127 (6) ◽  
pp. 761-768 ◽  
Author(s):  
Andres C. Garcia-Montero ◽  
Maria Jara-Acevedo ◽  
Ivan Alvarez-Twose ◽  
Cristina Teodosio ◽  
Laura Sanchez-Muñoz ◽  
...  
Keyword(s):  
Disease Progression ◽  
Progenitor Cell ◽  
Clinical Symptoms ◽  
Systemic Mastocytosis ◽  
Kit Mutation ◽  
Key Points ◽  
Marrow Mesenchymal Stem Cells ◽  
Early Acquisition ◽  
Indolent Systemic Mastocytosis ◽  
Kit D816v

Key Points Acquisition of the KIT D816V mutation in an early pluripotent progenitor cell confers ISM cases a greater risk for disease progression. Despite the early acquisition of the KIT mutation, onset of clinical symptoms of ISM is often delayed to middle adulthood.

Mesenchymal Stem Cells From Patients with Acute Myeloid Leukemia (AML) Can Differentiate Into B-Cells in NOD/SCID/IL-2Rγ-/- Mice.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4573-4573
Author(s):  
Rui-Yu Wang ◽  
Yue-Xi Shi ◽  
Zhihong Zeng ◽  
Wendy D. Schober ◽  
Teresa J. McQueen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
B Lymphocyte ◽  
Conflicts Of Interest ◽  
Lymphoid Cells ◽  
Proliferative Potential ◽  
Unexpected Finding ◽  
Nog Mice

Abstract Abstract 4573 Human mesenchymal stem cells (MSCs) derived from bone marrows are characterized by high proliferative potential and pluripotentiality to differentiate into multiple lineages such as osteo-, chondro-, and adipogenic cells. MSC express CD105, CD73 and CD90, but not CD45, CD34, CD14 or CD11b, CD79alpha or CD19 and HLA-DR surface molecules. In this study, we observed that MSC derived from the bone marrow of four AML patients differentiated into B-cell lymphoblasts with NOD/SCID/IL-2Rg-/- engraftment potential. MSC cell lines were established by culturing adherent cells from newly diagnosed AML (n=4) age 20 to 74 years in alpha-DMEM medium supplement with 20% fetal bovine serum. Surface antigen phenotype analysis and G-banding karyotype analysis were performed in passage 2 to 4. FACS-sorted CD90 positive cells were then intravenously (I.V.) injected into NOD/SCID/IL-2Rg-/- (NOG) mice via tail vein (n=9) or into the bone marrow (n=3). Circulating cells were analyzed for CD19, CD33, CD34, and CD90 expression on day 36, 45, 60, 75 after injection of MSC. Results 1) G-banding showed normal karyotype in all MSC; 2) Injected MSC engrafted and differentiated in NOG mice. Surprisingly, CD19 positive cells were found in all samples starting on day 36 (table) and increased on day 60 and 75 (from d36: 6.9±3.5%, d45:0.7±0.1%, d60:2.6 ± 1.6% and d75: 9.3 ± 1.0%); 3) CD90 positive cells were found on day 45 (range from 0.07-3.96% and decreased to 0.1-0.5% on day 75). Low percentage of CD33 (day 45: 0.19-0.78% and day 60: 0.12-2.53%) and CD34 positive cells (day 45: 0.32-1.9% and day 60: 0.21-2.39%) were observed before day 60 and were undetectable by day 75. Table shows the percentages of CD19+ cells found in circulation in NOD/SCID/IL-2Rg-/- (NOG) mice after MSC I.V. or intra-bone marrow injection. (* Mice died after phlebotomy.) Conclusion Human MSC derived from AML bone marrows have the capacity to differentiate into CD19 positive B lymphocyte in NOG mice in vivo. It has previously been reported that AML can be propagated by a leukemic stem cell with lymphoid characteristics (Cancer Cell 2006, 10, 363-74). Data reported here suggest the possibility that AML-derived MSC give rise to lymphoid cells that engraft in NOG mice. This unexpected finding could shed light on the role of stroma cells in the pathogenesis and propagation of leukemias. Disclosures: No relevant conflicts of interest to declare.

Application of Mesenchymal Stem Cells Derived From the Umbilical Cord Instead of Bone Marrow In Hematopoietic Stem Cells Transplantation.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4544-4544
Author(s):  
Ching-Tien Peng
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Umbilical Cord ◽  
Acute Gvhd ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Proliferative Potential ◽  
Hematopoietic Stem ◽  
Cell Based Therapy

Abstract Abstract 4544 Bone marrow-derived mesenchymal stem cells (BMMSCs) have been found to enhance engraftment of hematopoietic stem cell transplantation (HSCT), plus show effect against graft-versus host disease (GVHD) because of their immunosuppressive properties. However, harvesting these cells is an invasive and painful procedure. To substitute BMMSCs from alternative sources is necessary. We intravenously infused ex vivo-expanded third-party umbilical cord-derived mesenchymal stem cells (UCMSCs) obtained from a bank 8 times in 3 patients who developed severe, steroid-resistant acute GVHD after allogeneic HSCT. The acute GVHD improved with each infusion of UCMSCs. Besides, after cotransplantation of cord blood and UCMSCs in 5 patients, we found UCMSCs enhanced absolute neutrophil counts and platelet counts recovery. No adverse effects after UCMSCs infusions were noted. We also found that UCMSCs had superior proliferative potential and greater immunosuppressive effects than BMMSCs in vitro. This is the first report of UCMSCs in human clinical application. These findings suggest UCMSCs are effective in treating aGVHD and can enhance hematopoiesis after HSCT. Considering that they are not only easy to obtain but also proliferate rapidly, UCMSCs would be the ideal candidate for cell-based therapy, especially for diseases associated with immune responses because of their immunosuppressive effects. Disclosures: No relevant conflicts of interest to declare.

A Phase II Study of Nilotinib, a Novel Inhibitor of c-Kit, PDGFR, and Bcr-Abl, Administered to Patients with Systemic Mastocytosis.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2703-2703 ◽  
Author(s):  
Andreas Hochhaus ◽  
Oliver G. Ottmann ◽  
STephanie Lauber ◽  
Timothy Hughes ◽  
Gregor Verhoef ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mast Cell ◽  
Adverse Events ◽  
Disease Progression ◽  
Phase Ii ◽  
Phase Ii Study ◽  
Systemic Mastocytosis ◽  
Clinical Indication ◽  
Kit Mutation ◽  
Cell Counts

Abstract Systemic mastocytosis is a clonal disorder associated with a constitutive activation of the c-kit tyrosine kinase based on point mutations and is characterized by mast cell infiltration of extracutaneous organs. Nilotinib is a novel aminopyrimidine which potently inhibits Bcr-Abl, as well as the PDGF-R, and c-kit tyrosine kinases. Preclinical data demonstrated the activity of nilotinib against D816V mutated c-kit in biochemical and cellular assays. This Phase II study was designed to evaluate the safety and efficacy of nilotinib administered at an oral dose of 400 mg twice daily to patients with systemic mastocytosis defined by specific disease criteria and with a clinical indication for treatment. Data are available for 60 patients (34 male, 26 female). The median age is 51 years (range, 29 to 79). Of the 60 patients 31 (52%) had extramedullary involvement at baseline. In 30/36 patients investigated (83%) D816V c-kit mutation was found by D-HPLC and/or conventional sequencing in bone marrow or extracutaneous organs. Two patients showed the c-kit I798I polymorphism. Treatment is ongoing for 38 (63%) patients; 22 (37%) have discontinued; ten (17%) for adverse events, seven (12%) withdrew consent, and one (2%) each for disease progression and lost to follow-up. There were two (3%) deaths related to disease progression. Based on investigators’ assessment of serum tryptase, bone marrow mast cell counts and improvement of clinical symptoms 12 patients (20%) had a documented clinical response including two (3%) complete, five (8%) incomplete, four (7%) minor, and one partial response. Adverse events occurring in >15% of patients included nausea in 28 (47%), headache in 26 (43%), fatigue in 25 (42%), vomiting in 22 (37%), diarrhea in 21 (35%), pruritis in 16 (27%), and rash in 15 (25%) patients, dizziness and muscle spasms in 14 (23%) patients each, bone pain in 12 (20%), pyrexia and myalgias in 11 (18%) patients each, and dyspnea, constipation, increased ALAT, and arthralgias in ten (17%) patients each. Most side effects occurred early after initiation of nilotinib therapy and were successfully treated with H1- and H2-blockers and/or corticosteroids, indicating a mast cell degranulation syndrome. Overall the most frequent Grade 3/4 adverse events included diarrhea in four (7%) patients, and thrombocytopenia and headache in three (5%) patients each. The data suggest that nilotinib has clinical activity and an acceptable safety and tolerability profile in patients with systemic mastocytosis with constitutive c-kit activation. Individual molecular characterization will help to guide targeted therapy in this disease.

Osteogenic- and Adipogenic- Differentiated Bone Marrow-Derived Mesenchymal Stem Cells Fail to Support Expansion of Adult Hematopoietic Stem Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4810-4810
Author(s):  
Olga Kulemina ◽  
Izida Minullina ◽  
Sergey Anisimov ◽  
Renata Dmitrieva ◽  
Andrey Zaritskey
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Cord Blood ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Feeder Layers

Abstract Abstract 4810 Ex vivo expansion and manipulation of primitive hematopoietic cells has become a major goal in the experimental hematology, because of its potential relevance in the development of therapeutic strategies aimed at treating a diverse group of hematologic disorders. Osteoblasts, mesenchymal stem/progenitor cells (MSC/MPC), adipocytes, reticular cells, endothelial cells and other stromal cells, have been implicated in regulation of HSC maintenance in endosteal and perivascular niches. These niches facilitate the signaling networks that control the balance between self-renewal and differentiation. In the present study, we evaluated and compared the effects of three different stromal feeder layers on expansion of HSPC derived from BM and cord blood (CB): BM mesenchymal stem cells (MSC), osteoblast-differentiated BM mesenchymal stem cells (Ost-MSC) and adipocyte-differentiated BM mesenchymal stem cells (Ad-MSC). BM-MSC cultures were established from plastic adherent BM cell fractions and analyzed for immunophenotype, frequency of colony forming units (CFU-F), frequency of osteo- (CFU-Ost) and adipo- (CFU-Ad) lineage progenitors. Cultures with similar clonogenity (CFU-F: 26,4 ± 4,5%) and progenitors frequency (CFU-Ost: 14,7 ± 4,5%; CFU-Ad: 13,3 ± 4,5%) were selected for co-culture experiments. All MSC were positive for stromal cell-associated markers (CD105, CD90, CD166, CD73) and negative for hematopoietic lineage cells markers (CD34, CD19, CD14, CD45). CD34+ cells were separared from BM and CB samples by magnetic cell sorting (MACS) and analyzed for CD34, CD38 and CD45 expression. Feeder layers (MSC, Ost-MSC, Ad-MSC) were prepared in 24-well plates prior to co-culture experiments: MSCs (4×104 cells/well) were cultured for 24 h and either used for following experiments or stimulated to differentiate into either osteoblasts or adipoctes according to standard protocols. CD34+ cells (3500-10000 cells per well) were co-cultured in Stem Span media with or without a feeder layers and in the presence of cytokines (10 ng/mL Flt3-L, 10 ng/mL SCF, 10ng/mL IL-7) for 7 days. Expanded cells were analyzed for CD34, CD38 and CD45 expression. Results are shown on figures 1 and 2. As expected, CB-derived HSPC expanded much more effectively than BM-derived HSPC. The similar levels of expansion were observed for both, the total number of HSPC, and more primitive CD34+CD38- fraction in the presence of all three feeder layers. Ost-MSC supported CB-derived HSPC slightly better than MSC and Ad-MSC which is in a good agreement with data from literature (Mishima et.al., European Journal of Haematology, 2010), but difference was not statistically significant. In contrast, whereas BM-MSC feeder facilitated CD34+CD38- fraction in BM-derived HSPC, Adipocyte-differentiated MSC and osteoblast-differentiated MSC failed to support BM-derived CD34+CD38- expansion (11,4 ±.4 folds for MSC vs 0,9 ±.0,14 for Ad-MSC, n=5, p<0,01 and 0,92 ±.0,1 for Ost-MSC, n=5, p<0,01).Figure 1.Cord Blood HSPC ex vivo expansionFigure 1. Cord Blood HSPC ex vivo expansionFigure 2.Bone Marrow HSPC ex vivo expansionFigure 2. Bone Marrow HSPC ex vivo expansion Conclusion: BM- and CB-derived CD34+CD38- cells differ in their dependence of bone marrow stroma. Coctail of growth factors facilitate CB HSPC expansion irrespective of lineage differentiation of supporting MSC feeder layer. In contrast, primitive BM CD34+CD38- HSPC were able to expand only on not differentiated MSC. Disclosures: No relevant conflicts of interest to declare.

scholarly journals CXCL12 Knock-out Enhances Leukemia Stem Cell Response to Combination Chemotherapy Plus Tyrosine Kinase Inhibition in Flt3-ITD Acute Myeloid Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 7-8
Author(s):  
Nick R Anderson ◽  
Hui Li ◽  
Mason W Harris ◽  
Shaowei Qiu ◽  
Amanda K Mullen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Drug Resistance ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Conflicts Of Interest ◽  
Single Agent ◽  
Fold Increase ◽  
Long Term Effects ◽  
Leukemia Stem Cell

One of the most common mutations in adult AML is a constitutively activating internal tandem duplication in the juxtamembrane domain of the Flt3 receptor (Flt3-ITD), which portends poor prognosis due to high recurrence rates and defines a distinct subtype of disease with unique features and biology. Although several FLT3 TKIs have been developed for clinical use, responses to these drugs, especially as single agents, are limited and are not sustained. The objective of our study was to determine the contribution of bone marrow stromal populations to LSC resistance to Flt3-targeted TKI in Flt3-ITD AML. We utilized a newly generated Flt3-ITD TET2flox/flox Mx1-cre mouse model of AML, as well as primary Flt3-ITD TET2 mutant AML patient samples, to identify phenotypic populations with leukemia initiating capacity (LIC) in Flt3-ITD AML. In the animal model, administration of pIpC leads to deletion of TET2 and development of AML characterized by leukocytosis, accumulation of blasts, splenomegaly, anemia, thrombocytopenia, and lethality. Limiting dilution transplantation of FACS-sorted ST-HSC, MPP and GMP populations revealed that LIC were absent from GMPs and almost exclusively limited to the phenotypic ST-HSC population (calculated stem cell frequencies: &lt;1:180,000, 1:63,635, and 1:2,730 for GMP, MPP, and ST-HSC, respectively). We similarly found that in samples from human Flt3-ITD TET2 mutant AML patients LIC capacity was restricted to primitive HSPC populations (Lin-CD34+CD38-), and was not seen in committed GMP (Lin-CD34+CD38+CD123+CD45RA+). We characterized bone marrow stromal cells in Flt3-ITD AML mice by flow cytometry on collagenase digested bone fragments. We also transplanted murine AML cells into CXCL12-GFP mice to assess alterations in CXCL12-expressing stromal populations in AML bone marrow. We found expansion of several stromal populations in AML vs. WT mice, including a 3.5-fold increase in mesenchymal stem cells (CD45-Ter119-CD31-VECadherin-Sca1+CD51+) and a 1.5-fold increase in osteoprogenitors (CD45-Ter119-CD31-VECadherin-Sca1-CD51+). CXCL12 expression, however, was greater than 2-fold higher in osteoprogenitors and 2-fold lower in mesenchymal stem cells in AML vs. WT mice. We also showed that Flt3-ITD AML HSPCs have nearly 2-fold higher CXCR4 expression than WT HSPCs. These data taken together supported further exploration of the role of a CXCL12-expressing niche in supporting Flt3-ITD AML LSC. To assess the effect of CXCL12 deletion from the marrow microenvironment on AML TKI response, we transplanted murine AML cells into CXCL12flox/flox UBC-cre mice and control Cre-ve mice. We found that AML developing in Cre-ve control mice was resistant to single agent Flt3 TKI (AC220, Quizartinib) treatment, but that CXCL12 deletion modestly improved response to TKI. We next tested a combination of standard-of-care "7+3" chemotherapy (cytarabine + doxorubicin) and AC220, and found that this approach resulted in more effective and selective, but only partial, reduction of leukemia cells in this model. We found that control AML mice showed an initial response to combination chemo + TKI, but developed disease recurrence by 3 weeks of treatment. In contrast, CXCL12-deleted AML mice maintained peripheral blood response for up to 3 weeks, and showed enhanced suppression of LIC-containing populations compared to control mice. We are now performing secondary transplants using BM cells harvested from these treated mice to assess long-term effects on leukemia stem cell capacity. We are also testing the effect of the combination of chemotherapy and TKI following osteoblast-specific deletion of CXCL12, using CXCL12flox/flox BGLAP-cre mice, to assess whether osteoblastic cells are the source of CXCL12 responsible for this effect. In conclusion, our results suggest that LSC in Flt3-ITD AML are found within a primitive phenotypic ST-HSC population as opposed to GMP populations as seen in some other types of AML. Furthermore, CXCL12-expressing bone marrow microenvironmental cells contribute to drug resistance in AML LSC and global knockout of CXCL12 enhances drug response in these populations. Our studies support a potential role for a CXCL12-expressing osteoprogenitor niche in supporting Flt3-ITD AML LSC growth and drug resistance, targeting of which could improve responses and outcomes in Flt3-ITD AML. Disclosures No relevant conflicts of interest to declare.

Wnt/β-Catenin Signaling Promotes Neuronal Differentiation of Bone Marrow Mesenchymal Stem Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4814-4814
Author(s):  
Qin Yu ◽  
Lizhen Liu ◽  
Yanping Duan ◽  
Hui Dong ◽  
Wei Shan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Signaling Pathway ◽  
Neuronal Differentiation ◽  
Neural Differentiation ◽  
Conflicts Of Interest ◽  
Neuronal Marker ◽  
Immunocytochemical Analysis ◽  
Differentiation Efficiency

Abstract Abstract 4814 Introduction Mesenchymal stem cells (MSCs) have the potential to self-renew and differentiate into multiple cell types and have been used to replace damaged cells in the nervous system using animal models of neurological disorders or traumatic brain injury. Previous reports revealed that MSCs can differentiate into neuron-like cells in vitro. However, precise mechanisms controlling this process are unclear. Several lines of evidence suggest that Wnt signaling pathway plays pivotal roles in MSC differentiation. Here we explored the roles of Wnt/β-catenin signaling in neuronal differentiation of MSCs. Methods MSCs were obtained from rat bone marrow and cultured in proliferation medium containing DMEM /10%FBS. Passage 3–6 was used in this study. MSCs were induced neuronal differentiation as previously described. Twenty-four hours prior to neuronal induction, media were replaced with preinduction media consisting of DMEM/20% FBS and 1mM β-mercaptoethanol (BME). To initiate neuronal differentiation, the preinduction media were removed, and the cells were washed with PBS and transferred to neuronal induction media composed of DMEM/5mM BME and cultured for 5h. Cells after pre-induction for 6h, 12h, 24h or induction for 1h, 3h and 5h were collected. The expression of β-catenin, NSE, Nestin and Ngn1 was analyzed by Western blot, RT-PCR and immunocytochemical analysis. To further determine the roles of β-catenin, MSCs were transfected with β-catenin siRNA using Lipofectamine 2000. Cells after pre-induction for 24h and induction for 3h were collected and expression of β-catenin, NSE, Nestin and Ngn1 were detected. In addition, Wnt 3a were added to the differentiation system. Differentiation efficiency and expression of β-catenin and neuronal specific genes were analyzed. Results (1) MSCs could differentiate into neuron-like cells. After adding 5mM BME into MSCs culture system, cells showed neuron-like cells in morphology. Cytoplasm in the flat MSCs retracted towards the nucleus, forming a contracted multipolar. To characterize neuronal differentiation, we fixed BME-treated cultures after 5 h and stained them for the neuronal marker NSE and Nestin. The results showed that NSE+ cells were 76.00±6.50 % and Nestin+ cells were 70.52 ±5.02%. (2) Wnt/β-catenin signaling involved in the neural differentiation of MSC. β-catenin plays a central role in Wnt/β-catenin signaling pathway. Our data showed that the expression of β-catenin increased during the neural differentiation of MSCs both at mRNA and protein levels. Accordingly, the neuronal markers NSE and Nestin both increased during differentiation process. (3) β-catenin knockdown inhibited neuronal differentiation of MSCs. The western blotting and the immunocytochemical analysis revealed that the protein amount β-catenin decreased obviously after MSCs transfection with β-catenin siRNA for 72h. Subsequently, the expression of NSE, Nestin and Ngn1 decreased compared with MSCs transfected with control siRNA and non-transfected with siRNA. In addition, differentiation efficiency decreased signifufantly (P<0.05). (4) Wnt 3a promotes neuronal differentiation of MSCs. In comparison with that in control cells, Wnt3a, β-catenin protein were increased by Wnt3a (100 ng/ml)-conditioned medium, the neuronal differentiation was markedly improved in the Wnt3a treated cells in association with the increase in the protein and gene expression neural markers NSE and Nestin. Conclusion Our data firstly revealed that Wnt/β-catenin signaling promotes neuronal differentiation of bone marrow MSCs. β-catenin plays a pivotal role in this process and may be considered as a target to regulate in neurologic disease treatment. Disclosures: No relevant conflicts of interest to declare.

JAK2 Mutation Analysis and Function of Bone Marrow Mesenchymal Stem Cells in Myeloproliferative Neoplasms

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5179-5179
Author(s):  
Hong Tian ◽  
Yang Xu ◽  
Guanghua Chen ◽  
Man Qiao ◽  
Wu Depei
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Jak2v617f Mutation ◽  
Primary Study ◽  
Jak2 Mutation ◽  
Healthy Donors ◽  
Marrow Mesenchymal Stem Cells

Abstract Abstract 5179 Background: JAK2V617F and JAK2 exon12 mutations in haematopoietic cells were partially responsible for the pathogenesis of myeloproliferative neoplasms (MPN).But it was still unclear whether bone marrow mesenchymal stem cells (BMSCs), the significant component of hemopoiesis microenvironment, were participated in the pathogenesis of MPN. Objective: To study the physiopathology characteristics and analyze JAK2 mutation in BMSCs from MPN patients. Methods: By searched for the JAK2V617F mutation and exon 12 mutation in 135 MPN patients' blood /bone marrow samples, 20 patients with JAK2V617F mutation, 10 patients with JAK2 exon 12 mutation, 5 JAK2-mutation-negetive patients and 10 healthy donors were recruited. The phenotype, mesenchymal differentiation capacity, expression of hematopoietic and immune molecules and JAK2 mutation of isolated bone marrow BMSCs were detected. Results: BMSCs derived from the four groups were found to be similar in morphology, differentiation ability and expression of hematopoietic and immune molecules. Primary study indicated that the isolated BMSCs from patients groups were not able to harbor JAK2 mutation in spite of positive or negative JAK2 mutation in blood /bone marrow samples. Conclusion: BMSCs from MPN patients had similar biological characteristics to healthy donors, and BMSCs were not likely involved in pathogenesis of MPN. Disclosures: No relevant conflicts of interest to declare.

Functional Influence of CXCL12 Regulating miRNAs in Mesenchymal Stem Cells (MSCs).

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2350-2350
Author(s):  
Laleh S. Arabanian ◽  
Fernando Fierro ◽  
David M. Poitz ◽  
Ruth H. Strasser ◽  
Martin Bornhaeuser ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Transforming Growth Factor ◽  
Conflicts Of Interest ◽  
Luciferase Assay ◽  
Human Mscs ◽  
Bone Marrow Niche ◽  
Cxcl12 Expression ◽  
Hematopoietic Stem

Abstract Abstract 2350 CXCL12 is a chemokine known to be critical for the regulation of the interaction between hematopoietic stem cells (HSCs) and their niche in the bone marrow, e.g. mesenchymal stem cells (MSCs). MicroRNAs (miRNAs) are post-transcriptional regulators recently shown to mediate a variety of cellular processes in the bone marrow niche. However, identification of specific miRNAs and their regulatory role in the crosstalk between HSCs and MSCs are still poorly understood. From a library of 470 miRNAs, 26 miRNAs were shown to downregulate the levels of CXCL12 in the supernatant of the human MSC line SCP-1. Eight of them (miR-23, 130b, 135, 200b, 200c, 216, 222, 602) were chosen for further investigation according to their significant interaction with the 3'UTR of CXCL12 as determined by luciferase assay. Among them, miR-23a,130 and 222 were expressed in 46 human primary MSCs, whereas the other miRs show negligible expression in resting MSCs. However, we observed, that MSCs that underwent adipogenic and osteogenic differentiation showed strongly decreased CXCL12 protein values early (day 5) and at later stages (day 14). The later drop in CXCL12 expression was clearly associated with an increased expression of miR-23a and miR-200. We furthermore tested a subset of stimuli (proinflammatory cytokines, cytotoxic drugs, chemokines) for their ability to modulate the described miRNAs. Amongst them, exclusively the application of transforming growth factor ß1 (TGF-ß1), resulted in the induction of miR-23a and at the same time reduction of CXCL12. The effect was counteracted by transfection of anti-miR-23 molecules. Taken together, we have shown for the first time that CXCL12-targeting miRNAs (in particular miR23a) have a significant potential to regulate the properties of the stem cell niche. Moreover, miR-23 is implicated in the signalling pathway of TGF-ß1 in human MSCs. Disclosures: No relevant conflicts of interest to declare.

Phosphorylation Of GSK3β Is Associated With Inferior Survival In Acute Myeloid Leukemia and Is An Indicator Of AKT Activation In AML Blasts and Bone Marrow Mesenchymal Stem Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2551-2551
Author(s):  
Peter P. Ruvolo ◽  
Rui-yu Wang ◽  
Vivian R Ruvolo ◽  
Rodrigo Jacamo ◽  
Teresa McQueen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Survival Data ◽  
Conflicts Of Interest ◽  
Glycogen Synthase ◽  
Akt Activation ◽  
Mutational Status ◽  
Marrow Mesenchymal Stem Cells

Abstract Glycogen Synthase Kinase 3β (GSK3β) is a key regulator of cell metabolism, proliferation, survival, and differentiation. The kinase has abundant substrates including many proteins in the canonical WNT pathway. Considering that GSK3β phosphorylation of many pro-survival proteins result in their degradation (e.g MYC, MCL-1), it is not surprising that GSK3β activation by stress challenge leads to cell cycle arrest and/or apoptosis. GSK3β is negatively regulated by serine 9 phosphorylation mediated by Protein Kinase B (AKT). Since AKT activation supports survival of AML cells and inactivation of GSK3β could suppress stress signaling events, we hypothesize that serine 9 phosphorylation of GSK3β (p-GSK3β ) will be detrimental for AML patients. In the current study, we analyzed GSK3β expression by Reverse Phase Protein Analysis (RPPA) in a cohort of 511 AML patients. GSK3β expression was correlated with patient survival data and disease characteristics such as French-American-British (FAB) classification, cytogenetics, and mutational status. High levels of p-GSK3β were found to correlate with adverse outcome for survival and complete remission duration (CR) in patients with intermediate cytogenetics but not in those with unfavorable cytogenetics. CR was only 45 weeks in the third of patients with highest p-GSK3β levels compared to 98 weeks for patients with low levels (p = 0.008; N = 121). Even intermediate cytogenetic patients with FLT3 mutation fared better when p-GSK3β levels were lower (50 versus 24 weeks; p = 0.009; N = 35). Expression of GSK3β and its phosphorylated form was compared with expression of 229 other proteins using RPPA in the AML patient cohort. Consistent with p-GSK3β as an indicator of AKT activation, RPPA revealed that p-GSK3β is positively correlated with phosphorylation of AKT (S473), BAD (S136), and P70S6K. In addition, p-GSK3β negatively correlated with FOXO3A (which is degraded after phosphorylation by AKT). Bone marrow mesenchymal stem cells (BM MSCs) are a critical component of the leukemic microenvironment but how these cells modulate the survival of leukemia cells is not clear. RPPA analysis was performed on BM MSC from healthy donor (N = 71) and BM MSCs from AML patients (N = 106). Interestingly, both total and phosphorylated GSK3 were found to be elevated in the AML samples suggesting that AKT is also activated in the leukemic MSCs. In vitro models of the BM microenvironment suggest that the AKT pathway is activated in both the leukemic and supporting stromal cells so this finding is consistent with these models (Konopleva and Andreeff, Curr Drug Targets. 2007; 8: 685). Unlike in AML blast cells, FOXO3A was not correlated with GSK3β phosphorylation in the MSCs. Examination of miRs in normal versus AML BM MSCs using microarray analysis and validated by qRT-PCR indicate that miR-21 is elevated in the MSC of the normal individuals. As miR-21 is suppressed by FOXO3A, this finding supports the notion that AKT is active in the AML BM MSCs but FOXO3A may not be functional. This possibility is plausible as induction of FOXO3A results in apoptosis in MSC (Djouad Cloning Stem Cells. 2009; 11:407). Conclusion These findings suggest that AKT mediated phosphorylation of GSK3β may be detrimental to AML patients and p-GSK3β may serve as an important prognostic factor for at least a subset of AML patients. The results also suggest that activation of AKT can occur in both the malignant cells and MSC cells in the leukemic microenvironment. Disclosures: No relevant conflicts of interest to declare.

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