scholarly journals Identifying the Leukemia Stem Cell Niche Mediating Drug Resistance in Flt3-ITD Acute Myeloid Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 909-909
Author(s):  
Nick R Anderson ◽  
Hui Li ◽  
Mason W Harris ◽  
Shaowei Qiu ◽  
Andrew J Paterson ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Drug Resistance ◽  
Endothelial Cells ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Conflicts Of Interest ◽  
Single Agent ◽  
Fold Increase ◽  
Combination Regimen

The Flt3-ITD is one of the most common mutations in AML, and defines a distinct subtype of disease with unique features and biology, and with poor prognosis related to high rates of recurrence. Although several FLT3 TKIs have been developed for clinical use, responses are limited and are not sustained. The objective of our study was to determine the contribution of bone marrow stromal populations to LSC drug resistance to Flt3-targeted TKI in Flt3-ITD AML. We utilized a newly generated Flt3-ITD TET2flox/flox Mx1-cre mouse model of AML to identify phenotypic populations with leukemia initiating capacity (LIC) in Flt3-ITD AML. Administration of pIpC leads to deletion of TET2 and development of AML-like disease characterized by leukocytosis, accumulation of blasts, anemia and thrombocytopenia. Transplantation of selected STHSC, MPP and GMP populations revealed that LIC were almost exclusively found within the phenotypic ST-HSC population (calculated stem cell frequencies: <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 AML patients LIC capacity was similarly restricted to more primitive HSPC populations (Lin-CD34+CD38-) and was not seen in committed GMP (Lin-CD34+CD38+CD123+CD45RA+). We performed flow cytometry on collagenase-digested bone fragments from AML mice to characterize bone marrow stromal cells in Flt3-ITD AML mice. We also transplanted murine AML cells into CXCL12-GFP mice to assess alterations in CXCL12-expressing stromal populations in AML bone marrow. We found that several stromal populations are expanded in AML vs. WT mice, including a 3.5-fold increase in mesenchymal stem cells (CD45-Ter119-CD31-VECadherin-Sca1+CD51+), a 3.9-fold increase in endothelial cells (CD45-Ter119-CD31+), and a 1.5-fold increase in osteoprogenitors (CD45-Ter119-CD31-VECadherin-Sca1-CD51+). The expression of CXCL12, a key factor that mediates niche localization of HSC and LSC, was greater than 2-fold higher in osteoprogenitors, but not significantly different in endothelial cells, 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 osteoprogenitor niche in supporting Flt3-ITD AML LSC. We transplanted murine AML cells into CXCL12flox/flox UBC-cre or CXCL12flox/flox Osx-cre mice to assess the effect of global or osteoprogenitor-specific CXCL12-KO, respectively, on AML progression and TKI response. We found that this model of AML was resistant to single agent Flt3 TKI (AC220) treatment. Global CXCL12-KO using CXCL12flox/flox UBC-cre mice modestly improved response to TKI. We show that a combination regimen including standard-of-care "7+3" chemotherapy (cytarabine + doxorubicin) and a Flt3 TKI (AC220) results in more effective and selective targeting of leukemia cells in this model. We are currently treating osteoprogenitor-specific CXCL12-KO AML mice with the combination chemotherapy + TKI regimen to investigate the contribution of osteoprogenitors to disease progression and drug resistance in Flt3-ITD AML LSC. In conclusion, our results suggest that LSC in Flt3-ITD AML are present within a primitive phenotypic ST-HSC population more so than in MPP and GMP populations as often seen in some other types of AML. 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.

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: <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.

Growth Factor Independence 1 b (Gfi1b) as a New Regulator of Hematopoietic Stem Cell Fate

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 837-837
Author(s):  
Cyrus Khandanpour ◽  
Lothar Vassen ◽  
Marie-Claude Gaudreau ◽  
Christian Kosan ◽  
Tarik Moroy
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Cell Fate ◽  
Conflicts Of Interest ◽  
Fold Increase ◽  
Surface Proteins ◽  
Expression Array ◽  
Hematopoietic Stem

Abstract Abstract 837 Donor matched transplantation of bone marrow or hematopoietic stem cells (HSCs) are widely used to treat hematological malignancies, but are associated with high mortality. Methods for expansion of HSC numbers and their mobilization into the bloodstream of a donor could significantly improve therapy. We show here that the zinc finger transcriptional repressor Gfi1b is highly expressed in hematopoietic stem cells (defined as CD 150+, CD 48-, Lin-, Sca1+ and c-kit+) cells and is down-regulated more than 10 fold upon differentiation into multipotential progenitors (defined as CD 150+ or CD150-, CD 48+, Lin-, Sca1+ and c-kit+). Constitutive germline deletion of Gfi1b is lethal at midgestation due to impaired development of erythrocytes and megakaryocytes. We have therefore developed a conditional knock-out of Gfi1b to study its role specifically in the adult hematopoietic system. Deletion of Gfi1b leads to a 30-fold increase of HSC numbers in bone marrow and around a100 fold increase in spleen and peripheral blood. This was due to a higher rate of HSCs undergoing cell cycling. Concomitantly, the number of quiescent HSCs was reduced 5–6 times. We then performed an gene expression array of wt and Gfi1b deficient HSCs and observed that loss of Gfi1b leads to an altered RNA expression of integrins and adhesion molecules, for instance CXCR4, VCAM-1 and Tenascin C, which usually retain HSCs in a dormant state in the endosteal niche. These changes were also confirmed on protein level. Finally, we could observe a higher levels of Reactive Oxygen Species (ROS) in the Gfi1b deficient HSCs compared to wt HSCs. We verified whether elevated level of ROS are causative for the expansion of HSCs and noticed that application of N-Acetyl-Cystein, which counteracts the effects of ROS, limits significantly the expansion of HSCs, underscoring the important role of ROS in the expansion of Gfi1b deficient HSCs. Despite markedly increased proliferation, Gfi1b-/- HSCs can reconstitute lymphoid and myeloid lineages to the same extent as wt HSCs when transplanted in competition with wt HSCs. Furthermore, Gfi1b deficient HSCs also feature an expansion after transplantation and expand 5–10 fold more than wt HSC when transplanted initially in equal numbers with wt HSCs. It is possible that lower expression of CXCR4, VCAM-1 and other surface proteins leads to release and egression of Gfi1b deficient HSCs from the hypoxic endosteal stem cell niche and exposes the HSCs to more oxygen which in turn increases ROS levels. Elevated ROS could promote entry of Gfi1b-/- HSCs into cell cycle. In conclusion Gfi1b regulates HSC dormancy, pool size and potentially also the egress and mobilization of HSCs and might offer a new therapeutic approach to improve human HSC transplantation. Disclosures: No relevant conflicts of interest to declare.

Biological Characteristics of Bone Marrow-Derived Mesenchymal Stem Cells From a Patient with β-Thalassemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4579-4579
Author(s):  
Jinsun Yoon ◽  
Sung Heon Song ◽  
Seoju Kim ◽  
Eun Shil Kim ◽  
Gu Kong ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Healthy Donor ◽  
Conflicts Of Interest ◽  
Functional Characterization ◽  
Hematopoietic Stem ◽  
Normal Healthy Donor ◽  
Immunomodulatory Function

Abstract Abstract 4579 Thalassemia syndromes are considered as a heterogeneous group of inherited anemias leading to decrease or loss of synthesis of one or more globin chain subunits of the adult hemoglobin tetramer (Hb A). Beta-thalassemia results from a decrease in β-chain production of hemoglobin relative to alpha-chain production. Hematopoietic stem cell transplantation from HLA-identical siblings is curative for thalassemia syndrome. Bone marrow (BM)-derived mesenchymal stem cells (MSCs) are primitive, undifferentiated cells which are capable of self-renewal, and differentiating morphologically and functionally into different cell lineages including adipocytes, chondrocytes, myocytes, astrocytes, tenocytes, and hepatocytes. There have been contrasting data on whether BM derived MSCs are altered in various hematological disorders including leukemia, multiple myeloma, and myelodysplastic syndrome as well as autoimmune disease. To date, functional characterization of MSCs has never been performed in thalassemia syndrome. In the present study, we isolated BM-derivied MSCs from a patient with β-thalassemia in order to compare phenotypic and functional characteristics to those from normal healthy donor. No differences were observed between MSCs from β-thalassemia and those from normal healthy donor in terms of morphology, phenotype, karyotype, multi-differentiation capacity. In mitogen-stimulated T cell proliferation assay and mixed lymphocyte reaction, MSCs from β-thalassemia strongly inhibited the proliferation of allogeneic T cells in association with reduced proportion of CD3+, CD4+ and CD8+ cells. Furthermore, the fraction of CD4+CD25+Foxp3+ cells (Treg cells) was increased under the culture with MSCs from β-thalassemia, suggesting that MSCs from β-thalassemia exerts normal immunomodulatory function. In addition, β-thalassemia-derived MSCs expressed hematopoietic cytokines and supported hematopoiesis which were comparable to those from normal BM-dereived MSCs. In summary, β-thalassemia-derived MSCs exhibited normal phenotype, karyotype as well as normal immunomodulatory function, and autologous MSCs from patients with β-thalassemia may be an attractive source of stem cell in terms of hematopoietic support as well as immunomodulatory activity. Disclosures: No relevant conflicts of interest to declare.

Bone Marrow Derived Mesenchymal Stem Cells and Radiation Response: a Kinetic Study of Gene Response Using Microarray Analysis.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4575-4575
Author(s):  
Philippe Garrigou ◽  
Jean-Francois Mayol ◽  
Catherine Mouret ◽  
Christophe Delaunay ◽  
Michel Drouet ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Short Term ◽  
Hematopoietic Stem ◽  
Pai 1

Abstract Abstract 4575 Mesenchymal Stem cells (MSC) are an important radiosensitive component of the so called hematopoietic stem cell niche. Importantly this supportive microenvironment influences the stem cell repopulation capacity as well as the quiescent/non proliferative state of hematopoietic cells. Senescence is considered as a major process in MSC response following irradiation. However, other studies have reported in mice the reduction of the pool of bone marrow mesenchymal stem/progenitor cells following TBI independently of senescence. An altered osteoblastic differentiation was pointed out in these studies. Furthermore, MSC have been shown to be involved in the repair of ionizing radiation damage of distant epithelial sites which requires adherence genes mitigation. The aim of this study was to clarify some of these points using an in vitro model of irradiation and short term culture. Briefly, confluent human BM-MSC were irradiated at the dose of 2.5 Gy (dose rate: 95 cGy.min-1) and immediately put into culture (Minimum essential medium supplemented with 10% FCS and 10 μg/ml of ciprofloxacin, penicillin and streptomycin). Six, 12, 24, 48 and 72 hours after irradiation, cells were harvested and lysed. Total RNAs were purified using the automatic Qiacube system (Qiagen,Courtaboeuf, France) and processed on DNA microarray scanner (Agilent technologies Inc.) according to supplier's recommendations. Data were analyzed with GeneSpring GX Expresion Analysis software version 10.0 (Agilent) in order to identify the transduction pathways involved. No apoptosis was observed during this short term incubation. Among other genes we identified plasminogen activator inhibitor 1 (PAI-1) as a factor highly upregulated after irradiation, in addition to CD151. This is in accordance with MSC response to nutrient-poor, hypoxic stress environment (Copland et al, Stem cells 2009). As MSC are radiosensitive cells, this may indicate that PAI impacts MSC survival through the mitigation of their adhesiveness to surrounding matrices. As PAI-1 is an important factor involved in the balance of blood coagulation and fibrinolysis as well as in the regulation of angiogenesis, one may speculate the consequences of PAI-1 release from MSC on blood homeostasis. Work is going on to describe the main response target genes. This could allow us to identify therapeutic strategies based on ex-vivo or in vivo manipulation of MSC in a purpose of tissue remodelling. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Synergy of molecularly mobile polyrotaxane surfaces with endothelial cell co-culture for mesenchymal stem cell mineralization

RSC Advances ◽  
2021 ◽  
Vol 11 (30) ◽  
pp. 18685-18692
Author(s):  
Hiroki Masuda ◽  
Yoshinori Arisaka ◽  
Masahiro Hakariya ◽  
Takanori Iwata ◽  
Tetsuya Yoda ◽  
...  
Keyword(s):  
Stem Cells ◽  
Endothelial Cells ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Endothelial Cell ◽  
Mesenchymal Stem Cell ◽  
Molecular Mobility ◽  
Culture System

Molecular mobility of polyrotaxane surfaces promoted mineralization in a co-culture system of mesenchymal stem cells and endothelial cells.

scholarly journals Cytotoxic Effect of Hypoxic Environment in Mesenchymal Stem Cell

Proceedings ◽  
2018 ◽  
Vol 2 (25) ◽  
pp. 1592
Author(s):  
Sevil Özer ◽  
H. Seda Vatansever ◽  
Feyzan Özdal-Kurt
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Mesenchymal Stem Cell ◽  
Human Mesenchymal Stem Cells ◽  
Hypoxic Condition ◽  
Immunoperoxidase Technique ◽  
Cellular Functions ◽  
Marrow Mesenchymal Stem Cells

Bone marrow mesenchymal stem cells (BM-MSCs) are used to repair hypoxic or ischemic tissue. After hypoxic the level of ATP is decreases, cellular functions do not continue and apoptosis or necrosis occur. Apoptosis is a progress of programmed cell death that occurs in normal or pathological conditions. In this study, we were investigated the hypoxic effect on apoptosis in mesenchymal stem cell. Bone marrow-derived stem cells were cultured in hypoxic (1% or 3%) or normoxic conditions 24, 96 well plates for 36 h. Cell viability was shown by MTT assay on 36 h. After fixation of cells with 4% paraformaldehyde, distributions of caspase-3, Bcl-2 and Bax with indirect immunoperoxidase technique, apoptotic cells with TUNEL assay were investigated. All staining results were evaluated using H-score analyses method with ANOVA, statistically. As a result, hypoxic condition was toxic for human mesenchymal stem cells and the number of death cell was higher in that than normoxic condition.

Neuroprotective effect of bone marrow derived mesenchymal stem cell secretome in 6-OHDA-induced Parkinson's disease

2021 ◽  
Author(s):  
Dhruv Mahendru ◽  
Ashish Jain ◽  
Seema Bansal ◽  
Deepti Malik ◽  
Neha Dhir ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Stem Cells ◽  
Bone Marrow ◽  
Parkinson's Disease ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Neuroprotective Effect ◽  
Behavioral Parameters ◽  
Old Rats ◽  
6 Hydroxydopamine

Aim: The aim of the study was to evaluate the neuroprotective effect of bone marrow stem cell secretome in the 6-hydroxydopamine (6-OHDA) model of Parkinson's disease. Materials & methods: Secretome prepared from mesenchymal stem cells of 3-month-old rats was injected daily for 7 days between days 7 and 14 after 6-OHDA administration. After 14 days, various neurobehavioral parameters were conducted. These behavioral parameters were further correlated with biochemical and molecular findings. Results & conclusion: Impaired neurobehavioral parameters and increased inflammatory, oxidative stress and apoptotic markers in the 6-OHDA group were significantly modulated by secretome-treated rats. In conclusion, mesenchymal stem cells-derived secretome could be further explored for the management of Parkinson's disease.

scholarly journals Comparison of the Proliferation and Differentiation Potential of Human Urine-, Placenta Decidua Basalis-, and Bone Marrow-Derived Stem Cells

2018 ◽  
Vol 2018 ◽  
pp. 1-11 ◽  
Author(s):  
Chengguang Wu ◽  
Long Chen ◽  
Yi-zhou Huang ◽  
Yongcan Huang ◽  
Ornella Parolini ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Stem Cell Differentiation ◽  
Cell Types ◽  
Stem Cell Marker ◽  
Differentiation Potential ◽  
Multipotent Stem Cells ◽  
Decidua Basalis

Human multipotent stem cell-based therapies have shown remarkable potential in regenerative medicine and tissue engineering applications due to their abilities of self-renewal and differentiation into multiple adult cell types under appropriate conditions. Presently, human multipotent stem cells can be isolated from different sources, but variation among their basic biology can result in suboptimal selection of seed cells in preclinical and clinical research. Thus, the goal of this study was to compare the biological characteristics of multipotent stem cells isolated from human bone marrow, placental decidua basalis, and urine, respectively. First, we found that urine-derived stem cells (USCs) displayed different morphologies compared with other stem cell types. USCs and placenta decidua basalis-derived mesenchymal stem cells (PDB-MSCs) had superior proliferation ability in contrast to bone marrow-derived mesenchymal stem cells (BMSCs); these cells grew to have the highest colony-forming unit (CFU) counts. In phenotypic analysis using flow cytometry, similarity among all stem cell marker expression was found, excluding CD29 and CD105. Regarding stem cell differentiation capability, USCs were observed to have better adipogenic and endothelial abilities as well as vascularization potential compared to BMSCs and PDB-MSCs. As for osteogenic and chondrogenic induction, BMSCs were superior to all three stem cell types. Future therapeutic indications and clinical applications of BMSCs, PDB-MSCs, and USCs should be based on their characteristics, such as growth kinetics and differentiation capabilities.

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