Modeling the functional heterogeneity of leukemia stem cells: role of STAT5 in leukemia stem cell self-renewal

Blood ◽  
2009 ◽  
Vol 114 (19) ◽  
pp. 3983-3993 ◽  
Author(s):  
Michael Heuser ◽  
Laura M. Sly ◽  
Bob Argiropoulos ◽  
Florian Kuchenbauer ◽  
Courteney Lai ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Constitutive Expression ◽  
Leukemia Stem Cells ◽  
Functional Heterogeneity ◽  
Self Renewal ◽  
Leukemia Stem Cell ◽  
Gm Csf ◽  
Stat Signaling

Abstract Although the cancer stem cell (CSC) concept implies that CSCs are rare, recent reports suggest that CSCs may be frequent in some cancers. We hypothesized that the proportion of leukemia stem cells would vary as a function of the number of dysregulated pathways. Constitutive expression of MN1 served as a 1-oncogene model, and coexpression of MN1 and a HOX gene served as a 2-oncogene model. Leukemia-initiating cell (LIC) number and in vitro expansion potential of LICs were functionally assessed by limiting dilution analyses. LIC expansion potential was 132-fold increased in the 2- compared with the 1-oncogene model, although phenotypically, both leukemias were similar. The 2-oncogene model was characterized by granulocyte-macrophage colony-stimulating factor (GM-CSF) hypersensitivity and activated STAT/ERK signaling. GM-CSF hypersensitivity of the 2-oncogene model (MN1/HOXA9) was lost in Stat5b−/− cells, and the LIC expansion potential was reduced by 86- and 28-fold in Stat5b−/− and Stat1−/− cells, respectively. Interestingly, in 201 acute myeloid leukemia (AML) patients, coexpression of MN1 and HOXA9 was restricted to patients with the poorest prognosis and was associated with highly active STAT signaling. Our data demonstrate the functional heterogeneity of LICs and show that STAT signaling is critical for leukemia stem cell self-renewal in MN1- and HOXA9-expressing leukemias.

scholarly journals Anlotinib Induced Apoptosis and Regulated the Chemosensitivity and Immune-Related Properties of Leukemia Stem Cells By Inhibiting JAK2-STAT3/5 Signaling

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 12-12
Author(s):  
Long Liu ◽  
Long Yue Jiang ◽  
Bing Xu
Keyword(s):  
Stem Cells ◽  
Tyrosine Kinase ◽  
Immune Evasion ◽  
Tyrosine Kinases ◽  
Receptor Expression ◽  
The Self ◽  
Leukemia Stem Cells ◽  
Self Renewal ◽  
Stat Signaling ◽  
Apoptotic Proteins

Acute myeloid leukemia (AML) is derived from small populations of leukemia stem cells (LSCs) characterized by the self-renewal and chemoresistant properties. Residual LSCs after chemotherapy remain as the critical barriers to cure. Clearance of LSCs might rationally lead to an improvement of clinical outcome. Recently studies showed that JAK/STAT signaling play an important role in the self-renewal of AML-LSCs due to increased growth factor (GF) receptor expression such as c-kit, FLT3, CD123 and altered GF signaling by activating tyrosine kinases. Therefore, targeting such tyrosine kinases might be a strategy to eliminate LSCs. Anlotinib displayed its anti-tumor activity in lung cancer by targeting tyrosine kinase of VEGFR, FGFR, PDGFR and c-kit. However, whether anlotinib could inhibit the GF receptor-related tyrosine kinase overactivation and its downstream JAK-STAT signaling, and subsequently kill LSCs or regulate LSCs biology remains largely unknown. To explore whether anlotinib could exert effective ani-LSCs activity, we treated LSC like cell lines (CD34+CD38-KG-1 and Kasumi-1) with anlotinib, and found anlotinib could effectively induce apoptosis of LSC-like cells in a dose- and time-dependent manner. Similar results were observed in primary CD34+CD38-AML LSCs; notably, anlotinib did not significantly kill normal CD34+ cells in vitro. Additionally, the anti-LSC activity of anlotinib was further confirmed in the xenograft mouse model by injection of Kasumi cells (LSC-like cell line) into irradiated female BALB/c nude mice. To determine whether anlotinib could inhibit the over activation of the GF receptor-related tyrosine kinase, we performed western blot at 12h after anlotinib treatment when LSC-like cells did not showed significant apoptosis. As a result, anlotinib inhibit c-kit phosphorylation and JAK2 activation. Intriguingly, unlike JAK2 inhibitors, anlotinib could not only the inhibit phosphorylation of STAT3 and STAT5 but also downregulate their expression. Chemoresistance and immune evasion were the key features of LSCs, JAK2-STAT3/5 signaling was reported to involved in chemoresistance by upregulating anti-apoptotic proteins such as Bcl-2 ,Mcl-1 and also involved in immune escape by inducing immune suppressive molecules such as PD-L1 ,TGF-β.Thus we evaluated Bcl-2 expression and found a significant decrease in LSC-likes cells after anlotinib treatment. Similarly, PD-L1 and TGF-β were also significantly downregulated after anlotinib treatment. In conclusion, anlotinib not only displayed the effective anti-LSCs activity but also might regulate the chemoresistance and immune evasion of LSC by downregulating the anti-apoptotic proteins and suppressive molecules such as PD-L1, TGF-β respectively. Consequently, anlotinib might has the potential to contribute to a deeper clearance of LSCs by combining with chemotherapy or immunotherapy. Disclosures No relevant conflicts of interest to declare.

Inhibition of Chronic Myelogenous Leukemia Stem Cells with Novel Wnt Antagonists.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 238-238 ◽  
Author(s):  
Edward Kavalerchik ◽  
Jason Gotlib ◽  
Ifat Geron ◽  
Annelie Abrahamsson ◽  
Wolfgang Wrasidlo ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Chronic Myelogenous Leukemia ◽  
Reporter Gene ◽  
Myelogenous Leukemia ◽  
Beta Catenin ◽  
Self Renewal ◽  
Leukemia Stem Cell ◽  
Wnt Inhibitors

Abstract Introduction A growing proportion of chronic myelogenous leukemia (CML) patients show evidence of disease progression. Recent research suggests that leukemia stem cells (LSC) that share phenotypic characteristics with granulocyte-macrophage progenitors (GMP) are involved in CML progression. These LSC have aberrantly gained self-renewal capacity as a result of enhanced Wnt/beta-catenin signaling. We assayed the capacity of novel Wnt/beta-catenin antagonists to inhibit CML LSC. Methods To assay the efficacy of a novel Wnt inhibitor, MC-001, HEK293 cells were transfected with a Wnt-dependent reporter gene and expression plasmid for Dsh. After 16h, the cells were treated for 24 h with MCC-001, a novel marine sponge derived inhibitor, at varying concentrations and the reporter gene activity was measured. All cells were also transfected with a b-gal reporter gene to control for transfection efficiency. To assess the effects of MCC-001 and other Wnt inhibitors on Wnt/beta-catenin induced self-renewal, hematopoietic stem cells (HSC), GMP and lineage positive cells from normal (n=8) and advanced phase CML (n=8) peripheral blood and marrow (n=8) were clone sorted with the aid of a FACS Aria into methocult media (Stem Cell Technologies) with or without Wnt inhibitors including recombinant Dkk1, lentiviral axin or MCC-001. On day 10, individual colonies were plucked and replated in new methylcellulose and the replating efficiency determined at day 10. To establish an in vivo CML LSC model, HSC, GMP and lineage positive cells were transduced with a lentiviral luciferase GFP for 48 hours and transplanted intrahepatically into newborn immunocompromised mice (RAG2−/−gamma−/−) mice that facilitate high levels of human hematopoietic progenitor engraftment. Results The HEK293 beta-catenin reporter assay revealed that the MC-001 IC50 was 2.1 microM. In comparative Wnt inhibitor replating assays (n=8), recombinant Dkk1 did not inhibit CML HSC (n=8) while lentiviral axin and MCC-001 (at 2 and 10 microM) inhibited both CML HSC and CML GMP at doses that spared normal HSC replating (Figure 1). Transplantation of CML HSC, GMP and lineage positive cells into RAG2−/−gamma−/− mice demonstrated that only CML GMP provided serial transplantation potential and thus, were enriched for the LSC population (Figure 2). Conclusions Selective Wnt/beta-catenin inhibition with a marine sponge derived beta-catenin antagonist, MCC-001, blocks in vitro replating capacity of CML LSC at doses that spare normal HSC. Current experiments focus on in vivo inhibition of LSC self-renewal with novel Wnt inhibitors in a robust CML LSC bioluminescent imaging model (Figure 2). Figure 1. Chronic Myelogenous Leukemia Stem Cell Inhibition with MCC-001: A novel β-catenin Inhibitor Figure 1. Chronic Myelogenous Leukemia Stem Cell Inhibition with MCC-001: A novel β-catenin Inhibitor Figure 2. Bioluminescent Chronic Myelogenous Leukemia Stem Cell Transplantation Model. Figure 2. Bioluminescent Chronic Myelogenous Leukemia Stem Cell Transplantation Model.

Niche-Based Screening Reveals Leukemia Stem Cell Specific Therapeutics

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 760-760
Author(s):  
Kimberly A. Hartwell ◽  
Peter G. Miller ◽  
Alison L. Stewart ◽  
Alissa R. Kahn ◽  
David J. Logan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Drug Discovery ◽  
Small Molecules ◽  
Small Molecule ◽  
High Throughput ◽  
Hematopoietic Cells ◽  
Leukemia Stem Cells

Abstract Abstract 760 Recent insights into the molecular and cellular processes that drive leukemia have called attention to the limitations intrinsic to traditional drug discovery approaches. To date, the majority of cell-based functional screens have relied on probing cell lines in vitro in isolation to identify compounds that decrease cellular viability. The development of novel therapeutics with greater efficacy and decreased toxicity will require the identification of small molecules that selectively target leukemia stem cells (LSCs) within the context of their microenvironment, while sparing normal cells. We hypothesized that it would be possible to systematically identify LSC susceptibilities by modeling key elements of bone marrow niche interactions in high throughput format. We tested this hypothesis by creating and optimizing an assay in which primary murine stem cell-enriched leukemia cells are plated on bone marrow stromal cells in 384-well format, and examined by a high content image-based readout of cobblestoning, an in vitro morphological surrogate of cell health and self-renewal. AML cells cultured in this way maintained their ability to reinitiate disease in mice with as few as 100 cells. 14,720 small molecule probes across diverse chemical space were screened at 5uM in our assay. Retest screening was performed in the presence of two different bone marrow stromal types in parallel, OP9s and primary mesenchymal stem cells (MSCs). Greater than 60% of primary screen hits positively retested (dose response with IC50 at or below 5 μM) on both types of stroma. Compounds that inhibited leukemic cobblestoning merely by killing the stroma were identified by CellTiter-Glo viability analysis and excluded. Compounds that killed normal primary hematopoietic stem and progenitor cell inputs, as assessed by a related co-culture screen, were also excluded. Selectivity for leukemia over normal hematopoietic cells was additionally examined in vitro by comingling these cells on stroma within the same wells. Primary human CD34+ AML leukemia and normal CD34+ cord blood cells were also tested, by way of the 5 week cobblestone area forming cell (CAFC) assay. Additionally, preliminary studies of human AML cells pulse-treated with small molecules ex vivo, followed by in vivo transplantation, provided further evidence of potent leukemia kill across genotypes. A biologically complex functional approach to drug discovery, such as the novel method described here, has previously been thought impossible, due to presumed incompatibility with high throughput scale. We show that it is possible, and that it bears fruit in a first pilot screen. By these means, we discover small molecule perturbants that act selectively in the context of the microenvironment to kill LSCs while sparing stroma and normal hematopoietic cells. Some hits act cell autonomously, and some do not, as evidenced by observed leukemia kill when only the stromal support cells are treated prior to the plating of leukemia. Some hits are known, such as parthenolide and celastrol, and some are previously underappreciated, such as HMG-CoA reductase inhibition. Others are entirely new, and would not have been revealed by conventional approaches to therapeutic discovery. We therefore present a powerful new approach, and identify drug candidates with the potential to selectively target leukemia stem cells in clinical patients. Disclosures: No relevant conflicts of interest to declare.

A Highly Selective Anti-ROR1 Monoclonal Antibody Inhibits Human Acute Myeloid Leukemia CD34+ Cell Survival and Self-Renewal.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2560-2560
Author(s):  
Larissa Balaian ◽  
Anil Sadarangani ◽  
George F. Widhopf ◽  
Rui-kun Zhong ◽  
Charles Prussak ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Myeloid Leukemia ◽  
The Self ◽  
Self Renewal ◽  
Leukemia Stem Cell ◽  
Cd34 Cells ◽  
Selective Expression ◽  
Dose Dependent ◽  
Acute Myeloid

Abstract Abstract 2560 The mammalian orphan receptor tyrosine kinase-1 (ROR1) is expressed in a wide-variety of tissues during early embryonic development. By the late stages of embryogenesis the expression of this developmentally important protein is greatly diminished. Although not expressed in the tissues of post-partum animals, the ROR1 protein is expressed on neoplastic cells in chronic lymphocytic leukemia (CLL), some B-cell malignancies, and a variety of different carcinomas. We examined for expression of ROR1 in primary acute myeloid leukemia (AML) cells harvested from marrow aspirates and their normal counterparts by whole transcriptome paired-end RNA sequencing and by flow-cytometric analyses. These studies revealed selective expression of ROR1 in 62 (35%) of 179 AML samples examined. Many of these samples were found to have cells that co-expressed ROR1 and CD34, suggesting that ROR1 was present on the self-renewing leukemia stem-cell population, which resides in the marrow niche and potentially accounts for resistance to many cytotoxic drugs used in therapy. We investigated the activity of a chimeric anti-ROR1 mAb found effective in clearing CLL cells (UC99961) on AML expansion, growth, and renewal in a leukemia-stem-cell supportive niche assay. Mouse marrow cells lines SL/SL and M2–10B4 (transfected to produce hSCF,hIL3 and hIL3, hG-CSF respectively) were mixed 1:1 after mitomycin-C treatment, and used as a SLM2 stromal monolayer. CD34+ cells were selected from ROR1-positive (n=6) or negative (n=4) AML primary samples. As a normal control, CD34+ cells from cord blood (CB) were used (CB, n=3). In some experiments CD34+ cells were transfected with a GLP-lentivirus prior to co-culture. At the initiation of the co-culture, 10–50 μg/ml of the chimeric anti-ROR-1 mAb (UC99961) or control hIgG were added to the cultures. Two weeks after co-culture initiation, both stromal attached and floating cells were collected and their survival investigated by colony forming assay in methylcellulose. The UC99961 mAb was not cytotoxic to CB or ROR1-negative AML samples. In contrast, the UC99961 mAb provided a dose-dependent inhibition of colony formation for all ROR-1-positive AML samples examined. These results demonstrate the in vitro anti-leukemic specificity of this anti-ROR1 mAb in down-regulating AML stem and progenitor cell populations, without effecting normal CD34+ stem cells. To analyze the effect of ROR1 ligation on AML stem cell populations exclusively, AML self-renewal assays (2-ry colonies) were performed. In these studies, ROR1–positive AML samples were divided based on their response to mAb treatment. Half of the samples (n=3; 50%) demonstrated statistically significant (up to 90%) dose-dependent decreases in colony formation. However, another half was non-responsive and no correlation was found between ROR1 expression on leukemia CD34+ cells and response to anti-ROR1 mAb treatment in the self-renewal assays. Again UC99961 mAb treatment did not negatively impact CD34+ cells from CB or ROR1-negative AML, confirming the specificity and selective toxicity of the mAb for ROR1+ AML stem cells. These studies reveal selective expression of ROR1 on leukemia-stem-cells of large subset of AML patients. Furthermore, this study demonstrates that an anti-ROR1 mAb (UC99961) can inhibit survival and self-renewal in LSC supportive niche assays. Targeted ROR1 inhibition may represent a vital component of therapeutic strategies aimed at eradicating therapeutically recalcitrant malignant stem cells in AML and potentially other refractory cancer-stem-cell-driven malignancies. Disclosures: No relevant conflicts of interest to declare.

Long Term Maintenance of Myeloid Leukemia Stem Cell-Like Populations Cultured with Mesenchymal Stromal Cells (MSC)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3546-3546
Author(s):  
Sawa Ito ◽  
A. John Barrett ◽  
Andre Larochelle ◽  
Nancy F. Hensel ◽  
Keyvan Keyvanfar ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Stem Cell ◽  
Culture Conditions ◽  
Myelogenous Leukemia ◽  
Cell Cycle Analysis ◽  
Remission Induction ◽  
Leukemia Stem Cell

Abstract Abstract 3546 Because MSC support the growth and the differentiation of normal hematopoietic stem cells we hypothesized that MSC might also support leukemia cells, in particular leukemia stem cells (LSC) in vitro. We cultured blast cells from patients with acute myelogenous leukemia (AML) in liquid medium to study persistence of stem-cell-like and differentiated leukemia cell populations by flow cytometry, with and without MSC and additional growth factors. Cryopresrerved peripheral blood mononuclear cells (PBMC) were obtained from 6 AML patients (mean Age 47, range 23–74). Leukemia blasts were isolated by sorting live (propidium iodide (PI)-negative) CD34+ lineage (CD2+, CD3+, CD14+ and CD19+) -negative cells using a FACS ARIA II cell sorter (BD). Sorted blasts (2.5 ×105 cells) were co-cultured with an equal number of irradiated MSC derived from healthy donor bone marrow in RPMI medium supplemented with 10% human serum, with or without a human cytokine (CYTO) mixture (50 ng/ml interleukin 3, 150 ng/ml stem cell factor, and 150ng/ml Flt-3 ligand). MSC were replenished every two weeks. The phenotype of cultured cells was analyzed weekly using fluorescently-conjugated monoclonal antibodies against CD34, CD38, and CD45, plus the lineage panel and a dead cell exclusion dye Cell cycle analysis with Hoeschst 33342 and Pyronin Y was performed on cells co-stained with CD34, CD45 and PI. Primary leukemia samples were phenotypically heterogeneous with respect to proportions of cells (co-)staining for CD34 and CD38 as previously reported: three samples showed CD34+CD38- predominance (LSC-like leukemia), and three were CD34+CD38+ (common myeloid progenitor (CMP)-like leukemia). LSC-like leukemia maintained viable CD34+CD38- cells for at least 6 weeks when co-cultured with MSC alone, in contrast to cultures with cytokines or medium only which showed rapid decline in the LSC populations and no prolonged maintenance of viable cells (p=0.0005) (Figure, left panel). CMP-like leukemia maintained their CD34+CD38+ phenotype when co-cultured with MSC alone but persistence of this subset was not significantly different from the other culture conditions (p=0.5) and no culture remained viable after 4 weeks (Figure, right panel). Cell cycle analysis showed that co-culture with MSC maintained CD34+ blasts in G0 significantly more than other culture conditions (P<0.0001). We conclude that MSC support the maintenance of a leukemia stem cell phenotype in a long- term (6 week) in vitro culture system. The differential capacity of MSC to support LSC- like and CMP- like leukemia may be associated with the different frequency of leukemia initiating cells within each leukemic blast population. NSG mice xenotranplant model experiments are ongoing to confirm this hypothesis. Co-culture of LSC with MSC represents a simple approach to maintain LSC in vitro and could be utilized to screen the drug targeting LSCs. Further study of the effect of MSC on LSC would elucidate a potential mechanism whereby the marrow microenvironment serves as a reservoir of persisting leukemia after remission induction chemotherapy. Disclosures: No relevant conflicts of interest to declare.

scholarly journals All-trans retinoic acid synergizes with FLT3 inhibition to eliminate FLT3/ITD+ leukemia stem cells in vitro and in vivo

Blood ◽  
2016 ◽  
Vol 127 (23) ◽  
pp. 2867-2878 ◽  
Author(s):  
Hayley S. Ma ◽  
Sarah M. Greenblatt ◽  
Courtney M. Shirley ◽  
Amy S. Duffield ◽  
J. Kyle Bruner ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell Population ◽  
Leukemia Stem Cells ◽  
Synergistic Activity ◽  
Leukemia Stem Cell ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid ◽  
Key Points

Key Points ATRA and FLT3 TKIs have synergistic activity against FLT3/ITD+ AML cell lines and patient samples. Combination reduces the leukemia stem cell population and improves survival in genetic and xenograft AML mouse models.

scholarly journals Are MSCs Stem Cells? Demonstration of in Vitro and in Vivo Stem Cell Properties of Highly Enriched linneg/CD45neg/CD271pos/CD140alow/Neg Mesenchymal Stromal Cells from Adult Human Bone Marrow

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4374-4374
Author(s):  
Roshanak Ghazanfari ◽  
Hongzhe Li ◽  
Dimitra Zacharaki ◽  
Simón Méndez-Ferrer ◽  
Stefan Scheding
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Differentiation Potential ◽  
Self Renewal ◽  
Adult Human

Abstract Human bone marrow contains a rare population of non-hematopoietic mesenchymal stromal cells (BM-MSC) with multilineage differentiation capacity, which are essential constituents of the hematopoietic microenvironment. Self-renewal and differentiation are the two key properties of somatic stem cells, however, stem cell properties of human adult BM-MSC have not been demonstrated conclusively yet. We have previously shown that low/negative expression of PDGFRα on linneg/CD45neg/CD271pos cells identified a highly enriched population of primary BM-MSC in adult human bone marrow (Li et al. Blood, 2013, 122:3699). Based on this work, the current study aimed to investigate the in-vitro and in-vivo stem cell properties of this putative stromal stem cell population. The in-vitro clonogenic potential of freshly sorted human linneg/CD45neg/CD271pos/PDGFRlow/neg cells was evaluated by utilizing the CFU-F assay as well as the recently-developed mesensphere assay, which enables MSC amplification while preserving an immature phenotype (Isern et al, Cell Reports 2013, 30: 1714-24). Comparable colony frequencies were obtained with both assays (19.3 ± 2 and 17.5 ± 2.3 CFU-F and spheres per 100 plated cells, respectively, n=6, p=0.19). In order to test whether both assays identified the same population of clonogenic cells, colonies and spheres were replated under both conditions for up to three generations. The results showed comparable capacities of CFU-F and mesenspheres to form secondary and tertiary CFU-F and spheres. In-vitro self-renewal as indicated by increasing numbers of CFU-F and spheres (416.6 ± 431.7-fold and 49.5 ± 65.7-fold, respectively, n=3) was observed up to the third generation and decreased thereafter. The total number of generations was five (CFU-F) and six (spheres). In-vitro differentiation assays with both, CFU-F- and sphere-derived cells (tested until passage three) demonstrated tri-lineage differentiation potential (adipocytes, osteoblasts, chondrocytes). In addition, CFU-Fs and spheres had comparable surface marker profiles (CD73, CD90, CD105, and HLA-ABC positive; CD31, CD34 and HLA-DR negative), except for CD90, which was higher expressed on CFU-Fs. To investigate in-vivo self-renewal and differentiation potential of the putative stromal stem cells, linneg/CD45neg/CD271pos/PDGFRlow/neg -derived CFU-F and spheres were serially transplanted s.c into NSG mice. After 8 weeks, implants were harvested, human cells were FACS-isolated (CD90 and CD105 expression), and re-assayed under CFU-F and sphere conditions. Whereas in-vivo self-renewal of CFU-F could not be shown (111.5 ± 36 –fold decrease in total CFU-F numbers after primary transplantation, n=3), sphere self-renewal was clearly demonstrated by increased numbers of spheres after primary as well as secondary transplantation (1.13 ± 0.05 and 2.06 ± 0.26 –fold, respectively, n=3), which is remarkable given the fact that the number of recovered human cells is underestimated due to the isolation approach. Here, confirming GFP-marking experiments are ongoing. Finally, preliminary data indicate that linneg/CD45neg/CD271pos/PDGFRlow/neg –derived spheres display full in-vivo differentiation capacity in primary and secondary transplantations. Taken together, our data demonstrate - for the first time - that primary human linneg/CD45neg/CD271pos/PDGFRlow/neg cells meet stringent stem cell criteria, i.e. in-vitro and in-vivo self-renewal and differentiation. These findings answer the long-open question of the potential stem cell properties of adult human MSC and will enable to better understand the properties of native BM-MSC and their biological role in the bone marrow. Disclosures No relevant conflicts of interest to declare.

scholarly journals Advances and Applications in Stem Cell Biology

2012 ◽  
Vol 46 (2) ◽  
pp. 75-80
Author(s):  
Shamoli Bhattacharyya
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Biology ◽  
Adult Stem Cells ◽  
Great Promise ◽  
Stem Cell Biology ◽  
Self Renewal ◽  
Main Challenge ◽  
Basic Biology

ABSTRACT Mesenchymal stem cells have shown great promise as the source of adult stem cells for regenerative medicine. Present research efforts are directed at isolating these cells from various sources, growing them in vitro and maintaining their pluripotency as well as capacity for self renewal. It is crucial to identify the regulatory molecules which directly or indirectly control the proliferative status or influence the niche microenvironment. The main challenge is to understand the basic biology of the stem cells and manipulate them for further therapeutic applications. Considering their malignant potential, stem cells may be a double edged sword. While the benefits of these cells need to be harnessed judiciously, a significant amount of research is required before embarking on widespread use of this tool for the benefit of humanity. How to cite this article Bhattacharyya S. Advances and Applications in Stem Cell Biology. J Postgrad Med Edu Res 2012;46(2):75-80.

scholarly journals Loss of Phospholipase C Gamma 1 (PLCG1) Impairs AML1-ETO Induced Leukemia Stem Cell Self-Renewal

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 531-531
Author(s):  
Tina M. Schnoeder ◽  
Adrian Schwarzer ◽  
Florian Perner ◽  
Joanna Kirkpatrick ◽  
Anna Dolnik ◽  
...  
Keyword(s):  
Stem Cell ◽  
Phospholipase C ◽  
Median Survival ◽  
Colony Formation ◽  
Cellular Functions ◽  
Self Renewal ◽  
Leukemia Stem Cell ◽  
Gene Sets ◽  
Disease Penetrance

Chromosomal translocations found in acute myeloid leukemia (AML) can generate oncogenic fusions with aberrant epigenetic and transcriptional functions. However, direct therapeutic targeting of leukemia fusion proteins has not been accomplished so far. Although high remission rates can be induced in patients diagnosed with AML1-ETO/t(8;21)-positive AML only half of them achieve long-term disease-free survival (Papaemmanuiel et al., NEJM, 2016). In the other half of these patients, the disease maintaining leukemia stem cell (LSC) clone is not eliminated by chemotherapy. A functional characteristic of LSCs is unlimited self-renewal capacity and several signaling pathways have been identified that maintain stem cell self-renewal. Targeting the oncogene induced self-renewal capacity of LSCs has great potential to eliminate the malignant clone and prevent relapse. To identify oncogenic cellular functions with relevance for LSC self-renewal, we performed global proteome profiling in murine AML1-ETO9a (AE) compared to MLL-AF9 (MA9) driven LSCs. Gene set enrichment analyses revealed a significant enrichment of calcium-dependent cellular functions and Phospholipase C (PLC)-signaling in AE LSCs. These data could be confirmed in sorted CD34+ blasts from AE-positive AML when compared to non-AE-AML. All PLC family members are regulators of Ca2+ homeostasis. However, when analyzing published AML gene expression datasets we found exclusively PLCG1 to be highly expressed in t(8;21) AML. Conditional activation of AE in embryonic stem cells resulted in induction of PLCG1 expression and PLCG1 was identified as a direct target of the AE fusion by ChIP-sequencing in AE-positive Kasumi-1 cells.Here, PLCG1 depletion resulted in reduced Ca2+ release, impaired proliferation and reduced colony formation in vitro. In a xenograft model, inactivation of PLCG1 resulted not only in delayed disease development (median survival shNT vs. shPLCG1: 135 days vs. not reached, p=0.02) but also in reduction of disease penetrance by 87%. Consistent with these results, transcriptome analysis revealed strong induction of gene sets related to myeloid differentiation and down-regulation of gene sets linked to proliferation, stemness and c-Myc targets. To confirm the functional role of PLCG1-signaling in AE-driven LSCs, we generated a new conditional knockout mouse model for Plcg1 and induced leukemia using the oncogenes AE and KRAS-G12D (AE/K). Genetic inactivation of Plcg1in vivo after engraftment of leukemic cells resulted in significant reduction of LSC numbers (p=0.04) and a reduction of disease penetrance by 67% in primary recipients. Isolated LSCs revealed induction of differentiation, loss of cell cycle activity and failed to re-establish disease in secondary recipients (Plcg1+/+ vs. Plcg1-/-: median survival 12 days vs. not reached; p=0.0001). In contrast, genetic deletion of Plcg1 appeared to be dispensable for normal murine HSC function during primary and secondary transplantation. Primary human t(8;21) AML cells (derived from 4 different donors) showed impaired colony forming capacity following PLCG1 inactivation in vitro irrespective of co-occurring mutations while colony formation of human CD34+ BM cells was not affected to a major extent. As Ca2+ signaling appeared deregulated in t(8;21) AML, we aimed to investigate the effects of pharmacologic Ca2+ inhibition as a tractable target downstream of PLCG1. To assess specifically for LSC function, we treated primary recipient mice with established AE/K-driven leukemia with the clinically approved calcineurin inhibitor ciclosporin (CsA), a compound that blocks intracellular Ca2+ release. CsA-treated animals showed reduction in total leukemic burden (spleen weight diluent vs. CsA, p=0.01) and LSC numbers (p=0.02). This resulted in increased survival of secondary recipient hosts (diluent vs. CsA: median 15 vs. 29 days, p=0.0002). These effects could not be observed for other oncogenes (e.g. MA9), confirming its specificity for AE-induced disease. Consistently, CsA treated primary human t(8;21)-positive AML blasts failed to form colonies in methylcellulose. In summary, our findings identified PLCG1-dependent Ca2+ signaling as a critical pathway for t(8;21) LSC maintenance and self-renewal. Most importantly, as PLCG1 is dispensable for maintenance of normal HSPCs, PLCG1 could serve as a novel therapeutic target in t(8;21) AML. Disclosures Döhner: Daiichi: Honoraria; Jazz: Honoraria; Novartis: Honoraria; Celgene: Honoraria; Janssen: Honoraria; CTI Biopharma: Consultancy, Honoraria. Bullinger:Novartis: Honoraria; Menarini: Honoraria; Jazz Pharmaceuticals: Honoraria; Abbvie: Honoraria; Astellas: Honoraria; Amgen: Honoraria; Seattle Genetics: Honoraria; Sanofi: Honoraria; Janssen: Honoraria; Hexal: Honoraria; Gilead: Honoraria; Daiichi Sankyo: Honoraria; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria; Bayer: Other: Financing of scientific research; Pfizer: Honoraria.

scholarly journals Characterization in mice of the stromal niche maintaining AT2 stem cell self-renewal in homeostasis and disease

2021 ◽  
Author(s):  
Sara Taghizadeh ◽  
Monika Heiner ◽  
Jochen Wilhelm ◽  
Susane Herold ◽  
Chengshui Chen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Mesenchymal Cell ◽  
Mesenchymal Cells ◽  
The Self ◽  
Loss Of Function ◽  
Self Renewal ◽  
Alveolar Epithelial

AbstractResident mesenchymal cells (rMCs defined as Cd31NegCd45NegEpcamNeg) control the self-renewal and differentiation of alveolar epithelial type 2 (AT2) stem cells in vitro. The identity of these rMCs is still elusive. Among them, Axin2Pos mesenchymal alveolar niche cells (MANCs), which are expressing Fgf7, have been previously described. We propose that an additional population of rMCs, expressing Fgf10 (called rMC-Sca1PosFgf10Pos) are equally important to maintain AT2 stem cell self-renewal.The alveolosphere model, based on the AT2-rMC co-culture in growth factor reduced Matrigel, was used to test the efficiency of different rMC subpopulations isolated by FACS from adult murine lung to sustain the self-renewal and differentiation of AT2 stem cells.We demonstrate that rMC-Sca1PosFgf10Pos cells are efficient to promote the self-renewal and differentiation of AT2 stem cells. Co-staining of adult lung for Fgf10 mRNA and Sftpc protein respectively, indicate that 28% of Fgf10Pos cells are located close to AT2 cells. Co-ISH for Fgf7 and Fgf10 indicate that these two populations do not significantly overlap. Gene arrays comparing rMC-Sca1PosAxin2Pos and rMC-Sca1PosFgf10Pos support that these two cell subsets express differential markers. In addition, rMC function is decreased in diabetic and obese ob/ob mutant compared to WT mice with a much stronger loss of function in males compared to females.In conclusion, rMC-Sca1PosFgf10Pos cells play important role in supporting AT2 stem cells self-renewal and differentiation. This result sheds a new light on the subpopulations of rMCs contributing to the AT2 stem cell niche in homeostasis and in the context of COVID-19 pathogenesis.Key messageWhat is already known about the subject?Resident mesenchymal cells (rMCs defined as Cd31NegCd45NegEpcamNeg) control the self-renewal and differentiation of alveolar epithelial type 2 (AT2) stem cells in vitro. The identity of these rMCs is still elusive. Among them, Axin2Pos mesenchymal alveolar niche cells (MANCs), which are expressing Fgf7, have been previously described.What does this study add?Our study shows that an additional population of rMCs, expressing Fgf10 (called rMC-Sca1PosFgf10Pos) is equally important to maintain AT2 stem cell self-renewal. rMC-Sca1PosFgf10Pos are LipidToxHigh and are located close to AT2s. In addition, rMC-Sca1PosFgf10Pos cells support AT2 stem cell self-renewal and differentiation thereby identifying these cells as bone fide functional lipofibroblasts (LIFs). We have previously reported that LIF can transdifferentiate into activated MYF in the context of bleomycin-induced fibrosis in mice [1] and that activated MYF isolated from the lungs of end stage idiopathic fibrosis human patients can respond to Metformin to undergo transdifferentiation back to the LIF phenotype [2]. We also show that the function of rMCs-Sca1Pos is negatively impacted by gender and obesity, which represent two major aggravating factors for COVID-19 pathogenesis, leading to either death or major complications after infection recovery such as lung fibrosis.How might this impact on clinical practice and future development?By establishing that rMC-Sca1PosFgf10Pos are different from the MANCs, our study opens the way for a new key mesenchymal cell population that should be targeted to either prevent or reverse fibrosis. In addition, as this population maintains the AT2 stem cells self-renewal and differentiation, such targeting will also allow to progressively recover the loss in respiratory function.

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