Advanced flow cytometric analysis of nanoparticle targeting to rare leukemic stem cells in peripheral human blood in a defined model system

2015 ◽  
Author(s):  
Christy L. Cooper ◽  
James F. Leary
Keyword(s):  
Stem Cells ◽  
Human Blood ◽  
Flow Cytometric Analysis ◽  
Model System ◽  
Leukemic Stem Cells ◽  
Flow Cytometric

Flow Cytometric Analysis of Brain Tumor Stem Cells

2018 ◽  
pp. 69-77
Author(s):  
Minomi K. Subapanditha ◽  
Ashley A. Adile ◽  
Chitra Venugopal ◽  
Sheila K. Singh
Keyword(s):  
Stem Cells ◽  
Brain Tumor ◽  
Flow Cytometric Analysis ◽  
Tumor Stem Cells ◽  
Flow Cytometric ◽  
Brain Tumor Stem Cells

High-resolution tracking of cell division suggests similar cell cycle kinetics of hematopoietic stem cells stimulated in vitro and in vivo

Blood ◽  
2000 ◽  
Vol 95 (3) ◽  
pp. 855-862 ◽  
Author(s):  
Robert A. J. Oostendorp ◽  
Julie Audet ◽  
Connie J. Eaves
Keyword(s):  
Stem Cells ◽  
Fetal Liver ◽  
Flow Cytometric Analysis ◽  
Hematopoietic Stem ◽  
Murine Bone Marrow ◽  
Flow Cytometric ◽  
Differentiation Status ◽  
Kinetics Of

The kinetics of proliferation of primitive murine bone marrow (BM) cells stimulated either in vitro with growth factors (fetal liver tyrosine kinase ligand 3 [FL], Steel factor [SF], and interleukin-11 [IL-11], or hyper–IL-6) or in vivo by factors active in myeloablated recipients were examined. Cells were first labeled with 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) and then incubated overnight prior to isolating CFSE+ cells. After 2 more days in culture, more than 90% of the in vivo lymphomyeloid repopulating activity was associated with the most fluorescent CFSE+ cells (ie, cells that had not yet divided), although this accounted for only 25% of the repopulating stem cells measured in the CFSE+ “start” population. After a total of 4 days in culture (1 day later), 15-fold more stem cells were detected (ie, 4-fold more than the day 1 input number), and these had become (and thereafter remained) exclusively associated with cells that had divided at least once in vitro. Flow cytometric analysis of CFSE+ cells recovered from the BM of transplanted mice indicated that these cells proliferated slightly faster (up to 5 divisions completed within 2 days and up to 8 divisions completed within 3 days in vivo versus 5 and 7 divisions, respectively, in vitro). FL, SF, and ligands which activate gp130 are thus efficient stimulators of transplantable stem cell self-renewal divisions in vitro. The accompanying failure of these cells to accumulate rapidly indicates important changes in their engraftment potential independent of accompanying changes in their differentiation status.

Multiplexed Staining of Live Human Embryonic Stem Cells for Flow Cytometric Analysis of Pluripotency Markers

2009 ◽  
Vol 18 (8) ◽  
pp. 1135-1140 ◽  
Author(s):  
Andrew B.J. Prowse ◽  
John Wilson ◽  
Geoffrey W. Osborne ◽  
Peter P. Gray ◽  
Ernst J. Wolvetang
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Flow Cytometric Analysis ◽  
Embryonic Stem ◽  
Flow Cytometric ◽  
Pluripotency Markers

Biological Characteristics of ZUC3 Antigen Expressed on Rat Bone Marrow Mesenchymal Stem Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4254-4254
Author(s):  
He Huang ◽  
Jing Zheng ◽  
Xiaoyu Lai ◽  
Junli Cao ◽  
Jianling Fan
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Flow Cytometric Analysis ◽  
Osteoblastic Differentiation ◽  
Significant Decline ◽  
Human Mscs ◽  
Down Regulation ◽  
Flow Cytometric ◽  
Marrow Mesenchymal Stem Cells

Abstract Objective: Bone marrow mesenchymal stem cells (MSCs) are widely studied in recent years. As an important part of cell identification, specific surface markers of MSCs have been paid a lot of attention to for long, but no breakthrough as yet. Monoclonal antibodies (McAb) against surface of certain cells have been used to characterize cell lineages. ZUC3, a novel murine McAb was produced by hybridoma technology previously, which was specifically reactive with both human MSCs and rat MSCs. Studying the expression of ZUC3 antigen on rat MSCs after passage and differentiation, it was to define whether ZUC3 antigen would be available for the identification of rat MSCs or their differentiation lineages. Methods: Rat MSCs isolated by a single step of adhesion to cell culture plastic, and purified via replacement of medium and a serial of passage, then the cells were identified by surface molecules CD90, CD44 and CD45 by flow cytometry. Enzyme immunocytochemistry and indirect immunofluorescence were used to evaluate the availability of ZUC3 expression by rat MSCs as a surface marker. Then further exploratory researches were carried out concerning ZUC3 expression by rat MSCs during passages (P1 to P5) and multiple differentiation (neuron, osteoblasts and adipocytes) in the certain condition. Results: Homogeneous rat MSCs could be obtained in vitro, which were uniformly positive for adhesion molecules CD90, CD44, and negative for CD45. The McAb was specifically reactive with rat MSCs as the positive cells were more than 99% by immunohistochemistry and immunofluorescence staining, and ZUC3 antigen located on the membrane of rat MSCs. The flow cytometric analysis show ZUC3 antigen expression by rat MSCs from P1 to P5 were all more than 85%. Analysis by multiple comparison, it was found some differences between P2 and P1 (93.95±2.44% v.s. 86.90±1.80%, P<0.01). The maximal expression was reached at P3 (97.10±1.25%), and the flow cytometric analysis showed a single symmetrical peak. Data of P4 (94.50±2.23%) population were slightly lower than P3 (P>0.05). By contrast, P5 (88.35±2.99%) showed a significant decline comparing with the former passages (P<0.01). Furthermore, rat MSCs could be successfully induced to differentiate into neuron-like cells, osteoblasts, and adipocytes and there was to some extent a downward trend of ZUC3 expression after differentiation (P<0.01). More than 90% rat MSCs could transform to an neuron-like appearance which were positive for NeuN, NF-M after treated with alpha-thioglycerol, and there was some downward degree of ZUC3 expression (97.77±1.03% to 80.07±2.70%, P<0.01). During the osteoblastic differentiation, it was observed an obvious down-regulation of ZUC3 expression from the 10th day (96.63±1.03% to 90.07±2.40%, P<0.01 ) and percentage on the 10th (90.07±2.40%), 15th (84.43±2.80%), 20th (64.53±7.63%) and 25th (53.40±10.02%) day were significantly lower than their anterior time respectively (P<0.05). The results of adipogenic differentiation after MSCs incubated with proper medium were similar to what observed during osteoblastic differentiation and ZUC3 expression were down-regulation on the 7th (84.33±2.70%), 14th (75.90±2.00%) and 21st (70.57±0.47%) day compared with their anterior dots respectively (P<0.01). Conclusion: ZUC3 antigen could be used for identification of rat MSCs. Significant decline of ZUC3 expression had be observed after rat MSCs were induced to differentiate along neuronal, osteoblastic and adipogenic pathways, which indicated that ZUC3 antigen would be a marker of progenitor.

Epigenetic Profiling Of Leukemia Stem Cells In a Model Of TET2/FLT3-Mutant AML

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 476-476
Author(s):  
Alan H. Shih ◽  
Yanwen Jiang ◽  
Kaitlyn Shank ◽  
Suveg Pandey ◽  
Agnes Viale ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Targeted Therapies ◽  
Peripheral Blood ◽  
Flow Cytometric Analysis ◽  
Leukemia Stem Cells ◽  
Self Renewal ◽  
In Vivo Models ◽  
Flow Cytometric

Specific combinations of Acute Myeloid Leukemia (AML) somatic mutations are associated with distinct clinical and biologic features. However, in vivo models do not exist for the majority of common, poor-prognosis genotypes. Concurrent mutations in FLT3 and TET2 are associated with adverse outcome. We hypothesized that activating mutations in FLT3 would cooperate with inactivating mutations in TET2to induce AML in vivo, and that we could investigate AML pathogenesis and therapeutic response using a genetic model of this poor-risk AML genotype. To understand how these genes cooperate to induce AML, we generated Vav+Tet2fl/flFlt3-ITD mice, which resulted in fully penetrant, lethal disease in all recipient mice. Flow cytometric analysis revealed expansion of mac1+ cells in the peripheral blood, with progressive expansion of a c-Kit+, blast population which was apparent in the blood and bone marrow at 28 days, leading to lethal AML in all Vav+Tet2fl/flFlt3-ITD mice with a median survival of 12 months. Consistent with genetic data demonstrating most AML patients have monoallelic TET2 mutations, Vav+Tet2fl/+Flt3-ITD mice also develop AML, suggesting haploinsufficiency for Tet2 is sufficient to cooperate with the Flt3-ITD mutation to induce AML. All mice developed leukocytosis (median 85K/uL), splenomegaly (median 554mg) and hepatomegaly (median 2900mg) with evidence of extramedullary disease cell infiltration by leukemic blasts. Flow cytometric analysis of stem/progenitor populations revealed expansion of the granulocyte-macrophage progenitor (GMP) population and the lin- sca+ kit+ (LSK) stem cell population. Detailed analysis of the LSK population revealed a decrease in the LT-HSC population (LSK CD150+ CD48-) that was replaced by a monomorphic CD48+ CD150- multipotent progenitor population. Given previous studies have shown that LSK and GMP cells can contain leukemia stem cells (LSC) in other models of AML, we performed secondary transplant studies with LSK and GMP populations. LSK (CD48+ CD150-) cells, but not GMP cells, were able to induce disease in secondary and tertiary recipients in vivo. In order to assess the sensitivity of Tet2/Flt3-mutant AML and specifically the LSCs, to targeted therapies, we treated primary and transplanted mice with chronic administration of AC220, a FLT3 inhibitor in late-stage clinical trials. AC220 treatment inhibited FLT3 signaling in vivo, and reduced peripheral blood counts/splenomegaly. However, FLT3 inhibition did not reduce the proportion of AML cells in the bone marrow and peripheral blood. AC220 therapy in vivo reduced the proportion of GMP cells, but not LSK cells, demonstrating LSCs in this model are resistant to FLT3-targeted anti-leukemic therapy. We hypothesized that Tet2/Flt3-mutant LSCs possess a distinct epigenetic/transcriptional signature that contributes to leukemic cell self-renewal and therapeutic resistance. We performed RNA-seq using the Lifetech Proton sequencer to profile the expression landscape of Vav+Tet2fl/flFlt3-ITD mutant LSKs compared to normal stem cells. We were able to obtain an average of 62 million reads per sample. We identified over 400 genes differentially expressed in LSCs relative to normal hematopoietic stem cells (FC>2.5, padj <0.05). Of note, we found that genes involved in normal myelo-erythroid differentiation, including GATA1, GATA2, and EPOR, were transcriptionally silenced in LSCs relative to normal stem cells, consistent with their the impaired differentiation and increased self-renewal observed in LSCs. Enhanced representation bisulfite sequencing revealed a subset of these genes were marked by increased promoter methylation. The number of hyper differentially methylated regions (HyperDMRs, 10% methylation difference, FDR<0.2) was significantly greater in Vav+Tet2fl/flFlt3-ITD cells (787 HyperDMRs) compared to Vav+Tet2fl/fl cells (76 DMRs) suggesting FLT3 activation and TET2 loss cooperate to alter the epigenetic landscape in hematopoietic cells. Our data demonstrate that TET and FLT3 mutations can cooperate to induce AML in vivo, with a defined LSC population that is resistant to targeted therapies and characterized by site-specific changes in DNA methylation and gene expression. Current studies are aimed to assess the functional role of specific gene targets in LSC survival, and at defining therapeutic liabilities that can be translated to the clinical context. Disclosures: No relevant conflicts of interest to declare.

The CXCR4 Antagonist AMD 3100 Induces Rapid Mobilization of Facilitating Cells and Hematopoietic Stem Cells in Mice

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4694-4694
Author(s):  
Hong Xu ◽  
Ziqiang Zhu ◽  
Yiming Huang ◽  
Suzanne T. Ildstad
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Peripheral Blood ◽  
Flow Cytometric Analysis ◽  
Fold Increase ◽  
Dose Titration ◽  
Great Promise ◽  
Hematopoietic Stem ◽  
Flow Cytometric

Abstract Abstract 4694 Bone marrow transplantation (BMT) offers great promise for treating red blood cell disorders, inherited disorders of metabolism, autoimmune diseases, and inducing donor-specific tolerance to organ transplants. However, the widespread application of this approach is dependent upon the development of less toxic strategies for BMT and avoidance of graft-versus-host disease (GVHD). CD8+/TCR− facilitating cells (FC) facilitate engraftment of highly purified hematopoietic stem cells (HSC) across major histocompatibility complex barriers without causing GVHD. We previously reported that Flt3 ligand (FL) and granulocyte colony-stimulating factor (G-CSF) synergistically mobilize FC and HSC into the peripheral blood (PB). Recently, AMD 3100 has been found to be a rapid mobilizing agent whose effect occurs within hours after injection. It is a macrocyclic compound and potential fusion inhibitor that antagonizes CXCR4 alpha-chemokine receptor for its effect on HSC mobilization. CXCR4 and its ligand, stromal cell-derived factor-1 (SDF-1), are important in HSC homing and maintenance in the bone marrow microenvironment. Here, we investigated the effects of AMD 3100 on the mobilization of FC and HSC into PB in combination with FL and G-CSF. A dose titration of AMD 3100 was first performed. B6 mice were injected subcutaneously with AMD 3100 with the doses ranging from 1.25 mg/kg to 10 mg/kg. PB was obtained 0.5, 1, 3, and 6 hours post-injection. After individual count of peripheral blood mononuclear cells (PBMC), cells were stained for flow cytometric analysis to enumerate FC (CD8+/TCR−). The numbers of PBMC significantly increased even 0.5 hour after AMD 3100 treatment and peaked at 1 h. The maximal mobilization of PBMC was noted at 1 h with 5.0 mg/kg AMD 3100. Treatment with 5.0 mg/kg AMD 3100 caused a 3.1-fold increase of WBC at 1h compared with saline treated controls. An increase of FC was detectable with all doses of AMD 3100. The numbers of FC peaked between 1 and 3 h, and declined rapidly to resemble saline-treated controls at 6 h after. A 5.9-fold increase of FC was observed at 1 h with 5.0 mg/kg AMD 3100 (P = 0.012). These data suggest that AMD 3100 is a potent cell mobilizer from bone marrow to PB. We next investigated the effect of AMD 3100 in combination with FL and G-CSF on the mobilization of FC and HSC into PB. B6 mice were injected with FL (day 1 to 10), G-CSF (day 4 to 10), and AMD 3100 (day 10). PB was obtained 1 h after injection on day 10. After performing a count of peripheral WBC, cells were stained for flow cytometric analysis to enumerate FC (CD8+/TCR−) and HSC (Lin−/Sca-1+/c-kit+) mobilization. The maximal mobilization of PBMC was observed when animals were treated with AMD 3100/FL/G-CSF. The numbers of PBMC with AMD3100/FL/G-CSF treatment increased with 17.2-fold and 6.4-fold when compared with controls treated with saline or AMD 3100 alone (P < 0.00001), respectively. A maximal elevation of both FC and HSC was detected when AMD 3100 was added to FL/G-CSF treatment and reached 1.91 ± 0.42 × 103/μl (Figure 1A) and 1.89 ± 0.35 × 103/μl (Figure 1B), respectively. The increase of FC and HSC was significant. There was a 10.1-fold increase in FC and 230.8-fold increase in HSC when compared with recipients treated with AMD 3100 alone (P < 0.00001). AMD 3100/FL/G-CSF treatment resulted in a 1.7-fold of FC and 2.2-fold increase of HSC when compared with recipients treated with FL/G-CSF (P < 0.05). In summary, AMD 3100, FL, and G-CSF show a highly significant synergy on the mobilization of FC and HSC. This study may be clinically relevant in efforts to mobilize immunomodulatory FC and HSC to PB for transplantation, especially to induce tolerance for organ transplant recipients. Disclosures: Ildstad: Regenerex, LLC: Equity Ownership.

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