Mass Cytometric Analysis of AML Stem and Early Progenitor Cells Reveals Karyotype and Genotype-Specific Immunophenotypes That May Represent Targets for Antibody-Directed Therapies

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2380-2380
Author(s):  
Gregory K. Behbehani ◽  
Wendy J. Fantl ◽  
Bruno C Medeiros ◽  
Garry P. Nolan
Keyword(s):  
Stem Cells ◽  
Progenitor Cell ◽  
Expression Patterns ◽  
Fold Increase ◽  
Surface Marker ◽  
Leukemic Cells ◽  
Therapeutic Antibodies ◽  
Hematopoietic Stem ◽  
Surface Marker Expression ◽  
Marker Expression

Abstract Introduction: Leukemic stem cells (LSCs) are recognized as important mediators of leukemia relapse. Thus, therapeutic antibodies are in development to target antigens present on these cells. The success of this approach relies on a detailed understanding of the surface marker expression patterns of LSCs. To address this, we applied multi-parametric single-cell mass cytometry (MCM) to deeply profile the surface marker expression of all major immunophenotypic populations in bone marrow aspirates (BM) from patients with acute myeloid leukemia (AML). Methods: BMs from 41 leukemia patients (30 AML, 4 APL, 2 high-risk MDS, 5 AML in CR) and 5 healthy donors and were processed immediately after aspiration (<1min) and stored for pooled analysis with two overlapping 39-antibody MCM panels (50 markers total). All samples were barcoded, such that 20 samples (leukemia and healthy) could be combined into a single tube for simultaneous antibody staining and analysis, resulting in high precision (coefficient of variation = 10-20%). Results: Distinct AML subtype-specific patterns of cell frequencies across immunophenotypic populations were detected. Patients with core binding factor (CBF) AML (n=5) and those with adverse-risk karyotpic abnormalities (n=6) exhibited the greatest expansion of immunophenotypic hematopoietic stem cells (HSCs) and early progenitors (MPP, CMP), while patients with APL (n=4) and FLT3-ITD normal karyotype AML (NK-AML; n=11) exhibited reduced expansion of early progenitors and expansion in more mature myeloid progenitors (GMP, myelo-monoblasts). As a result of barcoding, high resolution (2-3 fold changes) measurement of surface marker expression detected multiple aberrancies across almost all identifiable immunophenotypic populations. Specifically, several genotype- and karyotype-specific trends in aberrant marker expression were observed in hematopoietic stem and progenitor cell populations (HSPCs; CD34+CD38low). FLT3-ITD samples were characterized by increased CD7, CD33, CD123, CD45, CD321, and CD99, as well as decreased CD34, CD117, and CD38. FLT3wt NK-AML samples (n=6) were characterized by increased CD99 as well as decreased CD71, CD47, CD34, and CD45. Adverse-risk karyotype samples (n=6) were characterized by increased CD99 and decreased CD47. All p values were significant (ranging from 0.02 to 4.5x10-7). While all 36 samples (AML or APL) displayed immunophenotypic abnormalities within the CD34+CD38low gated population (3.6 abnormalities on average), there were 9 samples in which an unambiguous separation into normal and leukemic populations was observed among the cells in this gate. Interestingly, among these samples, some markers were aberrantly high in leukemic cells of one subtype, and aberrantly low in leukemic cells of another, e.g. HLA-DR was extremely high in HSPCs of sample #22 (MLL rearrangement), and aberrantly low in all 4 APL samples. CD99 was most consistently elevated in the AML samples (28/36), but was normal in 6 samples, including a majority of those with t(8;21). These results suggest that therapeutic antibodies directed against molecules such as CD33 may be less effective for AML subtypes such as FLT3wt NK-AML (~40% decrease in CD33), and more effective for other subtypes such as FLT3-ITD NK-AML (7-fold increase in CD33). HSPCs from patients with FLT3-ITD mutations also displayed a 20-fold increase in CD123, which could also be therapeutically targeted. These findings, combined with our recent observation that HSPCs from FLT3-ITD NK-AML patients have an extremely low S-phase fraction, may provide a mechanistic basis for the improved disease-free survival recently reported for FLT3-ITD NK-AML patients treated with fractionated gemtuzumab ozogamicin in combination with standard therapy. Conclusions: We have developed an innovative MCM approach for the analysis of hematologic malignancies and demonstrated that different genotypic and karyotypic subsets of AML are highly varied in their immunophenotypic properties, particularly within the stem and progenitor cell compartment. These data also suggest that antigens that distinguish AML stem cells from normal stems cells are likely to be karyotype and genotype-specific. These findings have important implications for the design of therapeutic strategies in AML. Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Disclosures Behbehani: Fluidigm: Consultancy. Medeiros:Agios: Consulting - Ad board Other. Nolan:Fluidigm, Inc: Consultancy, Equity Ownership.

A High-Throughput Screen to Identify Compounds Preserving Primary Human AML Stem Cells Ex-Vivo,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3587-3587
Author(s):  
Caroline Pabst ◽  
Josee Hebert ◽  
Guy Sauvageau
Keyword(s):  
Stem Cells ◽  
High Throughput ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Surface Marker ◽  
Leukemic Cells ◽  
High Throughput Screen ◽  
Self Renewal ◽  
Surface Marker Expression ◽  
Marker Expression

Abstract Abstract 3587 Acute myeloid leukemia (AML) is the second most frequent leukemia in adults with still high relapse rates supposed to be due to a rare population of leukemia stem cells (LSC) within the leukemic bulk not responding to conventional anti-cancer drugs. Engraftment of human leukemic cells in immunocompromised mice mimicking the human disease is considered to prove the existence of stem cells in the sample; however, the engraftment potential of primary human AML cells is usually quickly lost upon ex-vivo culture indicating that LSC have undergone differentiation or apoptosis and thus lost their stem cell properties. This lack of appropriate culture conditions enabling ex-vivo maintenance of primary human LSC is a major obstacle in the development of LSC targeted therapies. Our aim is, therefore, to identify conditions which allow the maintenance and expansion of these rare cells ex-vivo to understand their unique properties and to further elucidate cellular pathways underlying the self-renewal program of human primary LSC. We established a flow cytometry based high-throughput screen in which we tested more than 6,000 selected compounds with regards to their potential to expand primary human AML cells while preserving their phenotypic and functional characteristics. We considered alteration of surface marker expression, i.e. loss of CD34 and acquisition of markers of myeloid maturation initially not expressed on the cells, as indicator of differentiation implicating at the same time a loss of stem cells in the culture. We hypothesized that compounds that would be able to prevent these alterations in surface marker expression could have the potential to promote self-renewal of primary human LSC ex vivo. A normal-karyotype AML representing the most frequent cytogenetic group of AML in adults with FAB M1 morphology was chosen to perform the screen. Moreover, the initial CD34+CD15- phenotype allowed us to monitor changes in surface marker expression. Cells were plated in 384 well plates with a density of 5,000 cells per well. 6,160 compounds were tested with and without an inhibitor to aryl hydrocarbon receptor (AhR) and the vehicle DMSO was used as negative control resulting in a final DMSO concentration of ∼0.1% in each well, which had been proven to be not toxic to the cells. After 5 days of incubation the cells were analyzed on a BD LSRII high-throughput flow cytometer with regards to CD34 and CD15 surface expression. Compounds showing an increase of ≥45% of CD34+ and CD34+CD15- cells over DMSO compared to the AhR antagonist were selected for further evaluation. In a secondary screen we retested 32 compounds in 5 different concentrations ranging from 1:34 to 3x based on the initial screen concentration after 5 and 9 days of incubation and confirmed an increase ≥50% of relative and absolute CD34+CD15- leukemic cells after 5 days for 13 compounds and after 9 days for 7 compounds. We have initiated functional in-vitro and in-vivo experiments to validate these results and to prove the existence of LSC in cultures where compounds have been added. The ability of the newly identified compounds to preserve human LSC ex-vivo will be further evaluated using a collection of primary AML samples comprising different morphological and cytogenetic groups. Together, our observations document the feasibility of finding anti-differentiation (pro-self-renewal?) compounds of primary human AML cells that can be expanded ex-vivo. These studies represent a first key step in the identification of specific LSC targeting compounds. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Shared Cell Surface Marker Expression in Mesenchymal Stem Cells and Adult Sarcomas

2012 ◽  
Vol 2 (1) ◽  
pp. 53-60 ◽  
Author(s):  
Stefan Wirths ◽  
Elke Malenke ◽  
Torsten Kluba ◽  
Simone Rieger ◽  
Martin R. Müller ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Cell Surface ◽  
Surface Marker ◽  
Cell Surface Marker ◽  
Surface Marker Expression ◽  
Marker Expression ◽  
Cell Surface Marker Expression

scholarly journals Transcriptional, Protein-Level and Functional Profiling of Human Fetal Liver-Derived Hematopoietic Stem Cells at Single Cell Resolution

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1187-1187
Author(s):  
Kim Vanuytsel ◽  
Carlos Villacorta-Martin ◽  
Wilfredo Garcia Beltran ◽  
Taylor Matte ◽  
Alejandro Balazs ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Surface ◽  
Single Cell ◽  
Protein Level ◽  
Fetal Liver ◽  
Surface Marker ◽  
Cell Surface Marker ◽  
Hematopoietic Stem ◽  
Nsg Mice ◽  
Surface Marker Expression

Intro: In the mouse, hematopoietic stem cells (HSCs) can be isolated and characterized at single cell resolution using a well-defined panel of markers. While it is possible to enrich for human HSCs using a panel of associated markers, similar resolution has not been attained. By profiling HSCs residing in the human fetal liver (FL) using a novel technique called CITE-Seq that combines single cell RNA sequencing (scRNAseq) and cell surface marker interrogation using oligo-tagged antibodies, we aimed to establish an accurate molecular signature of engraftable human HSCs shortly after they arise in development. As HSCs are defined functionally, we have coupled this transcriptomic and protein-level characterization with transplantation assays in immunocompromised NOD scid gamma (NSG) mice to connect expression profiles of cell subsets with functional engraftment. Methods: CITE-Seq was performed on human FL cells (week 19) that showed robust engraftment capability in NSG mice. CD34+ and CD34- cells were magnetically separated and stained with a panel of 19 oligo-tagged antibodies that were deemed relevant to characterize HSCs, including classical HSC markers but also novel targets that were identified in a previous pilot scRNAseq experiment conducted on CD34+ FL cells. From the CD34+ fraction, we sorted live-gated cells (CD34+bulk) as well as a population of cells that was further enriched based on the expression of GPI-80, a marker tightly linked to engraftment potential (CD34+GPI-80+, ~3%). CD34-GlycophorinA(GYPA)- cells were also sorted to assay for the presence of CD34- HSCs. These fractions were then loaded onto the 10x Genomics platform for capture of single cells and subsequent reverse transcription and amplification of both mRNAs and antibody-derived tags (ADTs). Results: Both mRNA and ADT libraries were successfully sequenced, yielding 29-43,000 reads/cell for the mRNA portion and >1,500 reads/cell for the ADT fraction. After quality control and filtering, this effort resulted in 8,775 CD34+bulk cells, 7,279 CD34+GPI-80+ cells, and 6,937 CD34-GYPA- cells available for further analysis. Simultaneous transplantation experiments of the fractions assayed by CITE-seq revealed superior engraftment potential of the CD34+GPI-80+ fraction, confirming enrichment for bona fide HSCs at the functional level. This was also reflected in the scRNAseq data where we found enrichment for known HSC markers such as VNN2 (GPI-80), PROM1 (CD133), PROCR (EPCR), THY1 (CD90), ITGA6 (CD49f), HMGA2, CLEC9A and HLF in the CD34+GPI-80+ fraction compared to CD34+bulk cells. As our pilot studies revealed considerable differences in transcriptional expression (via scRNAseq) as compared to protein-level expression (via cell surface marker expression), integration of the transcriptomic and cell surface marker expression data will further refine the signature of engraftable HSCs. Both layers of information at single cell resolution will allow for the identification of novel markers or unique combinations of markers that are directly correlated with engraftment potential. Conclusion: By isolating the GPI-80+ population within the CD34+ fraction in human FL, we have achieved unprecedented resolution of the signature of engraftable HSCs as confirmed by transplantation experiments. The in-depth characterization of this compartment as well as the surrounding CD34+ and CD34- cells within the FL is expected to yield valuable insights with respect to several biological questions. This data can be directly harnessed in improving the purification and expansion of engraftable HSCs as well as in guiding the in vitro generation of HSCs from pluripotent stem cells. Disclosures No relevant conflicts of interest to declare.

scholarly journals Comparison of Canine and Feline Adipose-Derived Mesenchymal Stem Cells/Medicinal Signaling Cells With Regard to Cell Surface Marker Expression, Viability, Proliferation, and Differentiation Potential

2021 ◽  
Vol 7 ◽  
Author(s):  
Metka Voga ◽  
Valerija Kovač ◽  
Gregor Majdic
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Mesenchymal Stem Cells ◽  
Surface Marker ◽  
Growth Media ◽  
Differentiation Potential ◽  
Surface Marker Expression ◽  
Proliferation And Differentiation ◽  
Marker Expression ◽  
In Cells

Remarkable immunomodulatory abilities of mesenchymal stem cells, also called multipotent mesenchymal stromal cells or medicinal signaling cells (MSCs), have entailed significant advances in veterinary regenerative medicine in recent years. Despite positive outcomes from MSC therapies in various diseases in dogs and cats, differences in MSC characteristics between small animal veterinary patients are not well-known. We performed a comparative study of cells' surface marker expression, viability, proliferation, and differentiation capacity of adipose-derived MSCs (ADMSCs) from dogs and domestic cats. The same growth media and methods were used to isolate, characterize, and culture canine and feline ADMSCs. Adipose tissue was collected from 11 dogs and 8 cats of both sexes. The expression of surface markers CD44, CD90, and CD34 was detected by flow cytometry. Viability at passage 3 was measured with the hemocytometer and compared to the viability measured by flow cytometry after 1 day of handling. The proliferation potential of MSCs was measured by calculating cell doubling and cell doubling time from second to eighth passage. Differentiation potential was determined at early and late passages by inducing cells toward adipogenic, osteogenic, and chondrogenic differentiation using commercial media. Our study shows that the percentage of CD44+CD90+ and CD34−/− cells is higher in cells from dogs than in cells from cats. The viability of cells measured by two different methods at passage 3 differed between the species, and finally, canine ADMSCs possess greater proliferation and differentiation potential in comparison to the feline ADMSCs.

scholarly journals Jaw Periosteum-Derived Mesenchymal Stem Cells Regulate THP-1-Derived Macrophage Polarization

2021 ◽  
Vol 22 (9) ◽  
pp. 4310
Author(s):  
Fang He ◽  
Felix Umrath ◽  
Siegmar Reinert ◽  
Dorothea Alexander
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Macrophage Polarization ◽  
Polarization State ◽  
Osteonecrosis Of The Jaw ◽  
Surface Marker ◽  
Surface Marker Expression ◽  
Periosteal Cells ◽  
Immunomodulatory Properties ◽  
Marker Expression

Mesenchymal stem cells from bone marrow have powerful immunomodulatory capabilities. The interactions between jaw periosteal cells (JPCs) and macrophages are not only relevant for the application of JPCs in regenerative medicine, but this understanding could also help treating diseases like osteonecrosis of the jaw. In previous studies, we analyzed, for the first time, immunomodulatory features of 2D- and 3D-cultured JPCs. In the present work, the effects of JPCs on the polarization state of macrophages in contact coculture were analyzed. To improve the macrophage polarization study, different concentrations of PMA (5 nM, 25 nM, and 150 nM) or different medium supplementations (10% FBS, 10% hPL and 5% hPL) were compared. Further, in order to analyze the effects of JPCs on macrophage polarization, JPCs and PMA-stimulated THP-1 cells were cocultured under LPS/IFN-γ or IL-4/IL-13 stimulatory conditions. Surface marker expression of M1 and M2 macrophages were analyzed under the different culture supplementations in order to investigate the immunomodulatory properties of JPCs. Our results showed that 5 nM PMA can conduct an effective macrophage polarization. The analyses of morphological parameters and surface marker expression showed more distinct M1/M2 phenotypes over FBS supplementation when using 5% hPL during macrophage polarization. In the coculture, immunomodulatory properties of JPCs improved significantly under 5% hPL supplementation compared to other supplementations. We concluded that, under the culture condition with 5% hPL, JPCs were able to effectively induce THP-1-derived macrophage polarization.

Identification Of Leukemia Suppressive Genes By Inducible Overexpression Of The DNA Methyltransferase DNMT3B During Leukemogenesis In Mice

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2493-2493
Author(s):  
Isabell Schulze ◽  
Petra Tschanter ◽  
Christian Rohde ◽  
Annika Krause ◽  
Heinz Linhart ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Stem Cells ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Expression Patterns ◽  
Leukemic Cells ◽  
Leukemic Stem Cells ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Serial Transplantation

Abstract DNA methyltransferases (DNMT) play an important role in regulation of DNA methylation and mutations of DNMT3A are frequently found in AML. In previous studies using a tetracycline-inducible DNMT3B mouse model, we could show that overexpression of DNMT3B affected leukemia initiation and maintenance upon retroviral transduction and serial transplantation of hematopoietic stem and progenitor cells with MSCV-MLL-AF9-GFP and MSCV-cmyc-bcl2-mcherry oncogenic vectors, respectively. Sublethally irradiated recipient mice of DNMT3B overexpressing MLL-AF9 and cmyc/bcl2 leukemic cells developed leukemia with a prolonged latency when compared to recipients of wildtype cells. We performed serial transplantation assays of MLL-AF9 leukemic stem cells, which were sorted for high expression of ckit. The life-prolonging effect of DNMT3B expression was stem cell-specific, as the potential to initiate leukemia was maintained upon serial retransplantation and recipients of DNMT3B overexpressing leukemic stem cells also died significantly later in secondary (p<0.001) and tertiary transplantations (p<0.001). Analysis of global DNA methylation levels in MLL-AF9 ckit+ leukemic stem cells and cmyc/bcl2 leukemic cells via Reduced Representation Bisulfite Sequencing (RRBS) revealed a strong hypermethylation in DNMT3B overexpressing cells, independent of the oncogene used for leukemia induction. Differentially methylated CpG sites were defined as CpGs with at least 20% methylation difference between wildtype and DNMT3B overexpressing samples. Hypermethylation in MLL-AF9 leukemic cells directly correlated with observed hypermethylation in cmyc/bcl2 leukemic cells and inversely correlated with hypomethylation in cmyc/bcl2 cells, indicating that in both leukemias, the same sites are prone to DNMT3B induced DNA methylation. To investigate, if these changes in DNA methylation resulted in different gene expression patterns, we performed microarray analysis of the same MLL-AF9 leukemic wildtype and DNMT3B expressing samples which were also used for DNA methylation analysis. In microarray analyses, we could identify several genes differentially expressed in DNMT3B overexpressing cells when compared to wildtype samples. Interestingly, changes in expression levels could not be attributed to differential DNA methylation in promoter regions. Instead, hypermethylation in exons and gene bodies resulted in downregulation of the respective genes, whereas genes with hypomethylated exons and gene bodies showed higher expression levels. Genes downregulated in DNMT3B overexpressing cells, were mainly cancer-associated genes, which are known to have functions in cellular growth and proliferation, as well as in the hematopoietic system development and maintenance. Gene Set Enrichment Analysis (GSEA) of wildtype cells revealed a strong enrichment of genes upregulated in different stages of hematopoietic stem and progenitor cells as well as in leukemic stem cells, whereas DNMT3B overexpressing samples were enriched in genes which have been shown to be downregulated in hematopoietic and leukemic stem cells and upregulated in mature hematopoietic cells. This strengthens our hypothesis that DNMT3B induced DNA methylation mainly influences the phenotype and function of hematopoietic stem cells and thereby, exerts its inhibitory function on leukemia initiation and maintenance. Taken together, these findings demonstrate that DNMT3B exerts its anti-leukemic effect mainly via induction of aberrant DNA methylation in hematopoietic and leukemic stem cells, thereby changing expression patterns of genes known to be important for stem cell function. The identification of differentially expressed DNMT3B target genes could help to find promising targets for new therapeutic strategies in AML. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Mesenchymal Stem/Stromal Cells Derived from Induced Pluripotent Stem Cells Support CD34posHematopoietic Stem Cell Propagation and Suppress Inflammatory Reaction

2015 ◽  
Vol 2015 ◽  
pp. 1-14 ◽  
Author(s):  
Mohsen Moslem ◽  
Irina Eberle ◽  
Iuliia Weber ◽  
Reinhard Henschler ◽  
Tobias Cantz
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Induced Pluripotent Stem Cells ◽  
Stromal Cells ◽  
Pluripotent Stem Cells ◽  
Surface Marker ◽  
Surface Marker Expression ◽  
Cell Source ◽  
Marker Expression ◽  
Induced Pluripotent

Mesenchymal stem/stromal cells (MSCs) represent a promising cell source for research and therapeutic applications, but their restrictedex vivopropagation capabilities limit putative applications. Substantial self-renewing of stem cells can be achieved by reprogramming cells into induced pluripotent stem cells (iPSCs) that can be easily expanded as undifferentiated cells even in mass culture. Here, we investigated a differentiation protocol enabling the generation and selection of human iPSC-derived MSCs exhibiting relevant surface marker expression profiles (CD105 and CD73) and functional characteristics. We generated such iPSC-MSCs from fibroblasts and bone marrow MSCs utilizing two different reprogramming constructs. All such iPSC-MSCs exhibited the characteristics of normal bone marrow-derived (BM) MSCs. In direct comparison to BM-MSCs our iPSC-MSCs exhibited a similar surface marker expression profile but shorter doubling times without reaching senescence within 20 passages. Considering functional capabilities, iPSC-MSCs provided supportive feeder layer for CD34+hematopoietic stem cells’ self-renewal and colony forming capacities. Furthermore, iPSC-MSCs gained immunomodulatory function to suppress CD4+cell proliferation, reduce proinflammatory cytokines in mixed lymphocyte reaction, and increase regulatory CD4+/CD69+/CD25+T-lymphocyte population. In conclusion, we generated fully functional MSCs from various iPSC lines irrespective of their starting cell source or reprogramming factor composition and we suggest that such iPSC-MSCs allow repetitive cell applications for advanced therapeutic approaches.

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