Functional Cytomics

  • Petriz group
Campus ICO-Germans Trias i Pujol

Josep Carreras Leukaemia Research Institute
Can Ruti Campus
Ctra de Can Ruti, Camí de les Escoles s/n
Edifici IMPPC
08916 Badalona, Barcelona, Spain

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Summary

An important and challenging problem in leukemia research is the limited ability for many laboratories to perform functional analyses of primary patient cells. In order to increase our understanding of the biology of human leukemic malignancies, we perform advanced experimentation using living-cell systems. Functional information extracted from single-cell analysis, provides crucial data to understand cell-to-cell heterogeneity. By enabling functional cytomics, we are able to evaluate the state of patients with leukemia as well as to examine the changes that occur in the accumulation of drugs into the cells over time. The Functional Cytomics Group is mainly focused in the basic mechanisms that regulate CD34+ and CD34- Side Population stem cells. Stem cells reside in most of tissues in a quiescent state, but rapidly become activated to both repair and regenerate the adjacent tissues. We are studying several genes involved in different aspects of stem cell activation, including some that encode for ABC multidrug resistance transporters, and others that regulate self-renewal and differentiation.

Research

Expression of primitive stem cell markers during origin, progression and maintenance of leukemia. Diagnostic implications.

Objective:
The goal of the proposed research is to learn more about the pathogenesis, natural history and treatment of human leukemia by analyzing the expression of primitive stem cell markers and their association with malignancy at a phenotypic and functional level, using a long series of leukemic malignancies and leukemic stem cells, with particular emphasis on self-renewal signaling pathways.

Design of consensus protocols for safety, quality and standardization of CD34+ cells after-thawing.

Upon the establishment of a convergence framework for the quality control between the Iberian Society of Cytometry (SIC) and the Spanish Society of Immunology (SEI), we will develop consensus strategies for the counting of CD34+ cells pre- and post-thaw through an external quality program and the CD34 intercalibration working group, now supported by both societies.

Objectives:
a) To analyze the intracenter variability of CD34+ cell counting; b) to standardize the counting of CD34+ after thawing and to analyze pre-infusion product quality by means of polychromatic cytometry in combination with metabolic functional assessment; c) to conduct "in silico" intercomparison studies between participating centers; d) to develop predictive models on pre-infusion product quality considering demographic analysis, pathology and graft-related variables in the study population; e) to agree a single protocol for CD34+ cell counting; f) to integrate different existing protocols for the cryopreservation of CD34+ cell-enriched products; g) to extend the convergence criteria within the EuroFlow framework.

Study of the mechanisms by which ABC transporters differentially activates low- or high-level transduction cell signaling: Potential role to both protect the stem cell compartment.

Objectives:
We study the role of ATP Binding Cassette (ABC) multidrug transporters on signal transduction in stem cells (SC). We analyze their protective potential on cell signaling pathways that are fundamental to many cell types, specially for SC function. We study how ABCB1 and ABCG2, two ABC transporters expressed in normal and leukemic cells, can regulate highly conserved stem cell signaling pathways, with implications for the development of therapeutic targets not only to treat leukemia, but also in other diseases. We analyze the regulation of Sonic Hedgehog, Notch, Wnt/β-catenin, epithelial-mesenchymal transition induced by TGF-β, PTEN/PI3K/Akt, and PPAR α/γ.

The use of anticancer drug libraries to study the reversal of multidrug resistance in Side Population leukemic stem cells.

Very recent data support that 25% of cancers are associated with the existence of cancer stem cells (CSCs), these being responsible for the spread of disease from a minority of cells with properties very similar to the normal stem cells. These CSCs lack a specific phenotype and can be only identified and isolated by functional characteristics, especially by the expression of multidrug transporters belonging to the ABC family (ATP Binding Cassette). The activity of one of these transporters, ABCG2, allows the isolation a very primitive type of stem cells, the so-called Side Population (SP). In turn, ABCG2 enables the SP to be highly refractory to many chemotherapeutic agents.

People

Selected publications

Avendaño A, Sales-Pardo I, García-Godoy MD, Rico LG, Marín P, Petriz J

Accuracy and reproducibility of stem cell side population measurements on clinically relevant products.

Curr Stem Cell Res Ther 2014, 9 (6) 526-34.
In 1996, Goodell et al. first described a rare subpopulation of bone marrow stem cells termed the Side Population (SP). SP cells are known to be CD34 negative and to have a high repopulating capability. The SP was identified by ultraviolet excitation based on the efflux of the DNA binding dye, Hoechst 33342 (Ho342). ABCG2, a halftransporter that belongs to the ATP binding cassette transporter superfamily, is the major contributor to the SP phenotype by actively pumping Ho432 selectively from stem cells. To date, very little is known about the identification of the SP in peripheral blood samples, and about its peripheral circulation, enrichment or isolation to evaluate its therapeutic potential. Due to the SP potential role in tissue regeneration, we studied the numbers of the SP in bone marrow and peripheral blood samples in regard to count accuracy and reproducibility.
More information
Petriz J

Flow cytometry of the side population (SP).

Curr Protoc Cytom 2013, Chapter 9 Unit9.23.
The side population (SP) has become an important hallmark for the definition of the stem-cell compartment, especially for the detection of stem cells and for their physical isolation by fluorescence-activated cell sorting (FACS). SP cells are CD34(-) and were discovered using ultraviolet excitation based on the efflux of Hoechst 33342 (Ho342). Although the method works as originally described, the protocol is difficult for most investigators to perform: first, because the ability to discriminate SP cells is based on the differential retention of Ho342 during a functional assay; second, because of the difficulties in setting the right experimental and acquisition conditions; and third, because analysis of the acquired data requires extensive expertise in flow cytometry to accurately detect the SP events. More recently, a new assay based on the efflux of Vybrant DyeCycle Violet stain (DCV) has been documented to discriminate SP cells. This unit contains many helpful pointers to aid the user in obtaining the best possible results with these assays.
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Balbuena J, Pachon G, Lopez-Torrents G, Aran JM, Castresana JS, Petriz J

ABCG2 is required to control the sonic hedgehog pathway in side population cells with stem-like properties.

Cytometry A Sep 2011, 79 (9) 672-83. Epub 19 Jul 2011
The Sonic Hedgehog (Hh) pathway has been implicated in the maintenance of stem or progenitor cells in many adult tissues. Importantly, abnormal Hh pathway activation is also associated with initiation of neoplasia, but its role in tumor growth is still unclear. Here, we demonstrate that cyclopamine, a plant-derived alkaloid product used to inhibit the Hh signaling pathway, reduces the Side Population (SP) obtained by Hoechst 33342 (Ho342) dye measurements. In addition, cyclopamine is able to modulate, along with oxysterols and other products, the ABCG2 transporter by increasing Ho342 and mitoxantrone uptake. Therefore, if the SP is solely measured as a Ho342 dye extruding fraction, this may be significantly modulated by the inhibition of ABCG2 transport fraction, independently from the action of cyclopamine on the Hh pathway. Our results indicate that ABCG2 may act in the upstream regulation of the Hh signaling pathway to protect the stemness of the SP compartment, giving support to the cancer stem cell hypothesis and suggesting that ABCG2 is not only critical for increased resistance to anticancer agents.
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Sales-Pardo I, Avendaño A, Martinez-Muñoz V, García-Escarp M, Celis R, Whittle P, Barquinero J, Domingo JC, Marin P, Petriz J

Flow cytometry of the Side Population: tips & tricks.

Cell. Oncol. 2006, 28 (1-2) 37-53.
The Side Population (SP) has become an important hallmark for the definition of the stem cell compartment, especially in the detection of these cells and in their physical isolation by fluorescence-activated cell sorting (FACS). SP cells are CD34neg and were discovered using ultraviolet excitation based on the efflux of Hoechst 33342 (Ho342). Although the method works as originally described, we believe that this method is difficult for most investigators. First, because the ability to discriminate SP cells is based on the differential retention of Ho342 during a functional assay; second, because of the difficulties in setting the right experimental and acquisition conditions; and third, because the analysis of the acquired data requires an extensive expertise on flow cytometry to accurately detect the SP events.
More information
García-Escarp M, Martinez-Muñoz V, Barquinero J, Sales-Pardo I, Domingo JC, Marin P, Petriz J

A rare fraction of human hematopoietic stem cells with large telomeres.

Cell Tissue Res. Mar 2005, 319 (3) 405-12. Epub 27 Jan 2005
The lack of specific markers for stem cells makes the physical identification of this compartment difficult. Hematopoietic stem cells differ in their repopulating and self-renewal potential. Our study shows that multiple classes of human hematopoietic CD34+ greatly differ in telomere length. Flow-cytometry-based fluorescent in situ hybridization and confocal microscopy of CD34+ cells has revealed remarkable telomere length heterogeneity, with a hybridization pattern consistent with different classes of human hematopoietic progenitor cells. These results also point to the existence of a significant clonal heterogeneity among primitive hematopoietic cells and provide the first evidence of a rare fraction of CD34+ cells with large telomeres in humans.
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