In the field of stem cells, the lack of surface markers, allowing the separation of stem cells subpopulations with a specific fate represent the major problem in therapies based on those cells. Thus the identification of such markers would significantly improve their clinical use. After an analysis of the most widely used methods in protein identification, we opted for a new way, and designed an approach to identify new markers, and consequently new cell subsets, based on a library of mouse polyclonal antisera specific for linear epitopes of poorly known human proteins, predicted to be either transmembrane or secreted. This library is versatile, can be used to screen any cell or tissue of interest, and allows screening of a large (1600) repertoire of “neglected” human proteins. Briefly, these proteins were selected in silico, expressed in bacteria (E. coli) as linear epitopes with a 10 Histidine tag; the recombinant proteins were then purified by Protein Affinity Chromatography and used to immunize a group of 5 mice each protein, to produce the polyclonal antisera. We focused our efforts on new stem cell markers identification on hematopoietic stem cells (HSC), due to their clinical relevance: HSC transplantation is, nowadays, the only widely used stem cell-based therapy. Despite this, and even though HSC have been extensively studied in the last 20 years, with the identification of a number of lineage-specific markers, the need of new subsets identification to better understand the hematopoiesis mechanism is still strong. We have shown that with our polyclonal antisera library is it possible to interrogate by flow cytometry the cell surface of hematic cells, and to identify new subsets of both mature and hematopoietic cells. This study identified three new proteins expressed by a subset of HSC: CRISP-1 and MOSC- 1, not previously outlined by classical molecular analysis, and TREML-1. We performed a phenotypic characterization of MOSC-1 and of TREML-1 expressing HSC, obtaining strong suggestion that these subsets could be, respectively, myeloid and megakaryocyte precursors. The newly identified stem cells markers were then used, in a secondary study, to better characterize the hematological and immunological reconstitution after HSC transplantation in pediatric patients, showing that they could provide useful information in the definition of engraftment prognosis. We now aim to expand the validation phase of the newly identified targets by increasing functional and differentiation studies. To that purpose, we have also started collaboration with other institution, to extend the possible investigations. Finally, we are starting to produce the identified proteins in mammalian cells, in order to generate a monoclonal antibody library for future studies.
(2010). Identification of new hematopoietic stem cell subsets with a polyclonal antibody library specific for poorly characterized proteins. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2010).
|Data di pubblicazione:||29-mar-2010|
|Tutor esterno:||MORO, MONICA|
|Titolo:||Identification of new hematopoietic stem cell subsets with a polyclonal antibody library specific for poorly characterized proteins|
|Settore Scientifico Disciplinare:||BIO/13 - BIOLOGIA APPLICATA|
|Corso di dottorato:||MEDICINA TRASLAZIONALE E MOLECOLARE (DIMET) - 45R|
|Citazione:||(2010). Identification of new hematopoietic stem cell subsets with a polyclonal antibody library specific for poorly characterized proteins. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2010).|
|Parole Chiave (Inglese):||stem cells, hematopoietic stem cells, flow cytometry, polyclonal antibody, proteomics, cell subsets|
|Appare nelle tipologie:||07 - Tesi di dottorato Bicocca post 2009|