Journal of Thrombosis and Haemostasis, 4: 848–860
ORIGINAL ARTICLE
Cord blood in vitro expanded CD41+ cells: identification of novel components of megakaryocytopoiesis A. BALDUINI,* M. D’APOLITO, D. ARCELLI,à V. CONTI,§ A. PECCI,– D. PIETRA,** M. DANOVA,* F . B E N V E N U T O , C . P E R O T T I , à à L . Z E L A N T E , S . V O L I N I A , § § C . L . B A L D U I N I – and A . S A V O I A § *Department of Biochemistry, IRCCS Policlinico S. Matteo, University of Pavia, Pavia; Medical Genetic Service, IRCCS ‘Casa Sollievo della Sofferenza’, S. Giovanni Rotondo, Foggia; à Molecular Oncology Laboratory, ‘Istituto Dermopatico dell’Immacolata’ (IDI) IRCCS, Rome; §Telethon Institute of Genetics and Medicine (TIGEM), Naples; –Department of Internal Medicine, **Division of Hematology, Biotechnology Research Laboratories, ààImmunohematology and Transfusion Service, IRCCS Policlinico S. Matteo, University of Pavia, Pavia and §§Department of Morphology and Embryology, University of Ferrara, Ferrara, Italy
To cite this article: Balduini A, d’Apolito M, Arcelli D, Conti V, Pecci A, Pietra D, Danova M, Benvenuto F, Perotti C, Zelante L, Volinia S, Balduini CL, Savoia A. Cord blood in vitro expanded CD41+ cells: identification of novel components of megakaryocytopoiesis. J Thromb Haemost 2006; 4: 848–60.
Summary. Background: Megakaryopoiesis represents a multistep, often unclear, process leading to commitment, differentiation, and maturation of megakaryocytes (MKs) that release platelets. Aim: To identify the novel genes that might help to clarify the molecular mechanisms of megakaryocytopoiesis and be regarded as potential candidates of inherited platelet defects, global gene expression of hematopoietic lineages was carried out. Methods: Human cord blood was used to purify CD34+ stem cells and in vitro expand CD41+ cells and burst-forming unit-erythroid (BFU-E). We investigated the expression profiles of these three hematopoietic lineages in the Affymetrix system and selected genes specifically expressed in MKs by comparing transcripts of the different lineages using the DCHIP and PAM algorithms. Results: A detailed characterization of MK population showed that 99% of cells expressed the CD41 antigen whereas 73% were recognizable as terminally differentiated fetal MKs. The profile of these cells was compared with that of CD34+ cells and BFU-E allowing us to select 70 transcripts (MK-core), which represent not only the genes with a well-known function in MKs, but also novel genes never detected or characterized in these cells. Moreover, the specific expression was confirmed at both RNA and protein levels, thus validating the ÔMK-coreÕ isolated by informatics tools. Conclusions: This is a global gene expression that for the first time depicts a well-characterized population of cord blood-derived fetal MKs. Novel genes have been detected, such as those encoding components of the
Correspondence: Anna Savoia, Telethon Institute of Genetics and Medicine (TIGEM), Via Pietro Castellino, 111, 80131 Napoli, Italy. Tel.: +39 081 6132228; fax: +39 081 5609877; e-mail
[email protected] Received 19 July 2005, accepted 17 November 2005
extracellular matrix and basal membrane, which have been found in the cytoplasm of Mks, suggesting that new physiological aspects of MKs should be studied. Keywords: CD41+ cells, cord blood, expression profiling, megakaryocytopoiesis. Introduction Platelet formation is the final event of a multi-step process involving commitment, proliferation, and differentiation of hematopoietic progenitors to megakaryocytes (MKs). Mature MKs extend long processes, called proplatelets, through holes in the endothelial lining of bone marrow vessels, budding off platelets into the blood [1,2]. The molecular and cellular mechanisms responsible for this unique process are largely unknown. One major limitation in the study of megakaryocytopoiesis and platelet formation is the relatively low number of MKs in hematopoietic tissues that prevent the purification of adequate samples. However, the development of in vitro culture techniques, using thrombopoietin stimulation, and cell-sorting with specific monoclonal antibodies have allowed the production of MKs in sufficient amount for most of the analyses [3,4]. In addition, microarray technology simultaneously depicts the transcription level of thousand genes, which is of fundamental importance in revealing the fluctuation of gene expression during normal and defective megakaryocytopoiesis. Moreover, comparison of expression profiling between adult and fetal MKs might explain the age-related differences observed in this process [5]. In this paper, we report the expression profile of a highly purified population of MKs expanded from human cord blood (CB) progenitor cells. In order to identify genes specifically expressed in MKs (ÔMK-coreÕ), we compared the MK transcripts with those expressed in burst-forming unit-erythroid 2006 International Society on Thrombosis and Haemostasis
Gene signature of cord blood-derived megakaryocytes 849
(BFU-E) and CD34+ cells. The MK-specific gene set consists not only of well-characterized elements of megakaryocytopoiesis or platelet function but also of novel products that have been localized within the MK cytoplasm. Materials and methods Cell preparations
Cells were obtained from normal human cord blood (CB) doomed to discard after approval of the Institutional Review Board. CD34+ preparation CD34+ cells obtained from CB nonadherent, low density (
