Cord blood in vitro expanded CD41 cells: identification of novel components of megakaryocytopoiesis.
BACKGROUND
Megakaryopoiesis represents a multi-step, often unclear, process leading to commitment, differentiation, and maturation of megakaryocytes (MKs) that release platelets.


OBJECTIVE
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 'MK-core' 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 extracellular matrix and basal membrane, which have been found in the cytoplasm of Mks, suggesting that new physiological aspects of MKs should be studied.
Balduini
A
A
Department of Biochemistry, IRCCS Policlinico S. Matteo, University of Pavia, Pavia, Italy.
d'Apolito
M
M
Arcelli
D
D
Conti
V
V
Pecci
A
A
Pietra
D
D
Danova
M
M
Benvenuto
F
F
Perotti
C
C
Zelante
L
L
Volinia
S
S
Balduini
C L
CL
Savoia
A
A
D000911:Antibodies, Monoclonal / Q000378:metabolism; D018952:Antigens, CD34 / Q000096:biosynthesis / Q000378:metabolism; D015672:Erythroid Precursor Cells / Q000378:metabolism; D005312:Fetal Blood / Q000166:cytology*; D005434:Flow Cytometry; D006801:Humans; D066298:In Vitro Techniques; D008856:Microscopy, Fluorescence; D005810:Multigene Family; D020411:Oligonucleotide Array Sequence Analysis; D040201:Platelet Membrane Glycoprotein IIb / Q000096:biosynthesis* / Q000737:chemistry; D012313:RNA / Q000737:chemistry / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D034061:Thrombopoiesis / Q000502:physiology*
D000911:Antibodies, Monoclonal; D018952:Antigens, CD34; D040201:Platelet Membrane Glycoprotein IIb; D012313:RNA