Study of gene expression profile during cord blood-associated megakaryopoiesis.
OBJECTIVE
To study the gene profile in cord blood (CB)-associated megakaryopoiesis.


METHODS
In vitro differentiation of megakaryocytes (Mks) was carried out using human CB CD34(+) cells under the stimulation of recombinant human interleukin-3, stem cell factor and thrombopoietin for 7 d, followed by thrombopoietin only for further 3 d. Lineage-specific differentiation of Mk was examined by the expression of CD41 using flow cytometry and confocal microscopy. Total cellular RNA was extracted from day-0 CD34(+), day-10 CD41(+) and CD41(-) populations were isolated by immunomagnetic sorting respectively. Microarray was performed, and the data were analyzed using the GeneChip Operating System, Spotfire software and Genomatix BiblioSphere.


RESULTS
Flow cytometric analysis showed 19.44 +/- 3.05% CD41(+) cells at day 10 of culture. The purity of CD41(+) population was enriched to 95.70 +/- 4.19% after sorting. Gene expression profiling revealed an upregulation of 285 and downregulation of 53 unique genes in the CD41(+) cells compared with CD41(-) and CD34(+) cells. Platelet-associated genes, such as thrombospondin 1, platelet glycoprotein IIIa, etc., were highly expressed in CD41(+) cells but not in CD41(-) cells and CD34(+) cells. Moreover, some genes that have not been reported to be associated with CB-derived megakaryopoiesis, such as Cbl-interacting proteins Sts-1, protocadherin 21, etc., are found to be highly expressed in the CD41(+) cells from this study.


CONCLUSIONS
This study reveals a global gene expression profile of in vitro human CB-derived megakaryopoiesis at day 10. Some of these genes may play regulatory roles during the development of CB-derived megakaryopoiesis.
Department of Clinical Research, Singapore General Hospital, Singapore, Republic of Singapore.
Che Kang
CK
Lim
William Y K
WY
Hwang
Swee Eng
SE
Aw
Li
L
Sun
D018952:Antigens, CD34 / Q000187:drug effects / Q000378:metabolism; D000070585:Cadherin Related Proteins; D015820:Cadherins / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics; D002448:Cell Adhesion / Q000187:drug effects / Q000502:physiology; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D005312:Fetal Blood / Q000166:cytology*; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D006801:Humans; D039661:Integrin beta3 / Q000235:genetics; D007377:Interleukin-3 / Q000494:pharmacology; D008055:Lipids / Q000096:biosynthesis; D008533:Megakaryocytes / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D018613:Microscopy, Confocal; D009419:Nerve Tissue Proteins / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D040201:Platelet Membrane Glycoprotein IIb / Q000187:drug effects / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods; D017027:Protein Tyrosine Phosphatases; D011994:Recombinant Proteins / Q000494:pharmacology; D019089:Stem Cell Factor / Q000494:pharmacology; D013926:Thrombopoietin / Q000494:pharmacology; D019700:Thrombospondin 1 / Q000235:genetics; D015854:Up-Regulation / Q000187:drug effects
D018952:Antigens, CD34; C442481:CDHR1 protein, human; D000070585:Cadherin Related Proteins; D015820:Cadherins; D002352:Carrier Proteins; D039661:Integrin beta3; D007377:Interleukin-3; D008055:Lipids; D009419:Nerve Tissue Proteins; D040201:Platelet Membrane Glycoprotein IIb; D011994:Recombinant Proteins; D019089:Stem Cell Factor; D019700:Thrombospondin 1; D013926:Thrombopoietin; D017027:Protein Tyrosine Phosphatases; C489397:UBASH3B protein, human