Study of gene expression profile during cord blood-associated megakaryopoiesis

Aims: 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((TM)) 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 megakaryopoeisis.