Study of gene expression profile during cord blood-associated megakaryopoiesis
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 GeneChipTM 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.
Document Type: Research Article
Affiliations: 1: Department of Clinical Research, Singapore General Hospital 2: Department of Haematology, Singapore General Hospital; Singapore Cord Blood Bank; Duke-NUS Graduate Medical School Singapore, Singapore
Publication date: September 1, 2008