Differentiation of murine embryonic stem cells into megakaryocytic lineage cultured in feeder layer-free media

Background and objectivesEmbryonic stem cells are potential pluripotent stem cells derived from inner cell mass of embryonic blastocyst stage. So far, differentiation of embryonic stem cells to megakaryocyte lineage has resulted from coculture with bone marrow stromal cells with disadvantages such as possibility of stem cell contamination from feeder layer and secretion of unknown factors from this layer ending in unwanted differentiation and genetic changes in embryonic stem cells. In this study, while excluding feeder layer, we differentiated murine bone marrow cells to megakaryocyte lineage. Materials and MethodsIn this experimental study, embryoid bodies resulted from R1 murine embryonic stem cells. The embryoid bodies were then cultured in two groups of test (containing IMDM with FBS, L-glutamin, non-essential aminoacids, beta mercaptoethanol, and TPO and IL-3 as growth factors) and control (the same culture medium used in test group without growth factors). After four days the colony assay test was performed, and after eight days immunohistochemistry staining for CD41 molecule, and finaly transcription test for PF4 gene using RT-PCR to demonstrate differentiation to megakaryocyte lineage. ResultsResults of the colony assay test indicated that the cells considered had the capacity to give rise to colonies. The total number of colonies and banzidine positive colonies were 57 ± 6.1 and 18 ± 3.6 respectively. On the other hand, immunocytochemistry analysis and PCR showed that the CD41 molecule and PF4 gene are expressed in differentiated cells. ConclusionsOur results indicated that murine embryonic stem cells differentiate into megakaryocyte lineage cells without utilizing feeder layer, and embryonic stem cells can be used as an immortal new source for producing megakaryocyte lineage cells on which basic biological research can be conducted.