The Effect of Humanized Cell Culture Media in Early Stage of Megakaryocyte Differentiation

The Effect of Humanized Cell Culture Media in Early Stage of Megakaryocyte Differentiation
Majid Zamani,¹ Yoda Yaghoubi,² Alireza Mohammadzaeh,³ Adel Naimi ,⁴ Parisa Samadi,^5,*
1. Department of Medical Laboratory Sciences, Faculty of Allied Medicine, Gonabad University of Medical Sciences, Gonabad, Iran
2. Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
3. Department of Microbiology, School of Medicine, Gonabad University of Medical Sciences, Gonabad, Iran
4. Cellular and Molecular Research Center, Sabzevar University of Medical Sciences, Sabzevar, Iran
5. Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
Introduction: Hematopoietic Stem cells (HSCs) are small population of cell in bone marrow which can impaired for a different reasons, such as leukemia or tumors. HSCs transplantation is useful method for treatment this disorders. Umbilical Cord Blood (UCB) is a suitable source for this purpose. Number of HSCs in one unit of UCB is insufficient for a cell therapy so this can lead to a delayed or failed reconstruction of the cell and platelet recoveries. So it’s necessary to generate megakaryocyte-committed cells for reduce period of platelet reconstruction and reduce high-cost, bacteremia, febrile reactions, and alloimmunization in allogenic platelet transplantation after HSCs transplantation. HSCs expansion in invitro need the cell culture media, which supplemented with FBS. FBS use has disadvantages such as limited resources, high cost and risk of xenovirouses transmission. Therefore, FBS should be replaced with products with human resources to reduce the risks of disease transmission and costs. Platelet lysates are a good source of enrichment for culture media. PL have different growth factors and cytokine, such as Insulin-like Growth Factor (IGF)-1, Platelet-derived Growth Factor (PDGF), Vascular Endothelial Growth Factor (VEGF), Hepatocyte Growth Factor (HGF), which can improve cell proliferation and differentiation. The effect of PL on different cell lines as well as the differentiation of erythroid cells from HSCs has been studied, although so far no studies have been performed on the effect of platelet lysate on early stage differentiation of megakaryocytes.
Methods: The human umbilical cord blood was obtained from consenting parturients then Mononuclear cells (MNCs) fraction was separated within 8 hours after delivery and MNCs were enriched by Ficoll/Histopaque (density: 1.077 g/mL). CD34+ cells were isolated by positive selection using immuno-magnetic microbeads and MiniMACS columns. CD34+ cells were cultured in Iscove’s Modified Dulbecco’s medium supplemented with 10% PL or FBS, thrombopoietin, stem cell factor, Interleukin 6 and Interleukin 3 in 12-well plates. The cells were incubated in 5% CO2 humidified air at 37 °C for 3 days. Cells were harvested after 3 days from PL or FBS-containing media and megakaryocyte linage markers (CD41 and CD42b) and gene (GATA1, GATA2, FLI1, RUNX1) expression was assessed by flow cytometry and Real-time PCR, respectively. The cell expansion was also evaluated.
Results: Flowcytometry data showed expression of CD41 (16.34% and 13.51%), CD42b (5.12% and 3.46%), CD41/CD42b (4.22% and 2.67%) at the day 3 in cells cultivated in the PL containing medium and FBS containing medium containing media, respectively. RT-PCR results indicated increase GATA1, GATA2, FLI1, and RUNX1 gene expression in the PL containing medium than FBS containing medium. In addition, mean of 13 and 9 fold cell expansion were observed in PL and FBS containing medium, respectively.
Conclusion: In this study, we evaluated the effect of Platelet lysate substitution for Fetal Bovine serum in early stage of Megakaryocyte differentiation. CD41 and CD42 are related to megakaryocyte differentiation. CD41 is expressed in the early stage of megakaryocyte differentiation and CD42 is expressed in more mature megakaryocytes. In addition, some genes like GATA2, GATA1, RUNX1, and FLI1 are related to promote megakaryopoiesis and megakaryocyte markers expression. In our study, megakaryocyte markers and gene expression was increased in cells cultured PL containing medium than FBS containing medium. Additionally, cell expansion was increased in cells cultured PL containing medium than FBS containing medium. Our results showed that PL have not worrying about the transition of xenovirus and ethical problems and can be a suitable substitution for FBS in HSCs culture and differentiation into megakaryocytes. Although, the use of PL in HSCs culture and differentiation needs long-term cultivation and more evaluations.
Keywords: Platelet lysate, Fetal Bovine serum, Stem Cell, Growth Factor, Megakaryocyte