Targeting RNA helicase DDX3 in stem cell maintenance and teratoma formation

Abstract

DDX3 is an RNA helicase that has antiapoptotic properties, and promotes proliferation and transformation. Besides the role of DDX3 in transformed cells, there is evidence to indicate that DDX3 expression is at its highest levels during early embryonic development and is also expressed in germ cells of adults. Even though there is a distinct pattern of DDX3 expression during embryonic development and in adults, very little is known regarding its role in embryonic stem cells and pluripotency. In this work, we examined the relationship between DDX3 and human embryonic stem cells and its differentiated lineages. DDX3 expression was analyzed by immunohistochemistry in human embryonic stem cells and embryonal carcinoma cells. From the data obtained, it was evident that DDX3 was overexpressed in undifferentiated stem cells compared to differentiated cells. Moreover, when DDX3 expression was abrogated in multiple stem cells, proliferation was decreased, but differentiation was facilitated. Importantly, this resulted in reduced potency to induce teratoma formation. Taken together, these findings indicate a distinct role for DDX3 in stem cell maintenance.

Keywords: DDX3; RK-33; differentiation; stem cells; teratoma.

Figure 1. Expression of DDX3 in pluripotent and differentiated cell lines

DDX3 expression is lower in differentiated cells (FF: human fetal fibroblasts; ECC Neuro: Neural differentiated hECCs) and higher in pluripotent stem cells (hEGCs, hECCs and hESCs). Relative expression of DDX3 was compared to β-actin as the endogenous control. ΔΔCt method was also employed using the unipotent germ cell progenitor cells, PGCs as the baseline value (N = 3, P < 0.05).

Figure 2. Immunofluorescence detection of DDX3 in undifferentiated human ECCs

A. Oct4 (green), B. DDX3 (red). C. A and B overlaid. Reduced DDX3 in differentiated ECCs showing reduced expression of the pluripotent marker. D. Cell surface expression of Tra-1-60 (green) and E. DDX3 (Red) is reduced in ECCs cultured under neural inducing differentiation. F. Overlay of D and E shows a few remaining undifferentiated ECCs that are TRA-1-60+/DDX3+. DAPI was used as nuclear stain (blue).

Figure 3. DDX3 expression correlates with pluripotency markers in hESC

A. (Left) hESCs express DDX3 in cells expressing the stem cell markers OCT4, NANOG, and SOX2. (Right) Knock-down of DDX3 activity by RK-33 reduces the expression of NANOG, OCT4 and SOX2 in hESCs. Nucleus was stained with DAPI (Blue). B. Quantitative analysis of the percent of cells, which expressed each marker are presented. While the majority or >95% of the undifferentiated hESC expressed all four markers. After inhibition of DDX3 activity, the percent of cells expressing DDX3 was significantly reduced and to a similar extent as the stem cell markers. (P < 0.05). C. Bar graph of cell number with DDX3 inhibitor versus control demonstrating survival appeared unaffected by DDX3 inhibition.

Figure 4. DDX3 inhibition promotes hESC differentiation after 4 days in culture

Left Column vehicle only controls (DMSO). Right Column with 0.1uM RK-33. hESCs exposed to DMSO consisted of more undifferentiated OCT4+, SOX2+ cells and reduced numbers of cells expressing differentiation markers A. mesoderm: Podo, CD34 B. neuroectoderm: Nestin+, SSEA1+ compared to hESC cultured with DDX3 inhibitor RK-33. DAPI (Blue) is nuclear stain.

Figure 5. Dose response of the DDX3 inhibitor RK-33 on undifferentiated and differentiated hESCs

A. Proliferation assay using hESCs in the presence of different concentrations of RK-33 (n = 2). B. Effect of RK-33 on undifferentiated and differentiated hESCs determined by MTT assays (n = 2). C. Effect of RK-33 on human embryonic fibroblast determined by cell counts (n = 2).

Figure 6. RK-33 enhances differentiation and reduces teratoma formation

Five million of the respective cells in matrigel were injected into the flanks of NOG mice. Following 8 weeks of incubation, tumor volumes were measured and the samples fixed, sectioned and stained with H&E. The pictures shown represent the actual teratoma volume as related to pixel ratio. The pictures were taken using a 1X lens. Four animals per group were used for this study.