218. Homeobox gene HLX is expressed in choriodecidua mesenchymal stem cells and regulates their ability to migrate

Mesenchymal stem cells (MSCs) can be prepared from the placenta (PMSC) and the choriodecidua component of the fetal membranes (CDMSC). PMSCs and CDMSCs share basic stem cell properties with adult MSCs but differ in their gene expression profiles and ultrastructure, showing features of more primitive and metabolically quiescent stem cells (1). Homeobox gene transcription factors are critical markers for identifying stem cells and they regulate important stem cell functions. Our laboratory showed the homeobox gene HLX is expressed in the placenta and the choriodecidua component of the fetal membranes, and is a regulator of proliferation in placental cells (2). In this study, our aim was to determine whether HLX was expressed in CDMSCs and to use short interfering RNAs (siRNAs) to specifically inactivate HLX and determine the effect on CDMSC function. Complementary DNA was prepared from CDMSCs and RT–PCR using HLX-specific primers generated the expected band size of 485bp following agarose gel electrophoresis (n = 3). At the protein level, HLX expression was detected in the nuclei of CDMSCs using immunocytochemistry. The expected HLX protein product was detected at ~50kDa using western blotting (n = 3). Conditions were optimised for the use of short interfering RNAs (siRNA) to decrease HLX expression in CDMSCs with 5nM giving the most efficient downregulation. Two independent siRNAs were tested (HLXsi3–4) and of these, HLXsi4 resulted in significantly decreased HLX mRNA levels in CDMSCs as shown by real-time PCR (0.66 ± 0.08, P = 0.03, n = 3). Functional assays to measure stem cell migration were carried out in quadriplicates on two samples. 10000 cells were placed on one side of a filter and the number of cells that migrated to the other side of the filter was stained and densitometric analysis was carried out using Axiovision image analysis software. These results suggest that the HLXsi4-mediated decrease in HLX expression resulted in reduced CDMSC migration (2.6x103 ± 401 v. 1.3x103 ± 225 densitometric units, P = 0.02). Therefore, HLX may play a role in stem cell migration. (1) Pasquinelli G, Tazzari P, Ricci F, Vaselli C, Buzzi M, Conte R, Orrico C, Foroni L, Stella A, Alviano F, Bagnara GP and Lucarelli E., Ultrastructural characteristics of human mesenchymal stromal. (2) Rajaraman G, Murthi P, Quinn L, Brennecke SP, Kalionis B. Homeodomain protein HLX is expressed primarily in cytotrophoblast cell types in the early pregnancy human placenta. Reprod Fertil Dev. 2008. (3) Rajaraman G, Murthi P, Leo B, Brennecke SP and Kalionis B. Homeobox gene HLX1 is a regulator of colony stimulating factor-1 dependent trophoblast cell proliferation. Placenta. 2007. 28(10):991–8.