Next, we studied the effects of leukemia cells on BMSCs co cultured in direct contact. BMSCs from three healthier donors were co cultured using the three distinct leukemia cell lines in direct speak to. The cells have been har vested at 4 h, 10 h and 24 h and total RNA was ex tracted. The total RNA from BMSC mono cultures was mixed together with the total RNA from TF 1, TF 1 or K562 cell mono cultures and also the resulting three mixed total RNA samples had been employed as a mono culture control in the gene expression profiling analysis. The RNA from BMSCs co cultured with the TF 1, TF 1a and K562 cells were ex tracted along with the gene expression profiles have been analyzed by microarrays. The analysis of microarray information applying Partek Genomic Suite revealed that 544 genes have been differentially expressed between co cultured and mono cultured control cells.
Hierarchical clustering analysis of these genes clearly separated selleck chemical the samples into two groups, co cultures and mono cultures. The outcomes have been equivalent towards the analysis of BMSCs co cultured in transwells with the leukemia cells. We identified that CXCL1, CXCL6, TEP1, IL8, CCL2 and PTGS2 genes were the most up regulated genes in BMSCs co cultured within the direct get in touch with with leukemia cells. Ingenuity Path way Analysis from the differentially expressed genes revealed that the top rated canonical pathways involved have been the gluco corticoid receptor signaling, IL 17 signaling and acute phase response signaling. Gene expression analysis of BMSCs co cultured with CD34 cells revealed modifications in metabolism connected genes To evaluate whether or not the observed BMSC gene induction was particularly induced by leukemia cells, BMSCs were co cultured in transwells with CD34 cells from healthful donors.
The BMSCs were harvested at four h, 10 h and 24 h and total RNA was extracted. The gene expres sion profiles of BMSC mono cultures and co cultured with the CD34 cells were analyzed by microarrays. Analysis of the microarray information revealed that 4904 genes were differentially expressed among the two groups. Hierarchical clustering evaluation of these genes separated the BMSCs into two mTOR cancer groups however the separation between co cultured and mono cultured cells was not excellent. A single group consisted of eight co cultured samples and two mono cultures, the sec ond group consisted of 7 mono cultured samples and 1 co cultured sample.
We found that the most up regulated genes in BMSCs co cultured with CD34 cells compared with BMSC mono cultures had been SERPINB2, IL1B, RTP3, CCL7 and IL8. Ingenuity pathway evaluation revealed that the leading ca nonical pathways involved had been the purine metabolism, mTOR signaling and EIF2 signaling. To valid ate the microarrays information, we performed a quantitative RT PCR evaluation which confirmed the higher expression of IL8 in BMSCs co cultured with CD34 cells compared with BMSC mono cultures.