Next, we studied the effects of leukemia cells on BMSCs co cultured in direct make contact with. BMSCs from three healthier donors have been co cultured using the three distinctive leukemia cell lines in direct speak to. The cells had been har vested at four h, 10 h and 24 h and total RNA was ex tracted. The total RNA from BMSC mono cultures was mixed with the total RNA from TF 1, TF 1 or K562 cell mono cultures and the resulting 3 mixed total RNA samples have been applied as a mono culture handle inside the gene expression profiling analysis. The RNA from BMSCs co cultured using the TF 1, TF 1a and K562 cells had been ex tracted and the gene expression profiles had been analyzed by microarrays. The analysis of microarray information working with Partek Genomic Suite revealed that 544 genes have been differentially expressed amongst co cultured and mono cultured control cells.
Hierarchical clustering analysis of these genes clearly separated kinase inhibitor Microtubule Inhibitors the samples into two groups, co cultures and mono cultures. The results had been equivalent to the evaluation of BMSCs co cultured in transwells with all the leukemia cells. We located that CXCL1, CXCL6, TEP1, IL8, CCL2 and PTGS2 genes have been one of the most up regulated genes in BMSCs co cultured inside the direct get in touch with with leukemia cells. Ingenuity Path way Analysis of your differentially expressed genes revealed that the top canonical pathways involved were the gluco corticoid receptor signaling, IL 17 signaling and acute phase response signaling. Gene expression evaluation of BMSCs co cultured with CD34 cells revealed changes 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 have been 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 all the CD34 cells were analyzed by microarrays. Evaluation of the microarray data revealed that 4904 genes were differentially expressed in between the two groups. Hierarchical clustering evaluation of these genes separated the BMSCs into two selleck chemicals groups but the separation between co cultured and mono cultured cells was not ideal. One particular group consisted of eight co cultured samples and 2 mono cultures, the sec ond group consisted of 7 mono cultured samples and 1 co cultured sample.
We discovered that by far the most up regulated genes in BMSCs co cultured with CD34 cells compared with BMSC mono cultures were SERPINB2, IL1B, RTP3, CCL7 and IL8. Ingenuity pathway analysis revealed that the prime ca nonical pathways involved were 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.