In this study, we aim to perform gene expression profiling of undifferentiated human pluripotent stem cells (hIPSC) and macrophages and their progenitors derived from hIPSC. The aims of the experiment is to identify a set of genes uniquely expressed in hIPSC but not in hIPSC derived monocytes/macrophages (hIPSdM) and define them as "stemness" genes. We will also identify genes upregulated in hIPSC-derived monocytes/macrophages but not hIPSC to provide insights into the regulatory switches of hIPSC differentiation.
We established protocols to generate monocytes and macrophages from hIPSC CRL1 line in vitro. Briefly, hIPSC were grown in bacteriological dishes in the presence of hIPSC base medium to generate embryoid bodies (EBs). Subsequently, EBs were transferred to gelatinized tissue culture dish in medium supplemented with M-CSF and IL-3. Supernatants containing macrophage progenitors were collected, spun down and plated onto bacteriological dishes to differentiate into macrophages. Two or three biological replicates of undifferentiated hIPSCs, macrophage progenitors and macrophages were collected and their RNA were isolated via the Qiagen RNA Isolation Kit. The RNA were treated with RNAse free DNase and eluted in RNase-free water. THe RNA integrity (RIN >8) were obtained for all samples by Agilent 2100 Bioanalyzer. We aim to sequence 5GBp per replicate per lane on an Illumina platform.