Transcriptional profiling of human embryonic stem cells and their functional derivatives

Abstract: Human    embryonic    stem    cells    (hESCs)    represent    populations    of    pluripotent, undifferentiated  cells  with  unlimited  replication  capacity,  and  with  the  ability  to differentiate into any functional cell type in the human body. Based on these properties, hESCs  and  their  derivatives  provide  unique  model  systems  for  basic  research  on embryonic development. Also, industrial in vitro applications of hESCs are now beginning to  find  their  way  into  the  fields  of  drug  discovery  and  toxicology.  Moreover,  hESC-derivatives are anticipated to be promising resources for future cell replacement therapies. However, in order to fully utilize the potential of hESCs it is necessary to increase our knowledge about the processes that govern the differentiation of these cells. At present, some  of  the  major  challenges  in  stem  cell  research  are  heterogeneous  cell  populations, insufficient  yield  of  the  differentiated  cell  types  and  immature  derivatives  with  limited functionality.  To  address  these  problems,  a  better  understanding  of  the  regulatory mechanisms  that  control  the  lineage  commitment  is  needed.  The  aim  of  this  thesis  has been to increase the knowledge of the global transcriptional programs which are activated when  cells  differentiate  along  specific  pathways,  and  to  identify  key  genes  that  show differential expression at specific stages of differentiation. The results indicate that hESCs express a unique set of housekeeping genes that are stably expressed in this specific cell type  and  in  their  derivatives,  which  highlights  the  importance  of  proper  validation  of reference genes for usage in hESCs. Furthermore, an extensive characterization of hESCs and differentiated progenies of the cardiac and hepatic lineages has been conducted, and sets  of  differentially  expressed  genes  were  identified.  Two  different  protocols,  which mediate  definitive  and  primitive  endoderm  respectively,  were  studied,  and  important discrepancies  between  these  two  cell  types  were  identified.  Moreover,  the  global expression profile of hESC-derived cardiomyocyte clusters were thoroughly investigated and compared to that of foetal and adult heart. To further study regulatory mechanisms of  importance  during  stem  cell  differentiation,  the  global  expression  of  microRNAs (miRNAs) was also investigated. Putative target genes of differentially expressed miRNAs were  identified  using  computational  predictions,  and  their  mRNA  expression  was analysed. Notably, an interesting correlation between the miRNA and mRNA expression was observed, which supports the general notion that miRNAs bind to and degrade their target mRNAs, and thus act as fine-tuning regulators of gene expression. Taken together, the results described in this thesis provide important information for further studies on regulatory mechanisms that control the differentiation of hESCs into functional cell types such as cardiomyocytes and hepatocytes.