In vitro expansion of haematopoietic stem and progenitor cells (HSPCs) is a challenging task since these cells tend to differentiate under culture conditions resulting in loss of their stem cell character. Possibilities to enhance HSPC self-renewal involve intrinsic regulators such as transcription factors or microRNAs (miRNAs) but also extrinsic stimulation by signalling molecules. Most attempts for intrinsic stimulation of HSPC proliferation utilise stable gene modification such as viral vectors that are still controversial in clinical settings as they harbour the risk of insertional mutagenesis. Therefore, our study aimed to investigate new approaches to modify HSPC gene expression at the post-transcriptional level by using novel and established RNA-based molecules. First, various transfection methods were tested to deliver non-integrating RNA molecules into HSPCs. Standard chemical transfection methods including lipofection for delivery of siRNAs targeting surface markers did not lead to protein knockdown although more than 90% of fluorescent cells were detected using a fluorescently labelled control siRNA. By means of confocal microscopy, we showed that the siRNA-reagent complexes stuck to or inside the cell membrane after transfection, thereby mimicking a transfection event. In contrast, efficient siRNA-mediated protein knockdown was achieved using electroporation, proving that this is the method of choice for HSPC transfection. For RNA-mediated HSPC expansion, the function of selected candidate miRNAs was modulated by electroporation of specific inhibitors or mimics. Enhancement of miRNA 125b and miRNA-99a function or inhibition of miRNA 142 3p slightly increased the expansion of HSPCs compared to a scrambled negative control. Serum taken from patients after chemotherapy has previously been shown to stimulate HSPC expansion. In this study, microarray analysis of miRNAs circulating in serum indicated that miRNAs are differentially expressed in serum before and after therapy. However, a combination of different soluble factors rather than a single miRNA probably is responsible for the observed growth-promoting effect. Overexpression of regulatory proteins such as HOXB4 using mRNAs was efficient but presumably too short-lived to significantly impact on HSPC behaviour. Novel, self-replicating RNAs resulted in prolonged protein expression, offering new opportunities for DNA-free gene modulation in HSPCs. Preliminary data suggest that overexpression of the epigenetic modifier BMI1 by self-replicating RNAs could prevent stem cell loss in vitro, but its effect on HSPC expansion has to be investigated more in detail in future studies.As an extrinsic approach for HSPC expansion, the Notch ligand Delta like 4 (Dll4) was coupled to beads and added to long-term cultures. This system promoted the expansion of primitive HSPC subpopulations in a stromal cell- and serum-free culture system. In conclusion, non-integrating intrinsic HSPC modulation in combination with extrinsic stimulation might be the key to successful in vitro expansion of HSPCs
