RNAi Screens for Maintenance of Hematopoietic Stem Cell Phenotype During Culture
Authors: Larsson Jonas 
Published: April 2021

Successful ex vivo expansion of hematopoietic stem cells (HSCs) could greatly enhance cell- and gene therapy applications in treatment of malignant and inherited hematological disorders. It has therefore been a long-standing goal in the field to gain a better understanding of the genes and pathways that regulate the self-renewal ability of HSCs. We have developed RNAi screening strategies to identify modifiers of self-renewal/proliferation in human hematopoietic stem- and progenitor cells (HSCPs). Employing pooled lentiviral shRNA libraries targeted to human cord blood derived CD34+ cells, we use the limited persistence of HSPCs under ex vivo culture conditions as a basis for functional selection of shRNAs conferring prolonged maintenance or expansion of undifferentiated cells. From the screens we have been able to identify directly targetable genes such as p38 MAPK, which can be pharmacologically inhibited to enhance the stem cell output of cultured cord blood cells. Additionally, we have found several epigenetic regulators among the top-scoring genes, including genes of the cohesin family, which modulate chromatin architecture, and JARID2, an important modifier of polycomb repressive complex 2 (PRC2). Depletion of either the cohesin genes or JARID2 by shRNA impairs differentiation and enhances both in vitro expansion and the in vivo reconstitution capacity of human HSPCs. However, whether these genes can be targeted pharmacologically to support HSC expansion remains to be explored. The screens have further identified a number of shRNAs that display a remarkable ability to expand HSPCs in culture, but whose gene targets have not been validated, indicating that they are affecting one or more genes in a non-specific manner. We performed gene expression profiling of cells transduced with these off-target shRNAs to gain insight about the molecular context under which HSPCs are propagated ex vivo. A common expression signature was the down-modulation of cellular stress response genes, such as p38 MAPK (previously identified in the screens) as well as genes involved in NF-□B signaling. Indeed, we found that pharmacological inhibition of NF-□B signaling leads to a significant improvement of stem cell function, from ex vivo cultured cord blood derived CD34+ cells, as assessed by transplantation to NSG mice. The effect of NF-□B inhibition was most critical early during the culture where it reduced the levels of several inflammatory and stress-related cytokines that are induced as an immediate response to culture initiation. Taken together, findings from our RNAi screens indicate that ex vivo HSC expansion is facilitated by targeting two distinct gene categories: cellular stress response genes activated by the culture conditions, as well as epigenetic regulators controlling the balance between renewal and differentiation. In order to define the most optimal expansion conditions we are currently assessing the combined targeting of several of these factors in conjunction with other compounds such as SR1 and UM171 using cellular barcoding. Here, cells are genetically marked by lentivirally delivered barcode sequences prior to expansion culture, which enables a precise detection and comparison of the stem cell output from multiple different expansion conditions in a directly competitive manner within a single xenografted animal.

Disclosures

No relevant conflicts of interest to declare.