Strength in Numbers

For decades, there has been considerable interest in understanding the molecular mechanisms governing hematopoietic stem cell (HSC) self-renewal versus differentiation. In particular, the concept of maintaining “stemness” during ex vivo culture conditions has been attractive to researchers hoping to expand smaller sources of HSCs for transplantation, such as those acquired from umbilical cord blood, or for those researchers interested in genetically manipulating HSCs for curative therapies of genetic disorders.

Several different experimental systems have been used to identify regulators of HSC self-renewal, some of which have advanced to early therapeutic interventions in transplantation. One approach has been the use of small molecule chemical screens, either on nonhuman cells like zebrafish embryos or on primary human CD34+ cells. These screens have identified molecules such as prostaglandin E₂, UM171, or the aryl hydrocarbon receptor (AHR) antagonist SR1.¹  Other studies have used genetic approaches in mice like RNAi screens, retroviral insertion screens, or hypothesis driven genetic alteration of HSCs. Intriguingly, using all of these methods, several groups have previously reported^2-5  that the RNA binding protein Musashi-2, originally discovered in Drosophila as a regulator of asymmetric cell division, is a key regulator of murine hematopoiesis. The mechanisms governing Musashi-2 regulation, and the exact hematopoietic effects, have been mixed depending on the mouse models used and assays performed.

A recent report by Dr. Stefan Rentas and colleagues sought to characterize the role of Musashi-2 overexpression in regulating human HSCs and to develop a mechanistic understanding of Musashi-2 regulation of hematopoiesis. They demonstrated that lentiviral overexpression of Musashi-2 enhanced the number of human short-term repopulating cells, as demonstrated in xenotransplantation assays in mice, as well as a suggestion of enhanced long-term repopulating HSCs after ex vivo culture. The authors then performed RNA sequencing of the Musashi-2 overexpressing CD34+ cells and of CD34+ cells with a lentiviral shRNA knockdown of Musashi-2. Surprisingly, pathway analysis revealed that targets involved in AHR signaling were suppressed in Musashi-2 overexpressing cells, while the inverse was true after Musashi-2 knockdown. Given that the AHR is the same target of SR1, which is currently being clinically explored for HSC expansion,⁶  the authors performed gene set enrichment analysis (GSEA) and showed that genes that were downregulated by Musashi-2 overexpression significantly matched those downregulated with SR1 treatment.

Protein-RNA interaction analysis then found that two of the primary RNA targets regulated by Musashi-2 are the 3'UTRs of HSP90 and CYP1B1, both AHR pathway components. The authors thus suggest a model whereby Musashi-2 post-transcriptionally downregulates HSP90 and then continuously represses CYP1B1. The resulting suppression of the AHR signaling pathway, much like SR1 treatment, enforces stem cell self-renewal programming, facilitating ex vivo expansion of human HSCs (Figure).