(T1330) SINGLE-CELL TRANSCRIPTOMICS OF GENETICALLY-MATCHED IN VIVO AND IN VITRO-DERIVED HUMAN BLOOD STEM/PROGENITOR CELLS REVEALS NOVEL ROLE OF MICRORNA-MODULATED REGULATORY PATHWAY GENES
Assistant Professor Cedars-Sinai Medical Center Los Angeles, California, United States
Abstract: Prominent researchers throughout the world have been striving to develop HSCs in vitro that could be used to reconstitute the entire human hematopoietic system. Although there have been recent impactful advances towards this goal, improvements are still needed to obtain and optimize fully functional HSCs in vitro that have the robust long-term secondary hematopoietic transplantation capability that is required for their use in a clinical setting. What molecular differences distinguish fully functional human HSCs obtained in vivo from their in vitro iPSC-derived counterparts? While comparative transcriptomic studies of in vivo HSCs and in vitro iPSC-derived populations have led to significant insights as to the differences between these populations, they have also been hindered by a number of potentially confounding effects. The inclusion of non-functional cells due to the previous absence of a pure HSC cell surface marker profile, and the effect of individual genetic background variation on differential gene expression in genetically-diverse samples, might all lead to the detection of differentially-expressed genes that may not be affecting cell function, or the masking of more subtle changes that are critical for cell function. We hypothesized that a more focused view of molecular variation underlying functional differences could be achieved by performing comparative analysis on genetically matched cells. To accomplish this goal, we performed the first single-cell transcriptomic analysis of in vivo pure HSCs and their genetically matched in vitro iPSC-derived HSPC counterparts. By eliminating genetic background as a source of variability, we discovered novel differences in regulatory pathways modulated by miRNAs that were only seen when genetic background was taken into account. Furthermore, when the identified miRNAs were modulated during differentiation, this led to a significant increase in both overall hematopoietic differentiation efficiency, as well as an increase in several reported key HSC genes. Further analyses are ongoing, but we propose that modulation of these identified key miRNAs during hematopoietic differentiation provides a novel potential strategy towards the successful and reliable derivation of clinically viable HSCs in vitro from human pluripotent stem cells.
Funding Source: This research was funded in part by the National Institute on Aging, National Institutes of Health, grant number R21AG078872-01A1 (to ADP).
