Tracking ex vivo hematopoietic stem cell function using Fgd5 and EPCR reveals molecular regulators of expansion.

Hematopoietic stem cells (HSCs) cultured outside the body are the fundamental component of a wide range of cellular and gene therapies. Recent efforts have achieved more than 200-fold expansion of functional HSCs, but their molecular characterization has not been possible due to the substantial majority of cells in these cultures being non-HSCs and the fact that single cell-initiated cultures display substantial clone-to-clone variability. Using the Fgd5 reporter mouse in combination with EPCR, we have identified an in vitro reporter strategy capable of exclusively identifying HSC-containing clones and prospectively separating HSCs from non-HSCs. Linking single clone functional transplantation data with single clone gene expression profiling, we also reveal the molecular profile of expanded HSCs which is more similar to actively cycling fetal liver HSCs than quiescent adult bone marrow HSCs from which they were derived. A gene expression signature derived from the molecular profiling identifies core molecular components of HSCs, including Prdm16, Fstl1 and Palld, that can further predict the functional outcome in transplantation assays. Taken together, this system represents a new tool to be used for a wide-range of functional screening and molecular experiments previously not possible to carry out due to limited HSC numbers Overall design: We performed bulk RNA-sequencing on expanded clones, derived from single murine LT-HSCs. For each clone, the stem cell population (EPCR+Lin-Sca1+cKit+ [ELSK]) and all remaining cells (nonELSK) were separated by flow sorting and sequenced. In total, we collected and sequenced 11 ELSK and 12 nonELSK samples. Both ELSK and nonELSK groups are further divided by their repopulation outcome - repopulating ELSK (PosELSK), repopulating nonELSK (PosNonELSK), non-repopulating ELSK (NegELSK) and non-repopulating nonELSK (NegNonELSK). We also performed scRNA-seq on hibernating HSCs (n=36) and fetal liver HSCs (n=54).