For years, regenerative medicine experts and physicians have never stopped developing new sources of hematopoietic stem cells (HSCs) for curative therapy against malignant blood diseases including immunodeficiency and leukaemia. Multiple groups attempted to artificially obtain HSCs from induced pluripotent stem cells (iPSCs). However, this strategy struggled since these HSC-like cells do not have long-term hematopoietic reconstitution ability in vivo, implying that the molecular mechanism regulating HSC function is extremely complex.

This led Fuchou Tang group and Bing Liu’s lab from Affiliated Hospital of Academy of Military Medical Sciences to assume that they should take lessons from the HSC emergence from scratch at embryonic stage. HSC originates from hemogenic endothelium and subsequently forms immature HSC precursors (pre-HSCs). To accurately understand the endothelial-HSC transition, the very first bottleneck is how to accurately identify these rare, naive but powerful pre-HSCs. They found that existing strategies of in vitro HSC induction and functional identification (e.g. re-/co- aggregation assays by Alexander Medvinsky group) was time-consuming, requiring high skills, and of low efficiency. Therefore, the researchers developed a new simplified robust strategy named OP9/DL1 co-culture system. They proved that this unique system could successfully induce hemogenic endothelial cells into functional HSCs with standard transplantation ability (Li et al., JGG, 2013). Interestingly, this simplified but with high induction efficiency assay greatly made it possible to conduct HSC induction with a small number of cells (even one individual cell).

Fig. 1 Identification of pre-HSCs at single-cell resolution (OP9-DL1 co-culture system)

Combined with this efficient functional detection system, Dr. Tang and his colleagues then screened and eventually discovered potent surface markers to capture the nascent pre-HSCs at high purity (>30%, approaching to that of adult HSCs), as rigorously validated by newly developed single-cell-initiated serial transplantation. Therefore, the highly purified pre-HSCs enabled, for the first time, further analysis of the elusive nascent HSC precursors in mouse embryos at single-cell resolution (almost 20 years after the identification of adult HSCs). After that, they profiled the cells at the nodal stage of HSC development to explore the molecular mechanisms regulating endothelial-to-pre-HSC transition with single-cell transcriptome sequencing technology. Unsupervised dimensionality reduction and clustering algorithm of the transcriptome data showed that multiple signalling pathways and transcription factor networks might play critical roles during the HSC emergence.

Fig 2 Pseudotime analysis of single cells during HSC emergence by the Monocle method

With the in-depth data mining and further functional evidence, we also found that HSC at the initial stage of origin has clear heterogeneity in cell cycle and reconstitution potential by transplantation. Based on clues from the mining of sequencing data and the evidences form the conditioned knockout mouse assay, we verified that Rictor (a key molecule in mTOR signaling) could accurately regulate the pre-HSC formation and HSC generation in vivo.

Collectively, this work paves the way for dissection of complex molecular mechanisms regulating stepwise generation of HSCs in vivo, informing future efforts to engineer HSCs for clinical applications.