In preprints: insights into the regulation of haematopoietic stem cell emergence in vitro and in vivo

Haematopoietic stem cells (HSCs) are a rare but essential population of cells responsible for the lifelong production of blood cells. HSCs emerge in the aorta-gonad-mesonephros (AGM) region between Carnegie stages (CS) 13 and 17 (4-6 weeks post-conception) (Ivanovs et al., 2011; Tavian et al., 1999). HSC emergence involves a complex developmental ontogeny that remains incompletely understood, with a key step being the endothelial-to-haematopoietic transition (EHT) (Calvanese and Mikkola, 2023; Ivanovs et al., 2017). EHT is a transient developmental process in which functionally immature HSC precursors extrude from haemogenic endothelium (HE) in the AGM, forming intra-aortic haematopoietic clusters (Bertrand et al., 2010; Boisset et al., 2010; Kissa and Herbomel, 2010). At CS17, nascent HSCs migrate to the fetal liver, where they mature and expand before engrafting in the bone marrow (Calvanese and Mikkola, 2023).

Despite its importance, the regulation of EHT remains poorly understood, which presents significant challenges for generating HSCs in vitro that can be therapeutically harnessed. In a recent preprint, Wellington and colleagues offer crucial insight into the molecular regulation of EHT by constructing an integrated single-cell transcriptomic map of embryonic and induced pluripotent stem cell (iPSC)-derived EHT (Wellington et al., 2024 preprint). This high-resolution map identifies key molecular determinants of EHT that are not fully recapitulated in vitro, revealing that fibroblast growth factor (FGF) signalling can be modulated to improve the efficiency of haematopoietic stem and progenitor cell (HSPC) generation.

Although iPSC-derived haematopoiesis recapitulates key stages of blood development, including forming HE with haemogenic potential, generating long-term HSCs that are capable of multilineage engraftment remains inefficient (Ditadi et al., 2017). This inefficiency may be driven by deficiencies in EHT, but a lack of high-resolution single-cell data has impeded the investigation of shortcomings in iPSC-derived EHT. To address this, Wellington et al. performed single-cell RNA sequencing (scRNA-seq) on embryoid bodies (EBs) cultured under haematopoietic differentiation conditions, profiling cells from days 7 to 21 and capturing 166,085 cells. Their analysis revealed a transient ‘bridge’ of cells connecting endothelial and haematopoietic clusters, peaking at day 8, corresponding to HE undergoing EHT.

To compare EHT dynamics in vitro with in vivo development, the authors built a high-resolution transcriptomic map of purified endothelial and haematopoietic cells from day 8 iPSC-derived EBs integrated with published human embryo AGM scRNA-seq datasets (CS10-17) (Calvanese et al., 2022; Crosse et al., 2020; Zeng et al., 2019). The map revealed that both iPSC- and AGM-derived cells formed a shared trajectory of endothelial and haematopoietic clusters connected by HE in the EHT state. The map provides a powerful resource for investigating the molecular differences that underlie the distinct haematopoietic outcomes from iPSC- and AGM-derived EHT.

To uncover molecular pathways important for HSC generation, Wellington et al. identified differentially expressed genes (DEGs) between iPSC- and embryo-derived cells in the integrated arterial endothelium, HE and emergent HSPC clusters. Their analysis revealed that, during EHT, iPSC-derived cells fail to upregulate genes related to haematopoiesis, including key HSC-specifying transcription factors such as HLF and SPINK2, and also fail to downregulate genes associated with endothelial identity, such as KDR. These findings suggest that insufficient suppression of endothelial transcriptional programs, alongside inadequate activation of haematopoietic programs, could explain the inefficiency of iPSC-derived HSC generation.

While characterising the molecular shortcomings of iPSC-derived haematopoiesis is a valuable outcome of the integrated dataset, a key question remains: how can this knowledge be leveraged to improve HSC generation? Reasoning that modulation of signalling pathways upstream of DEGs could improve the haemogenic potential of iPSC haematopoiesis, the authors applied NicheNetR, a computational tool for predicting ligand-receptor interactions, to their integrated dataset (Browaeys et al., 2020). Several ligand-receptor pairs were predicted to be differentially active; notably, FGF23 was identified as an upstream signal for 36 DEGs between iPSC- and embryo-derived HE. FGF signalling is known to regulate endothelial identity and HE formation, including upregulating KDR, and both FGF23 and the more canonical FGF2 ligand are not suppressed during iPSC-derived EHT (Simons et al., 2016).

To explore the potential of modulating FGF signalling to augment the haematopoietic potential of EHT, the authors used zebrafish embryos as a model system. After confirming that FGF signalling is attenuated in endothelial cells of the dorsal aorta (a structure analogous to the AGM in mammals) at the onset of EHT, the authors perturbed the pathway using the inhibitor erdafitinib and the agonist BCI. The inhibitor significantly increased the generation of CD41⁺ HSPCs, while activation of FGF signalling with BCI reduced haematopoietic output. The authors also tested the effect of modulating FGF signalling in iPSC-derived EBs at days 7-8 of differentiation. Encouragingly, assessment of human iPSC-derived haematopoietic output at day 10 using flow cytometry revealed a significant increase in CD34⁺CD43⁺ HSPCs from FGF-inhibited EBs compared to control DMSO-treated EBs.

By constructing comprehensive single-cell transcriptomic maps of iPSC differentiation dynamics and integrating with in vivo AGM datasets, the authors provide a valuable resource for the field. The integrated map uncovers key deficiencies in the suppression of endothelial transcriptional programs and activation of haematopoietic identity during in vitro EHT. By demonstrating that inhibition of FGF signalling during EHT improves HSPC generation, the study suggests that iPSC differentiation can be optimised through targeted interventions. Overall, this study represents an important advancement towards the goal of efficiently producing HSCs in vitro for therapeutic applications, complementing recent progress in iPSC differentiation to generate long-term engrafting multilineage haematopoietic progenitors that better resemble AGM-derived HSCs (Ng et al., 2024).