A Link between Blood Flow and the Inflammatory Networks That Govern Hematopoietic Stem Cell Specification

Hematopoietic stem and progenitor cells (HSPCs) are multipotent, self-renewing cells that maintain the blood-forming system throughout the life of all individuals. The unique properties of these cells make HSPC transplantation an effective therapy for treatment of many blood disorders. The advent of induced-pluripotent stem cells (iPSCs) led to great optimism in the field as a potentially limitless source of immunologically-matched, clinical-grade hematopoietic cells. Despite success in producing in vitro hematopoietic progenitors and cells that are capable of limited in vivo reconstitution, there are no robust, reproducible protocols that can generate long-term reconstituting HSPCs. Having a more complete understanding of the signals and molecular pathways required to develop HSPCs in their native environment will move the field forward, leading to more robust protocols and the development of novel HSPC-based therapeutics.

Years of investigation have revealed that HSPCs require multiple molecular inputs for proper specification, including activity of the Notch, Wnt, FGF, and BMP signaling pathways. In addition, we have reported inflammatory signaling (Tnfa and NF-kB) as a novel group of HSPC fate modulators. However, the spatial temporal requirement of NF-kB activity has been enigmatic due to: (1) lack of tools that faithfully report the dynamic activation of NF-kB; (2) difficulty on assessing NF-kB dynamics in vivo in a mammalian system during embryonic development due to artifactual inflammatory conditions triggered by invasive techniques required for in utero experimentation. Addressing this knowledge gap will be critical for optimizing in vitro protocols for the generation of patient-specific HSPCs.

To date, all existing zebrafish NF-kB reporter lines fail to report dynamic cellular activation of NF-kB in real time due to the long half-life of their fluorescent reporter proteins. To overcome this limitation, we have generated a novel NF-kB reporter zebrafish line driving a destabilized version of eGFP (D2eGFP) containing a PEST domain. This modified eGFP has a short half-life of two hours, enabling us to pinpoint the precise developmental window these signals are required. Time-lapse confocal imaging, in conjunction with flow cytometry, of double transgenic kdrl:mCherry, NF-kB:D2eGFP embryos revealed that NF-kB was activated in vivo within the hemogenic endothelium (HE) just prior to the establishment of circulation (18-24 hpf) and was deactivated soon after its initiation (26 hpf). When this window of NF-kB activity was disrupted by inhibiting the translocation of the NF-kB p65 subunit to the nucleus using Caffeic acid phenethyl ester (CAPE), HSPCs failed to specify. qPCR analysis from FACS-isolated HE with activated NF-kB kdrl:mCherry⁺ ,NF-kB:D2eGFP⁺ and the surrounding endothelium kdrl:mCherry⁺ ,NF-kB:D2eGFP^- showed a significant enrichment of the early HSPC markers gfi1 and runx1 in the NF-kB⁺ endothelial population at 22 hpf. In addition, mRNAseq of kdrl:mCherry⁺ HE of NF-kB⁺ showed hematopoietic GO terms enriched, demonstrating that NF-kB is a contributing activator of the endothelial to hematopoietic transition.

Blood flow is a well-known conserved regulator of HSPC formation. To investigate the correlation between NF-kB deactivation and circulation, blood flow was inhibited by morpholino knockdown of tnnt2a in Tg(kdrl:mCherry, NF-kB:D2eGFP) embryos and assessed by confocal microscopy and flow cytometry for NF-kB activity. In the absence of circulation, NF-kB displayed sustained activity throughout the HE. To determine if this prolonged NF-kB activity was contributing to the loss of HSPCs observed in tnnt2a morphants, we blocked NF-kB at the developmental time circulation would normally occur using CAPE and observed a significant rescue of HSPC numbers. These data provide evidence of a link between the extrinsic biomechanical forces of blood flow and the intrinsic molecular regulation of the inflammatory networks that govern HSPC specification. Altogether, our NF-kB reporter line provides one of the earliest markers that can be utilized to isolate HE in vivo. These data pinpoint the early NF-kB requirement to form HSPCs and could be applied in vitro to improve current protocols of hematopoietic differentiation from human PSCs.

No relevant conflicts of interest to declare.