DLK1 regulates HSC quiescence through phase separation mediated inhibition of receptor signaling

While much is known about factors that stimulate the proliferation and differentiation of stem cells, less is known about the factors that prevent their differentiation and preserve their function. One factor implicated in the maintenance of an undifferentiated stem cell state is the non-canoncial Notch ligand, Delta like homologue 1 (DLK1). DLK1 is widely expressed during embryogenesis where it regulates stem cells to control organ size. In the adult, DLK1 expression is limited to select tissues, including hematopoietic stem cells (HSC), where it has been shown to enhance the generation of primitive murine hematopoietic precursors. Yet, the precise effects of DLK1 on stem cell proliferation and differentiation remain elusive. In preliminary experiments using cord blood CD34+ cells, we have found that DLK1 knockdown decreased CD34+ precursors following in vitro culture and reduced their engraftment in immune deficient mice. In contrast, DLK1 overexpression led to an increased proportion of the least mature CD34+CD90lo precursors in vitro and an increased proportion of CD34+ cells in vivo. Moreover, exposure of CD34+ cells to exogenous DLK1 extracellular domain (ECD) increased longer-term engraftment. Using scRNAseq, we observed that DLK1 knockdown in highly enriched HSC shifted the transcriptional state from a quiescent HSC (qHSC) to an activated HSC (aHSC) during short-term culture. These findings compel further assessment of DLK1 effects on HSC quiescence in EC co-culture, where previous studies have shown expansion of repopulating cells and preliminary studies suggest the expansion of transcriptionally-defined qHSC following cell division in vitro. However, the mechanism by which DLK1 elicits these effects is unclear. DLK1, an intrinsically disordered protein, has been reported to inhibit multiple receptor mediated cell signaling pathways activated by extracellular ligand, including the TGFb, FGF, Notch, and insulin pathways, each relevant to hematopoietic stem cell function. In preliminary studies using the TGFb and Notch pathways as models, we have shown that DLK1 inhibits receptor activation via its ECD. Additional studies suggest that ECD phase separation induces clusters in the plasma membrane, termed condensates, that co-localize with TGFb and Notch receptor components, leading to altered receptor dynamics. Herein, we test our hypothesis that DLK1 regulates HSC quiescence by elucidating: whether DLK1 maintains and/or expands qHSC by inducing aHSC to return to quiescence (Aim 1); how DLK1 interactions with receptor components within condensates alters receptor signaling (Aim 2); and whether inhibition of multiple signaling pathways is required for the effects of DLK1 on HSC (Aim 3). If successful, these findings will suggest that DLK1 maintains qHSC by inhibiting the ability of receptors to respond to multiple environmental signals that induce HSC proliferation and differentiation through the alteration of receptor dynamics within DLK1 condensates. These findings will provide the foundation for novel strategies to enhance stem cell engineering, including the expansion of true repopulating HSC for transplantation. Project Number: 5R01HL173520-02 | Fiscal Year: 2026 | NIH Institute/Center: National Heart Lung and Blood Institute (NHLBI) | Principal Investigator: IRWIN BERNSTEIN | Institution: FRED HUTCHINSON CANCER CENTER, SEATTLE, WA | Award Amount: $646,305 | Activity Code: R01 | Study Section: Basic Biology of Blood, Heart and Vasculature Study Section [BBHV] View on NIH RePORTER: https://reporter.nih.gov/project-details/5R01HL17352002