Inhibition of Canonical Wnt Signaling Promotes Ex Vivo Maintenance and Proliferation of Hematopoietic Stem Cells in Zebrafish

Abstract

The maintenance and proliferation of hematopoietic stem cells (HSCs) are tightly regulated by their niches in the bone marrow. The analysis of niche cells or stromal cell lines that can support HSCs has facilitated the finding of novel supporting factors for HSCs. Despite large efforts in the murine bone marrow, however, HSC expansion is still difficult ex vivo, highlighting the need for new approaches to elucidate the molecular elements that regulate HSCs. The zebrafish provides a unique model to study hematopoietic niches as HSCs are maintained in the kidney, allowing for a parallel view of hematopoietic niches over evolution. Here, using a stromal cell line from the zebrafish kidney, zebrafish kidney stromal (ZKS), we uncover that an inhibitor of canonical Wnt signaling, IWR-1-endo, is a potent regulator of HSCs. Co-culture assays revealed that ZKS cells were in part supportive of maintenance, but not expansion, of gata2a:GFP+runx1:mCherry+ (gata2a+runx1+) HSCs. Transcriptome analysis revealed that, compared to candidate niche cells in the kidney, ZKS cells weakly expressed HSC maintenance factor genes, thpo and cxcl12, but highly expressed canonical Wnt ligand genes, wnt1, 7bb, and 9a. Thpo supplementation in ZKS culture slightly increased, but inhibition of canonical Wnt signaling by IWR-1-endo treatment largely increased the number of gata2a+runx1+ cells (> 2-fold). Moreover, we found that gata2a+runx1+ cells can be maintained by supplementing both IWR-1-endo and Thpo without stromal cells. Collectively, our data provide evidence that IWR-1-endo can be used as a novel supporting factor for HSCs.

Synergistic effect and molecular mechanism of PVA and UM171 in ex vivo expansion of primitive hematopoietic stem cells

“…The above results indicate that upregulation of the SIRT1-HIF1A pathway promotes cell proliferation and thereby promoting differentiation. Therefore, we compared the expression of downstream genes in the Notch and Wnt pathways between the PVA and PVA + UM171 groups to determine whether UM171 is involved in the regulation of these two classic stem cell pathways, [34][35][36] and the results suggest that UM171 may promote selfrenewal of HSCs by activating the Notch and Wnt pathways. In conclusion, the antioxidant properties of PVA can delay differentiation, while UM171 can promote selfrenewal by regulating the stem cell pathways, and the combination of them is beneficial for the maintenance and expansion of HSCs in vitro.…”

“…The above results indicate that upregulation of the SIRT1‐HIF1A pathway promotes cell proliferation and thereby promoting differentiation. Therefore, we compared the expression of downstream genes in the Notch and Wnt pathways between the PVA and PVA + UM171 groups to determine whether UM171 is involved in the regulation of these two classic stem cell pathways, 34–36 and the results suggest that UM171 may promote self‐renewal of HSCs by activating the Notch and Wnt pathways.…”

Osteoblastic Wls Ablation Protects Mice from Total Body Irradiation-Induced Impairments in Hematopoiesis and Bone Marrow Microenvironment

“…It is suggested that non-canonical Wnt signaling maintains quiescent HSCs via inhibition of canonical Wnt signaling [ 49 ]. A recent report highlights that the suppression of canonical Wnt signaling accelerated the ex vivo maintenance and proliferation of HSCs and increased HPC numbers in zebrafish [ 50 ]. Although the role of canonical Wnt in the fate and function of BM HSCs remains unclear, these reports together with our findings strongly indicate that deficiency of canonical Wnt ligands and decreased activation of canonical Wnt signaling are required for hematopoietic radioprotection.…”

“…It is suggested that non-canonical Wnt signaling maintains quiescent HSCs via inhibition of canonical Wnt signaling [49]. A recent report highlights that the suppression of canonical Wnt signaling accelerated the ex vivo maintenance and proliferation of HSCs and increased HPC numbers in zebrafish [50].…”

Extremely low-frequency electromagnetic fields facilitate both osteoblast and osteoclast activity through Wnt/β-catenin signaling in the zebrafish scale

“…Immunofluorescence of isolated cells was performed as previously described ( Kimura et al, 2022 ). Briefly, cells were collected from zebrafish scales as described above, smeared with Cyto-tek 2500 (Sakura), and fixed with 4% PFA in PBS for 20 min at room temperature.…”