NOTCH-mediated ex vivo expansion of human hematopoietic stem and progenitor cells by culture under hypoxia

Abstract

Summary Activation of NOTCH signaling in human hematopoietic stem/progenitor cells (HSPCs) by treatment with an engineered Delta-like ligand (DELTA1 ext-IgG [DXI]) has enabled ex vivo expansion of short-term HSPCs, but the effect on long-term repopulating hematopoietic stem cells (LTR-HSCs) remains uncertain. Here, we demonstrate that ex vivo culture of human adult HSPCs with DXI under low oxygen tension limits ER stress in LTR-HSCs and lineage-committed progenitors compared with normoxic cultures. A distinct HSC gene signature was upregulated in cells cultured with DXI in hypoxia and, after 21 days of culture, the frequency of LTR-HSCs increased 4.9-fold relative to uncultured cells and 4.2-fold compared with the normoxia + DXI group. NOTCH and hypoxia pathways intersected to maintain undifferentiated phenotypes in cultured HSPCs. Our work underscores the importance of mitigating ER stress perturbations to preserve functional LTR-HSCs in extended cultures and offers a clinically feasible platform for the expansion of human HSPCs.

Hematopoietic Stem Cell Factors: Their Functional Role in Self-Renewal and Clinical Aspects

“…The hypoxic condition also supports the stability of HIF-2α heterodimerization with HIF-1β. Since unbound HIF-1β under normoxia binds to aryl hydrocarbon receptor (AhR) that triggers transcription of cellular differentiation genes CYP1A1 and CYP1B1, confirming the crucial role of AhR pathway in self-renewal ( Araki et al, 2021 ). AhR antagonists like Stem-Reginin1 (SR1) can increase the HSCs numbers by 50-fold and establish long-term engraftment in immunodeficient mice ( Boitano et al, 2010 ).…”

“…One method to go about it is by Notch signalling activation within HSCs. Treating HSCs with an engineered Delta-like ligand like DELTA1 ext−IgG (DXI) under normoxic conditions activates the Notch pathway in ST-HSCs by triggering the transcriptional activity of HES and HEY genes responsible for self-renewal ( Araki et al, 2021 ). However, this engraftment did not sustain for long since it was ineffective on the LT-HSCs.…”

“…However, this engraftment did not sustain for long since it was ineffective on the LT-HSCs. Therefore, to counter the oxidative stress implicated on the endoplasmic reticulum under normoxic conditions, HSCs expanded ex vivo with exogenously provided DXI in hypoxia manifested an almost five fold increase in LT-HSCs compared to the untreated cells, offering a clinical potential for the long term HSCs sustenance ex vivo ( Araki et al, 2021 ). The hypoxic condition also supports the stability of HIF-2α heterodimerization with HIF-1β.…”

Solute Carrier Nucleoside Transporters in Hematopoiesis and Hematological Drug Toxicities: A Perspective

“…The importance of BA-regulated ER stress signaling in HSCs is further supported by seminal works from the Miharada lab which demonstrated that fetal HSCs exhibit low levels of adaptive responses to ER stress that significantly depend on BA chaperone activity for expansion and differentiation [203,204]. As such, the involvement of ER stress signaling in HSC homeostasis is gaining momentum in research, as seen in several new reports on this topic [205][206][207][208][209]. Importantly, the mechanism that allows ENT3 to regulate erythroid pool size introduces a new concept into the understanding of stress-adaptive ER stress signaling in HSCs [207].…”

New Insights into Hematopoietic Stem Cell Expansion to Stimulate Repopulation of the Adult Blood System for Transplantation

“…The Notch signaling pathway can be proved to mediate the differentiation of HSCs. It has been found that Notch1 can facilitate T-cell differentiation and inhibit B-cell differentiation [ 37 ]. Notch2 signaling affects HSC self-renewal by inhibiting the differentiation of HSCs to multipotent progenitors (MPP) and MPP differentiation to myeloid/monocytic (M) cell lineage [ 16 ].…”