Abstract Patients with GATA2 deficiency are predisposed to developing myelodysplastic syndrome (MDS), which can progress to acute myeloid leukemia (AML). This progression is often associated with the acquisition of additional cytogenetic and somatic alterations. Mutations in SETBP1 and ASXL1 genes are frequently observed in pediatric GATA2 patients, but their roles in disease progression remain poorly understood. Genome editing of induced pluripotent stem cells (iPSCs) enabled precise reconstruction of mutation combinations found in patients. Here we developed a human hiPSC-based model to study the impact of SETBP1 and ASXL1 mutations in context of GATA2 deficiency. We show that germline heterozygous GATA2 mutation alone showed no significant effect on myeloid development, while the addition of SETBP1 and ASXL1 mutations impaired myelopoiesis, resulting in monocytopenia. We identified a key role of the SETBP1 mutation in promoting chromatin remodeling near genes involved in myeloid neoplasms, which likely initiated the blockage of myeloid differentiation observed in vitro. Motif analysis of more accessible chromatin regions in the SETBP1 and SETBP1/ASXL1 mutant background highlighted an enrichment for MEIS1, PU.1, RUNX1, and HOXA9, implicating these factors in the disease progression. Our study establishes a novel humanized model system for studying GATA2 deficiency. We demonstrate that SETBP1 mutations act as a primary driver in hematopoietic impairment, providing insights that may inform future therapeutic strategies for patients progressing to MDS/AML

The developmental origin of blood-forming hematopoietic stem cells (HSCs) is a longstanding question. Here, our non-invasive genetic lineage tracing in mouse embryos pinpoints that artery endothelial cells generate HSCs. Arteries are transiently competent to generate HSCs for 2.5 days (∼E8.5-E11) but subsequently cease, delimiting a narrow time frame for HSC formation in vivo. Guided by the arterial origins of blood, we efficiently and rapidly differentiate human pluripotent stem cells (hPSCs) into posterior primitive streak, lateral mesoderm, artery endothelium, hemogenic endothelium, and >90% pure hematopoietic progenitors within 10 days. hPSC-derived hematopoietic progenitors generate T, B, NK, erythroid, and myeloid cells in vitro and, critically, express hallmark HSC transcription factors HLF and HOXA5-HOXA10, which were previously challenging to upregulate. We differentiated hPSCs into highly enriched HLF+ HOXA+ hematopoietic progenitors with near-stoichiometric efficiency by blocking formation of unwanted lineages at each differentiation step. hPSC-derived HLF+ HOXA+ hematopoietic progenitors could avail both basic research and cellular therapies.
Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.

Human induced pluripotent stem cell (hiPSC)-derived macrophages provide a valuable tool for disease modeling and drug discovery. Here, we present a protocol to generate functional macrophages from hiPSCs using a feeder-free hematopoietic differentiation technique. We describe steps for preparing hiPSCs, mesodermal differentiation, hematopoietic commitment, and macrophage differentiation and expansion. We then detail assays to characterize their phenotype, polarization, and phagocytic functions. The functional macrophages generated here could be used to generate organoids for disease modeling and drug discovery studies. For complete details on the use and execution of this protocol, please refer to Jeong et al.1 and Heo et al.2.
Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.

Glioblastoma (GBM) is an aggressive brain tumor giving a poor prognosis with the current treatment options. The advent of chimeric antigen receptor (CAR) T-cell therapy revolutionized the field of immunotherapy and has provided a new set of therapeutic options for refractory blood cancers. In an effort to apply this therapeutic approach to solid tumors, various immune cell types and CAR constructs are being studied. Notably, macrophages have recently emerged as potential candidates for targeting solid tumors, attributed to their inherent tumor-infiltrating capacity and abundant presence in the tumor microenvironment.
In this study, we developed a chemically defined differentiation protocol to generate macrophages from human pluripotent stem cells (hPSCs). A GBM-specific CAR was genetically incorporated into hPSCs to generate CAR hPSC-derived macrophages.
The CAR hPSC-derived macrophages exhibited potent anticancer activity against GBM cells in vitro.
Our findings demonstrate the feasibility of generating functional CAR-macrophages from hPSCs for adoptive immunotherapy, thereby opening new avenues for the treatment of solid tumors, particularly GBM.
© 2023 The Author(s).

Oxidative protein folding occurs in the endoplasmic reticulum (ER) to generate disulfide bonds, and the by-product is hydrogen peroxide (H2 O2 ). However, the relationship between oxidative protein folding and senescence remains uncharacterized. Here, we find that the protein disulfide isomerase (PDI), a key oxidoreductase that catalyzes oxidative protein folding, accumulated in aged human mesenchymal stem cells (hMSCs) and deletion of PDI alleviated hMSCs senescence. Mechanistically, knocking out PDI slows the rate of oxidative protein folding and decreases the leakage of ER-derived H2 O2 into the nucleus, thereby decreasing the expression of SERPINE1, which was identified as a key driver of cell senescence. Furthermore, we show that depletion of PDI alleviated senescence in various cell models of aging. Our findings reveal a previously unrecognized role of oxidative protein folding in promoting cell aging, providing a potential target for aging and aging-related disease intervention.
© 2023 The Authors.