Highly efficient derivation of functional human induced pluripotent stem cell-derived macrophages under serum-free conditions to study innate immune responses.

Induced pluripotent stem cell–derived macrophages (iMacs) in translational medicine: Disease models, drug testing, and therapeutic applications

Immune cell development from human pluripotent stem cells.

This review summarizes research with a focus on clinical translation of iPSC-derived immune cells, as well as highlights continued challenges and prospects of this field.

Phenotypic and functional patterns of murine bone marrow-derived dendritic cells influenced by in vitro differentiation conditions

The selection of an appropriate differentiation protocol for murine bone marrow-derived dendritic cells (BM-DCs) still presents a challenge due to the inherent plasticity of dendritic cells (DCs) and the high diversity of generation methods. As such, it is essential to evaluate in vitro BM-DCs characteristics during initial steps of experimental model development and tailor the protocols according to specific research goals while preserving the functional and phenotypic properties of DCs. In our study, we observed relevant variations in phenotypic and functional characteristics of BM-DCs differentiated with GM-CSF alone (GDCs) compared to those differentiated with GM-CSF and IL-4 (G4DCs). We found that GDCs exhibited high levels of costimulatory molecules, indicating a phenotypically mature state, but preserved the ability to respond to LPS (lipopolysaccharide) maturation stimuli by cytokine production. Moreover, cytokine secretion patterns observed in prime/ boost co-culture systems indicate the potential of Ag-pulsed BM-DCs to prime naive T cells. In contrast, while addition of IL-4 resulted in cells with a more immature phenotype that are responsive to LPS-driven maturation, it also promoted nonspecific T cell signaling that could interfere with in vitro evaluation of priming and antigen-specific T cell responses. These results highlight the impact of specific differentiation conditions that impose on the phenotypic and functional patterns of BM-DCs. Furthermore, the study emphasizes the significance of reassessing, even widely, the already employed protocols for DCs differentiation, and moreover, our study can provide valuable insights and guidance for novel in vitro experimental designs based on BM-DCs.

Preliminary transcriptome profiling of induced pluripotent stem cell-derived hematopoietic stem and progenitor cells in Kawasaki disease

Background The etiology of Kawasaki disease (KD) remains unclear. Hematopoietic stem and progenitor cells (HSPCs) serve as the precursor cells for a multitude of immune cells. Investigating their initial transcriptional status may help uncover the aberrant immune mechanisms underlying KD. Methods Induced pluripotent stem cells (iPSCs) were reprogrammed from peripheral blood mononuclear cells (PBMCs) isolated from patients with KD and febrile individuals, followed by directed differentiation into HSPCs. We performed bulk RNA-sequencing to compare transcriptomic profiles of iPSC-derived HSPCs between the two groups, with further comparison against integrated HSPC data from public KD single-cell datasets. Results We recruited three patients with KD prior to Intravenous immunoglobulin (IVIG) therapy and three febrile control patients, and successfully established an iPSC-derived HSPCs disease model. Transcriptomic profiling revealed elevated immune and inflammatory response signatures in iPSC-derived HSPCs from patients with KD compared with those from febrile individuals. In addition, in vitro-generated KD iPSC-HSPCs exhibited partial transcriptional features similar to in vivo HSPCs isolated from PBMCs of patients with KD. Gene Set Enrichment Analysis (GSEA) further revealed that gene sets associated with B-cell developmental processes were transcriptionally downregulated in iPSC-derived HSPCs from patients with KD relative to febrile controls. Conclusions iPSC-derived HSPCs from patients with KD display altered immune-inflammatory transcriptional profiles and suppressed B-cell developmental signatures at the transcriptomic level. These preliminary findings suggest that early hematopoietic immune dysregulation may contribute to KD pathogenesis. We propose that these iPSC-derived HSPCs could be a good cellular model for studying the etiology of KD in vitro. These findings are preliminary, constrained by the small sample size and limited to transcriptomic analysis only. Further studies with larger cohorts and functional experiments are needed to verify these results.

iPSC-Derived Microglia-like Cells Exhibit Protocol-Dependent Transcriptomic Features and Robust Phagocytosis of Glioma Cells

Microglia are the brain-resident macrophages and key regulators of the brain tumor microenvironment. Although induced pluripotent stem cell-derived microglia (iMG) provide a valuable model for studying human microglial, systematic comparisons of differentiation protocols are limited, and their utility for modeling microglia-tumor cell interactions remains underexplored. Here, we analyzed 54 public RNA-seq datasets representing 22 iMG differentiation protocols, including embryoid body (EB)-based, two-dimensional (2D), transcription factor-induced, and coculture-based approaches. Most iMG closely resembled primary human microglia, although substantial protocol-dependent differences were observed. iMG generated using EB-based protocols showed higher TMEM119 expression, whereas those generated using 2D-based protocols showed higher P2RY12 expression. A widely adopted EB-based protocol showed the highest phagocytosis gene signature. Using this protocol, we generated iMG that efficiently phagocytosed patient-derived glioma stem-like cells and upregulated inflammatory and immunoregulatory genes following phagocytosis. These findings provide a transcriptomic benchmark for current iMG models and support their use in investigating microglia-glioma interactions.

Expansion of functional human long-term HSCs through restraining excessive cell cycle activation.

Ex vivo expansion of human hematopoietic stem cells (HSCs) holds promise for overcoming their limited availability, a major barrier to broader clinical application. Although recent advances in culture systems can increase HSC numbers, these conditions frequently impair self-renewal and induce myeloid bias, and the underlying molecular mechanisms remain poorly understood. Here, we performed single-cell multiome sequencing (scMultiome-seq) on human umbilical cord blood-derived CD34⁺ hematopoietic stem and progenitor cells to co-profile transcriptional and epigenetic adaptations within the same cells during ex vivo culture. Our analyses revealed reduced transcriptional and epigenetic HSC signatures, accompanied by markedly increased activity of myeloid-associated transcription factor motifs, providing molecular insight into the functional decline and myeloid bias of cultured HSCs. We further observed substantial functional heterogeneity among phenotypically defined HSCs following culture. To address these limitations, we established a niche-mimetic culture system that integrates intrinsic and extrinsic bone marrow regulatory cues, including pharmacologic inhibition of the m6A reader YTHDF2 using the small molecule Y13-27, a three-dimensional microenvironment, and N-cadherin-mediated adhesion. This condition (3D-NcadP-Y) robustly preserved long-term repopulating capacity. When combined with the self-renewal agonist UM729, the resulting platform (3D-NcadP-Y-UM) uniquely enabled the expansion of serially transplantable long-term HSCs with balanced multilineage potential. scMultiome-seq and cellular analyses demonstrated that this condition preserves transcriptional and epigenetic long-term HSC signatures, maintains multilineage-associated transcription factor motifs, and limits excessive cell-cycle activation. Together, these findings elucidate molecular mechanisms underlying culture-induced HSC dysfunction and establish a niche-mimetic strategy for expanding functional human long-term HSCs while preserving key features of stemness.