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Optimized Method to Generate Well-Characterized Macrophages from Induced Pluripotent Stem Cells

ABSTRACT: Background: Macrophages play a pivotal role in various pathogenic processes, necessitating the development of efficient differentiation techniques to meet the high demand for these cells in research and therapy. Human macrophages can be obtained via culturing of peripheral blood monocytes; however, this source has limited yields and requires patient contact for each proposed use. In addition, it would be difficult to perform gene editing on peripheral blood monocytes. The differentiation of induced pluripotent stem cells (iPSCs) into macrophages can meet these needs for recurrent studies with high yields with the possibility of gene editing. Methods: We refined the traditional embryoid body-based differentiation strategy to create a novel three-phase method that optimizes yield, consistent quality, and reproducibility. This approach incorporates the use of microwell plates and cell filtration to standardize the production of embryoid bodies and subsequent macrophage progenitors. Using up to five independent iPSC donors, we performed several assays for macrophage functions and polarization. such as marker protein staining by flow cytometry, lipoprotein uptake, phagocytosis, cytokine release, inflammasome activation, and the effects of M1-like and M2-like polarization. RNA sequencing was performed to determine segregation of cells at different stages of differentiation and by iPSC donor, as well as to identify marker genes for each stage of differentiation. Results: The iPSC-derived macrophages generated through this method exhibit characteristic features and cell marker proteins, as well as classical macrophage activities including lipoprotein uptake, bacterial phagocytosis, cytokine release, and inflammasome activation. We demonstrate the effects of M1-like and M2-like polarization on cytokine release. The first three principal components of the RNA sequencing data showed clear clustering by differentiation stage, while the fourth and fifth principal components clustered the differentiated macrophages by their respective iPSC donor. Marker genes were identified for each stage of differentiation and polarization. Conclusions: The methods provide an optimized and simplified procedure to produce iPSC-derived macrophages. Our results demonstrate the reproducibility of this method in generating high-quality macrophages suitable for a variety of biomedical applications.

ORGANISM(S): Homo sapiens

PROVIDER: GSE277609 | GEO | 2024/12/01

REPOSITORIES: GEO

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Project description:Background: Macrophage polarization programs, commonly referred to as âclassicalâ and âalternativeâ activation, are widely considered as distinct states that are exclusive of one another, and are associated with different functions such as inflammation and wound healing, respectively. In a number of disease contexts, such as traumatic brain injury (TBI), macrophage polarization influences the extent of pathogenesis, and efforts are underway to eliminate pathogenic subsets. However, previous studies have not distinguished whether the simultaneous presence of both classical and alternative activation signatures represents the admixture of differentially polarized macrophages, or if they have adopted a unique state characterized by components of both classical and alternative activation. Results: We analyzed the polarization of individual macrophages responding to TBI using single-cell RNA sequencing. Analysis of signature polarization genes revealed diverse activation states, including M(IL4), M(IL10), and M(LPS, IFNÎ³). However, the expression of a given polarization marker was no more likely than at random to predict simultaneous expression or repression of markers of another polarization program within the same cell, suggesting a lack of exclusivity in macrophage polarization states in vivo in TBI. Also unexpectedly, individual TBI macrophages simultaneously expressed high levels of signature polarization genes across two or three different polarization states, and in several distinct and seemingly incompatible combinations. Conclusions: Single-cell gene expression profiling demonstrated that monocytic macrophages in TBI are not comprised of distinctly polarized subsets, but are uniquely and broadly activated. TBI macrophage activation in vivo is deeply complex, with individual cells concurrently adopting both inflammatory and reparative features. These data provide physiologically relevant evidence that the early macrophage response to TBI is comprised of novel activation states that are discordant with the current paradigm of macrophage polarizationâa key consideration for therapeutic modulation. Monocyte derived macrophages were isolated from the ipsilateral hemisphere of mouse brains one day following traumatic brain injury elicited by control cortical impact in C57BL/6 adult male mice. Single-cells were isolated and processed for RNA sequencing using a Fluidigm C1 integrated fluidic circuit chip. 45 biological replicates were analyzed.

Project description:Background: Macrophage polarization programs, commonly referred to as “classical” and “alternative” activation, are widely considered as distinct states that are exclusive of one another, and are associated with different functions such as inflammation and wound healing, respectively. In a number of disease contexts, such as traumatic brain injury (TBI), macrophage polarization influences the extent of pathogenesis, and efforts are underway to eliminate pathogenic subsets. However, previous studies have not distinguished whether the simultaneous presence of both classical and alternative activation signatures represents the admixture of differentially polarized macrophages, or if they have adopted a unique state characterized by components of both classical and alternative activation. Results: We analyzed the polarization of individual macrophages responding to TBI using single-cell RNA sequencing. Analysis of signature polarization genes revealed diverse activation states, including M(IL4), M(IL10), and M(LPS, IFNγ). However, the expression of a given polarization marker was no more likely than at random to predict simultaneous expression or repression of markers of another polarization program within the same cell, suggesting a lack of exclusivity in macrophage polarization states in vivo in TBI. Also unexpectedly, individual TBI macrophages simultaneously expressed high levels of signature polarization genes across two or three different polarization states, and in several distinct and seemingly incompatible combinations. Conclusions: Single-cell gene expression profiling demonstrated that monocytic macrophages in TBI are not comprised of distinctly polarized subsets, but are uniquely and broadly activated. TBI macrophage activation in vivo is deeply complex, with individual cells concurrently adopting both inflammatory and reparative features. These data provide physiologically relevant evidence that the early macrophage response to TBI is comprised of novel activation states that are discordant with the current paradigm of macrophage polarization—a key consideration for therapeutic modulation.

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Optimized Method to Generate Well-Characterized Macrophages from Induced Pluripotent Stem Cells

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