Pluripotent Stem Cell-derived Macrophages Research Articles

Synergy between pluripotent stem cell-derived macrophages and self-renewing macrophages: Envisioning a promising avenue for the modelling and cell therapy of infectious diseases.

As crucial phagocytes of the innate immune system, macrophages (Mϕs) protect mammalian hosts, maintain tissue homeostasis and influence disease pathogenesis. Nonetheless, Mϕs are susceptible to various pathogens, including bacteria, viruses and parasites, which cause various infectious diseases, necessitating a deeper understanding of pathogen-Mϕ interactions and therapeutic insights. Pluripotent stem cells (PSCs) have been efficiently differentiated into PSC-derived Mϕs (PSCdMϕs) resembling primary Mϕs, advancing the modelling and cell therapy of infectious diseases. However, the mass production of PSCdMϕs, which lack proliferative capacity, relies on large-scale expansions of PSCs, thereby increasing both costs and culture cycles. Notably, Mϕs deficient in the MafB/c-Maf genes have been reported to re-enter the cell cycle with the stimulation of specific growth factor cocktails, turning into self-renewing Mϕs (SRMϕs). This review summarizes the applications of PSCdMϕs in the modelling and cell therapy of infectious diseases and strategies for establishing SRMϕs. Most importantly, we innovatively propose that PSCs can serve as a gene editing platform to creating PSC-derived SRMϕs (termed PSRMϕs), addressing the resistance of Mϕs against genetic manipulation. We discuss the challenges and possible solutions in creating PSRMϕs. In conclusion, this review provides novel insights into the development of physiologically relevant and expandable Mϕ models, highlighting the enormous potential of PSRMϕs as a promising avenue for the modelling and cell therapy of infectious diseases.

Induced pluripotent stem cell-derived macrophages as a platform for modelling human disease.

Macrophages are innate immune cells that are present in essentially all tissues, where they have vital roles in tissue development, homeostasis and pathogenesis. The importance of macrophages in tissue function is reflected by their association with various human diseases, and studying macrophage functions in both homeostasis and pathological tissue settings is a promising avenue for new targeted therapies that will improve human health. The ability to generate macrophages from induced pluripotent stem(iPS) cells has revolutionized macrophage biology, with the generation of iPScell-derived macrophages (iMacs) providing unlimited access to genotype-specific cells that can be used to model various human diseases involving macrophage dysregulation. Such disease modelling is achieved by generating iPScells from patient-derived cells carrying disease-related mutations or by introducing mutations into iPScells from healthy donors using CRISPR-Cas9 technology. These iMacs that carry disease-related mutations can be used to study the aetiology of the particular disease in vitro. To achieve more physiological relevance, iMacs can be co-cultured in 2D systems with iPScell-derived cells or in 3D systems with iPScell-derived organoids. Here, we discuss the studies that have attempted to model various human diseases using iMacs, highlighting how these have advanced our knowledge about the role of macrophages in health and disease.

Discovery of NRG1-VII: the myeloid-derived class of NRG1

The growth factor Neuregulin-1 (NRG1) has pleiotropic roles in proliferation and differentiation of the stem cell niche in different tissues. It has been implicated in gut, brain and muscle development and repair. Six isoform classes of NRG1 and over 28 protein isoforms have been previously described. Here we report a new class of NRG1, designated NRG1-VII to denote that these NRG1 isoforms arise from a myeloid-specific transcriptional start site (TSS) previously uncharacterized. Long-read sequencing was used to identify eight high-confidence NRG1-VII transcripts. These transcripts presented major structural differences from one another, through the use of cassette exons and alternative stop codons. Expression of NRG1-VII was confirmed in primary human monocytes and tissue resident macrophages and induced pluripotent stem cell-derived macrophages (iPSC-derived macrophages). Isoform switching via cassette exon usage and alternate polyadenylation was apparent during monocyte maturation and macrophage differentiation. NRG1-VII is the major class expressed by the myeloid lineage, including tissue-resident macrophages. Analysis of public gene expression data indicates that monocytes and macrophages are a primary source of NRG1. The size and structure of class VII isoforms suggests that they may be more diffusible through tissues than other NRG1 classes. However, the specific roles of class VII variants in tissue homeostasis and repair have not yet been determined.

Engineered CD147-Deficient THP-1 Impairs Monocytic Myeloid-Derived Suppressor Cell Differentiation but Maintains Antibody-Dependent Cellular Phagocytosis Function for Jurkat T-ALL Cells with Humanized Anti-CD147 Antibody.

CD147 is upregulated in cancers, including aggressive T-ALL. Traditional treatments for T-ALL often entail severe side effects and the risk of relapse, highlighting the need for more efficacious therapies. ADCP contributes to the antitumor response by enhancing the ability of phagocytic cells to engulf cancer cells upon antibody binding. We aimed to engineer CD147KO THP-1 cells and evaluated their differentiation properties compared to the wild type. A humanized anti-CD147 antibody, HuM6-1B9, was also constructed for investing the phagocytic function of CD147KO THP-1 cells mediated by HuM6-1B9 in the phagocytosis of Jurkat T cells. The CD147KO THP-1 was generated by CRISPR/Cas9 and maintained polarization profiles. HuM6-1B9 was produced in CHO-K1 cells and effectively bound to CD147 with high binding affinity (KD: 2.05 ± 0.30 × 10-9 M). Additionally, HuM6-1B9 enhanced the phagocytosis of Jurkat T cells by CD147KO THP-1-derived LPS-activated macrophages (M-LPS), without self-ADCP. The formation of THP-1-derived mMDSC was limited in CD147KO THP-1 cells, highlighting the significant impact of CD147 deletion. Maintaining expression markers and phagocytic function in CD147KO THP-1 macrophages supports future engineering and the application of induced pluripotent stem cell-derived macrophages. The combination of HuM6-1B9 and CD147KO monocyte-derived macrophages holds promise as an alternative strategy for T-ALL.

A Comparative Study of Human Pluripotent Stem Cell-Derived Macrophages in Modeling Viral Infections.

Macrophages play multiple roles in innate immunity including phagocytosing pathogens, modulating the inflammatory response, presenting antigens, and recruiting other immune cells. Tissue-resident macrophages (TRMs) adapt to the local microenvironment and can exhibit different immune responses upon encountering distinct pathogens. In this study, we generated induced macrophages (iMACs) derived from human pluripotent stem cells (hPSCs) to investigate the interactions between the macrophages and various human pathogens, including the hepatitis C virus (HCV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and Streptococcus pneumoniae. iMACs can engulf all three pathogens. A comparison of the RNA-seq data of the iMACs encountering these pathogens revealed that the pathogens activated distinct gene networks related to viral response and inflammation in iMACs. Interestingly, in the presence of both HCV and host cells, iMACs upregulated different sets of genes involved in immune cell migration and chemotaxis. Finally, we constructed an image-based high-content analysis system consisting of iMACs, recombinant GFP-HCV, and hepatic cells to evaluate the effect of a chemical inhibitor on HCV infection. In summary, we developed a human cell-based in vitro model to study the macrophage response to human viral and bacterial infections; the results of the transcriptome analysis indicated that the iMACs were a useful resource for modeling pathogen-macrophage-tissue microenvironment interactions.

Induced pluripotent stem cell-derived human macrophages as an infection model for Leishmania donovani.

The parasite Leishmania donovani is one of the species causing visceral leishmaniasis in humans, a deadly infection claiming up to 40,000 lives each year. The current drugs for leishmaniasis treatment have severe drawbacks and there is an urgent need to find new anti-leishmanial compounds. However, the search for drug candidates is complicated by the intracellular lifestyle of Leishmania. Here, we investigate the use of human induced pluripotent stem cell (iPS)-derived macrophages (iMACs) as host cells for L. donovani. iMACs obtained through embryoid body differentiation were infected with L. donovani promastigotes, and high-content imaging techniques were used to optimize the iMACs seeding density and multiplicity of infection, allowing us to reach infection rates up to 70% five days after infection. IC50 values obtained for miltefosine and amphotericin B using the infected iMACs or mouse peritoneal macrophages as host cells were comparable and in agreement with the literature, showing the potential of iMACs as an infection model for drug screening.

A second-generation M1-polarized CAR macrophage with antitumor efficacy.

Chimeric antigen receptor (CAR) T cell therapies have successfully treated hematological malignancies. Macrophages have also gained attention as an immunotherapy owing to their immunomodulatory capacity and ability to infiltrate solid tumors and phagocytize tumor cells. The first-generation CD3ζ-based CAR-macrophages could phagocytose tumor cells in an antigen-dependent manner. Here we engineered induced pluripotent stem cell-derived macrophages (iMACs) with toll-like receptor 4 intracellular toll/IL-1R (TIR) domain-containing CARs resulting in a markedly enhanced antitumor effect over first-generation CAR-macrophages. Moreover, the design of a tandem CD3ζ-TIR dual signaling CAR endows iMACs with both target engulfment capacity and antigen-dependent M1 polarization and M2 resistance in a nuclear factor kappa B (NF-κB)-dependent manner, as well as the capacity to modulate the tumor microenvironment. We also outline a mechanism of tumor cell elimination by CAR-induced efferocytosis against tumor cell apoptotic bodies. Taken together, we provide a second-generation CAR-iMAC with an ability for orthogonal phagocytosis and polarization and superior antitumor functions in treating solid tumors relative to first-generation CAR-macrophages.

Abstract P1127: Induced Pluripotent Stem Cell-derived Macrophages Modulate Human Engineered Cardiac Tissue Function

In the native human myocardium, cardiac resident macrophages are a critical mediator for normal cardiac function, performing a variety of biological roles in electrical conduction, regeneration, and phagocytosis and efferocytosis. However, most models of human engineered cardiac tissues (hECT), which aim to recapitulate the native adult myocardium for disease modeling and drug screening applications, do not include a macrophage population. Thus, in this study we introduced an iPSC-derived macrophage (iM0) population into an all iPSC-derived tissue engineered model of the human myocardium in order to assess its impact on tissue phenotype and function. CD14 + macrophages were differentiated from iPSCs and added as 7% of the total cell composition (7% iM0, 69.75% iCM, 23.25% iCF) in the hECT model. The hECTs were then cultured for 4 weeks and subjected to a ramped electrical stimulation protocol for matured function. The resulting tissues showed that the presence of a CD68 + macrophage population was maintained over this extended period of culture and significantly impacted hECT function, increasing contractile force production and modulating contraction and relaxation velocity. Interrogation of calcium signaling via isoproterenol revealed that iM0 presence in the tissues may be causing these functional differences via modulation of the cAMP signaling pathway. These observations demonstrate that macrophages significantly contribute to cardiac function in iPSC-derived hECT models and emphasize the need to further explore their contributions not only in healthy hECT models, but also in the contexts of disease and injury.

ATG7 and ATG14 restrict cytosolic and phagosomal Mycobacterium tuberculosis replication in human macrophages

Autophagy is a cellular innate-immune defence mechanism against intracellular microorganisms, including Mycobacterium tuberculosis (Mtb). How canonical and non-canonical autophagy function to control Mtb infection in phagosomes and the cytosol remains unresolved. Macrophages are the main host cell in humans for Mtb. Here we studied the contributions of canonical and non-canonical autophagy in the genetically tractable human induced pluripotent stem cell-derived macrophages (iPSDM), using a set of Mtb mutants generated in the same genetic background of the common lab strain H37Rv. We monitored replication of Mtb mutants that are either unable to trigger canonical autophagy (Mtb ΔesxBA) or reportedly unable to block non-canonical autophagy (Mtb ΔcpsA) in iPSDM lacking either ATG7 or ATG14 using single-cell high-content imaging. We report that deletion of ATG7 by CRISPR–Cas9 in iPSDM resulted in increased replication of wild-type Mtb but not of Mtb ΔesxBA or Mtb ΔcpsA. We show that deletion of ATG14 resulted in increased replication of both Mtb wild type and the mutant Mtb ΔesxBA. Using Mtb reporters and quantitative imaging, we identified a role for ATG14 in regulating fusion of phagosomes containing Mtb with lysosomes, thereby enabling intracellular bacteria restriction. We conclude that ATG7 and ATG14 are both required for restricting Mtb replication in human macrophages.

SARS-CoV-2 Delta (B.1.617.2) variant replicates and induces syncytia formation in human induced pluripotent stem cell-derived macrophages.

Alveolar macrophages are tissue-resident immune cells that protect epithelial cells in the alveoli from invasion by pathogens, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Therefore, the interaction between macrophages and SARS-CoV-2 is inevitable. However, little is known about the role of macrophages in SARS-CoV-2 infection. Here, we generated macrophages from human induced pluripotent stem cells (hiPSCs) to investigate the susceptibility of hiPSC-derived macrophages (iMΦ) to the authentic SARS-CoV-2 Delta (B.1.617.2) and Omicron (B.1.1.529) variants as well as their gene expression profiles of proinflammatory cytokines during infection. With undetectable angiotensin-converting enzyme 2 (ACE2) mRNA and protein expression, iMΦ were susceptible to productive infection with the Delta variant, whereas infection of iMΦ with the Omicron variant was abortive. Interestingly, Delta induced cell-cell fusion or syncytia formation in iMΦ, which was not observed in Omicron-infected cells. However, iMΦ expressed moderate levels of proinflammatory cytokine genes in response to SARS-CoV-2 infection, in contrast to strong upregulation of these cytokine genes in response to polarization by lipopolysaccharide (LPS) and interferon-gamma (IFN-γ). Overall, our findings indicate that the SARS-CoV-2 Delta variant can replicate and cause syncytia formation in macrophages, suggesting that the Delta variant can enter cells with undetectable ACE2 levels and exhibit greater fusogenicity.

Future prospects for cancer immunotherapy using induced pluripotent stem cell-derived dendritic cells or macrophages.

Cancer immunotherapy is now the first-line treatment for many unresectable cancers. However, it remains far from a complete cure for all patients. Therefore, it is necessary to develop innovative methods for cancer immunotherapy, and immune cell therapy could be an option. Currently, several institutions are attempting to generate immune cells from induced pluripotent stem cells (iPSCs) for use in cancer immunotherapy. A method for generating dendritic cells (DCs) and macrophages (MPs) from iPSC has been established. iPSC-derived DCs (iPS-DCs) can activate T cells via antigen presentation, and iPSC-derived macrophages (iPS-MPs) attack cancer. Since iPSCs are used as the source, genetic modification is easy, and various immune functions, such as the production of anti-tumour cytokines, can be added. Furthermore, when iPS-DCs and iPS-MPs are immortalized, cost reduction through mass production is theoretically possible. In this review, the achievements of cancer research using iPS-DCs and iPS-MPs are summarized, and the prospects for the future are discussed.

Human MD2 deficiency—an inborn error of immunity with pleiotropic features

Toll-like receptors (TLRs) are important pattern recognition receptors that sense microbes and control host defense. Myeloid differentiation protein 2 (MD2) is the indispensable coreceptor for TLR4, facilitating the binding to the gram-negative bacterial cell wall component LPS and activation of downstream signaling. We sought to provide phenotypic and mechanistic insights into human MD2 deficiency. To elucidate the genetic cause in a patient with very early onset inflammatory bowel disease, we performed whole-exome sequencing and studied the functional consequences of the identified mutation in LY96 (encoding for MD2) in genetically engineered induced pluripotent stem cell-derived macrophages with knockout of MD2 or knockin of the patient-specific mutation, including TLR4-mediated signaling, cytokine production, and bacterial handling. Whole-exome sequencing identified a homozygous in-frame deletion in the LY96 gene (c.347_349delCAA; p.Thr116del) in a patient with very early onset inflammatory bowel disease and a sibling presenting with pneumonia and otitis media. Induced pluripotent stem cell-derived macrophages with knockout of MD2 or expression of the Thr116del mutation showed impaired activation of nuclear factor kappa B and mitogen-activated protein kinase signaling as well as TLR4 endocytosis on challenge with LPS or bacteria. In addition, MD2-deficient macrophages showed decreased cytokine expression (eg, IL-6, TNF, and IL-10) in response to LPS or gram-negative but not gram-positive bacteria. Human MD2 deficiency causes defective TLR4 signaling in response to LPS or gram-negative bacteria. The clinical manifestations and expressivity might be variable due to unknown secondary risk factors. Because TLR4 represents a therapeutic target for multiple inflammatory conditions, our study may provide insights into potential side effects of pharmacological TLR4 targeting.

336 Induced pluripotent stem cell-derived macrophages in an inflammatory skin disease model to study granuloma formation

Granulomas are aggregates of immune cells that can form in various organs. In cutaneous sarcoidosis macrophages and T cells present inside granuloma cores are not able to eliminate the yet unknown antigen and arrest in an immune state causing tissue destruction. We aim to study the switch of a protective immune cell phenotype to a pathological, disease-driving population within granulomas. Therefore, we use a combination of direct visualization, RNA sequencing analysis of isolated cells and sophisticated co-culture models, including induced pluripotent stem cell-derived macrophages (iPSM) together with patient-derived T cells and fibroblasts.

Human pluripotent stem cell-derived macrophages host Mycobacterium abscessus infection.

SummaryHuman macrophages are a natural host of many mycobacterium species, including Mycobacterium abscessus (M. abscessus), an emerging pathogen affecting immunocompromised and cystic fibrosis patients with few available treatments. The search for an effective treatment is hindered by the lack of a tractable in vitro intracellular infection model. Here, we established a reliable model for M. abscessus infection using human pluripotent stem cell-derived macrophages (hPSC-macrophages). hPSC differentiation permitted reproducible generation of functional macrophages that were highly susceptible to M. abscessus infection. Electron microscopy demonstrated that M. abscessus was present in the hPSC-macrophage vacuoles. RNA sequencing analysis revealed a time-dependent host cell response, with differing gene and protein expression patterns post-infection. Engineered tdTOMATO-expressing hPSC-macrophages with GFP-expressing mycobacteria enabled rapid image-based high-throughput analysis of intracellular infection and quantitative assessment of antibiotic efficacy. Our study describes the first to our knowledge hPSC-based model for M. abscessus infection, representing a novel and accessible system for studying pathogen-host interaction and drug discovery.

Aberrant inflammatory responses to type I interferon in STAT2 or IRF9 deficiency

Inflammatory phenomena such as hyperinflammation or hemophagocytic lymphohistiocytosis are a frequent yet paradoxical accompaniment to virus susceptibility in patients with impairment of type I interferon (IFN-I) signaling caused by deficiency of signal transducer and activator of transcription 2 (STAT2) or IFN regulatory factor 9 (IRF9). We hypothesized that altered and/or prolonged IFN-I signaling contributes to inflammatory complications in these patients. We explored the signaling kinetics and residual transcriptional responses of IFN-stimulated primary cells from individuals with complete loss of one of STAT1, STAT2, or IRF9 as well as gene-edited induced pluripotent stem cell-derived macrophages. Deficiency of any IFN-stimulated gene factor 3 component suppressed but did not abrogate IFN-I receptor signaling, which was abnormally prolonged, in keeping with insufficient induction of negative regulators such as ubiquitin-specific peptidase 18 (USP18). In cells lacking either STAT2 or IRF9, this late transcriptional response to IFN-α2b mimicked the effect of IFN-γ. Our data suggest a model wherein the failure of negative feedback of IFN-I signaling in STAT2 and IRF9 deficiency leads to immune dysregulation. Aberrant IFN-α receptor signaling in STAT2- and IRF9-deficient cells switches the transcriptional output to a prolonged, IFN-γ-like response and likely contributes to clinically overt inflammation in these individuals.

Single-Cell RNA Sequencing of Human Pluripotent Stem Cell-Derived Macrophages for Quality Control of The Cell Therapy Product.

Macrophages exhibit high plasticity to achieve their roles in maintaining tissue homeostasis, innate immunity, tissue repair and regeneration. Therefore, macrophages are being evaluated for cell-based therapeutics against inflammatory disorders and cancer. To overcome the limitation related to expansion of primary macrophages and cell numbers, human pluripotent stem cell (hPSC)-derived macrophages are considered as an alternative source of primary macrophages for clinical application. However, the quality of hPSC-derived macrophages with respect to the biological homogeneity remains still unclear. We previously reported a technique to produce hPSC-derived macrophages referred to as iMACs, which is amenable for scale-up. In this study, we have evaluated the biological homogeneity of the iMACs using a transcriptome dataset of 6,230 iMACs obtained by single-cell RNA sequencing. The dataset provides a valuable genomic profile for understanding the molecular characteristics of hPSC-derived macrophage cells and provide a measurement of transcriptomic homogeneity. Our study highlights the usefulness of single cell RNA-seq data in quality control of the cell-based therapy products.

3043 – HUMAN MACROPHAGE MODELS OF CHIP REVEAL UNIQUE AND SHARED PHENOTYPES BETWEEN DNMT3A- AND TET2-MUTATED TERMINALLY DIFFERENTIATED IMMUNE CELLS

Age-related acquisition and expansion of leukemogenic mutations in hematopoietic stem cells, referred to as clonal hematopoiesis of indeterminate potential (CHIP), is associated with blood cancer and coronary artery disease as well as several other age-related diseases. The most frequently mutated genes in CHIP are DNMT3A and TET2, which encode enzymes catalyzing DNA methylation and DNA hydroxymethylation, respectively. Whether specific defects in DNA methylome or hydroxymethylome in human immune cells underlie the association between CHIP and diseases is unknown. Using human macrophages differentiated in vitro from pluripotent stem cells, we investigated the impact of CHIP-associated DNMT3A and TET2 mutations on cellular differentiation, DNA methylome/hydroxymethylome, and transcriptome. CHIP-associated DNMT3A and TET2 mutations are almost always heterozygous and predicted to negatively affect their function, indicating haploinsufficiency as the predominant pathogenic mechanism. We did not observe any gross macrophage differentiation defects associated with DNMT3A or TET2 haploinsufficiency, consistent with observations in humans and mice. Deep characterization of DNMT3A- and TET2-haploinsufficient macrophages demonstrated that DNMT3A and TET2 defects have limited overlapping impact on DNA methylome and hydroxymethylome, but drive shared transcriptomic changes, including increased cell cycle and checkpoint gene expression. Our results indicate that pluripotent stem cell-derived human macrophages are a powerful system to dissect the impact of CHIP mutations on terminally differentiated immune cells. Age-related acquisition and expansion of leukemogenic mutations in hematopoietic stem cells, referred to as clonal hematopoiesis of indeterminate potential (CHIP), is associated with blood cancer and coronary artery disease as well as several other age-related diseases. The most frequently mutated genes in CHIP are DNMT3A and TET2, which encode enzymes catalyzing DNA methylation and DNA hydroxymethylation, respectively. Whether specific defects in DNA methylome or hydroxymethylome in human immune cells underlie the association between CHIP and diseases is unknown. Using human macrophages differentiated in vitro from pluripotent stem cells, we investigated the impact of CHIP-associated DNMT3A and TET2 mutations on cellular differentiation, DNA methylome/hydroxymethylome, and transcriptome. CHIP-associated DNMT3A and TET2 mutations are almost always heterozygous and predicted to negatively affect their function, indicating haploinsufficiency as the predominant pathogenic mechanism. We did not observe any gross macrophage differentiation defects associated with DNMT3A or TET2 haploinsufficiency, consistent with observations in humans and mice. Deep characterization of DNMT3A- and TET2-haploinsufficient macrophages demonstrated that DNMT3A and TET2 defects have limited overlapping impact on DNA methylome and hydroxymethylome, but drive shared transcriptomic changes, including increased cell cycle and checkpoint gene expression. Our results indicate that pluripotent stem cell-derived human macrophages are a powerful system to dissect the impact of CHIP mutations on terminally differentiated immune cells.

3198 – HUMAN PLURIPOTENT STEM CELL-DERIVED MACROPHAGES FOR MODELLING MYCOBACTERIAL INFECTION

Human macrophages are a natural host of many mycobacterium species, including Mycobacterium abscessus (M. abscessus), an emerging pathogen affecting patients with lung diseases and immunocompromised individuals. There are few available treatments and the search for effective antibiotics against M. abscessus has been hindered by the lack of a tractable in vitro intracellular model of infection. Here, we established a reliable model for M. abscessus infection using human pluripotent stem cell-derived macrophages (hPSC-macrophages). hPSC differentiation permitted a reproducible generation of functional human macrophages that were highly susceptible to M. abscessus infection. Electron microscopy demonstrated that M. abscessus was present in the vacuoles of hPSC-macrophages. RNA-sequencing analysis revealed a time-dependent immune response to M. abscessus with different gene expression patterns. Engineered tdTOMATO-expressing hPSC-macrophages with GFP-expressing M. abscessus enabled rapid image-based analysis of intracellular infection and quantitative assessment of antibiotic resistance. Our study describes the first hPSC-based model for M. abscessus infection, which represents a novel platform for studying M. abscessus-host interaction and an accessible tool for drug discovery. Human macrophages are a natural host of many mycobacterium species, including Mycobacterium abscessus (M. abscessus), an emerging pathogen affecting patients with lung diseases and immunocompromised individuals. There are few available treatments and the search for effective antibiotics against M. abscessus has been hindered by the lack of a tractable in vitro intracellular model of infection. Here, we established a reliable model for M. abscessus infection using human pluripotent stem cell-derived macrophages (hPSC-macrophages). hPSC differentiation permitted a reproducible generation of functional human macrophages that were highly susceptible to M. abscessus infection. Electron microscopy demonstrated that M. abscessus was present in the vacuoles of hPSC-macrophages. RNA-sequencing analysis revealed a time-dependent immune response to M. abscessus with different gene expression patterns. Engineered tdTOMATO-expressing hPSC-macrophages with GFP-expressing M. abscessus enabled rapid image-based analysis of intracellular infection and quantitative assessment of antibiotic resistance. Our study describes the first hPSC-based model for M. abscessus infection, which represents a novel platform for studying M. abscessus-host interaction and an accessible tool for drug discovery.

ACVR1R206H extends inflammatory responses in human induced pluripotent stem cell-derived macrophages.

Macrophages play crucial roles in many human disease processes. However, obtaining large numbers of primary cells for study is often difficult. We describe 2D and 3D methods for directing human induced pluripotent stem cells (hiPSCs) into macrophages (iMACs). iMACs generated in 2D culture showed functional similarities to human primary monocyte-derived M2-like macrophages, and could be successfully polarized into a M1-like phenotype. Both M1- and M2-like iMACs showed phagocytic activity and reactivity to endogenous or exogenous stimuli. In contrast, iMACs generated by a 3D culture system showed mixed M1- and M2-like functional characteristics. 2D-iMACs from patients with fibrodysplasia ossificans progressiva (FOP), an inherited disease with progressive heterotopic ossification driven by inflammation, showed prolonged inflammatory cytokine production and higher Activin A production after M1-like polarization, resulting in dampened responses to additional LPS stimulation. These results demonstrate a simple and robust way of creating hiPSC-derived M1- and M2-like macrophage lineages, while identifying macrophages as a source of Activin A that may drive heterotopic ossification in FOP.

Human iPSC-derived macrophages for efficient Staphylococcus aureus clearance in a murine pulmonary infection model

AbstractPrimary or secondary immunodeficiencies are characterized by disruption of cellular and humoral immunity. Respiratory infections are a major cause of morbidity and mortality among immunodeficient or immunocompromised patients, with Staphylococcus aureus being a common offending organism. We propose here an adoptive macrophage transfer approach aiming to enhance impaired pulmonary immunity against S aureus. Our studies, using human-induced pluripotent stem cell-derived macrophages (iMφs), demonstrate efficient antimicrobial potential against methicillin-sensitive and methicillin-resistant clinical isolates of S aureus. Using an S aureus airway infection model in immunodeficient mice, we demonstrate that the adoptive transfer of iMφs is able to reduce the bacterial load more than 10-fold within 20 hours. This effect was associated with reduced granulocyte infiltration and less damage in lung tissue of transplanted animals. Whole transcriptome analysis of iMφs compared with monocyte-derived macrophages indicates a more profound upregulation of inflammatory genes early after infection and faster normalization 24 hours postinfection. Our data demonstrate high therapeutic efficacy of iMφ-based immunotherapy against S aureus infections and offer an alternative treatment strategy for immunodeficient or immunocompromised patients.