Summary of studies that have used iPSCs-derived monocytes and macrophages for
Abstract
The ability to derive macrophages from human-induced pluripotent stem cells (iPSCs) provides an unlimited source of genotype-specific cells with the potential to play a role in advancing our understanding of macrophage biology in both homeostasis and disease. While sharing many of the functional characteristics of monocyte-derived macrophages, iPSC-derived macrophages have also been shown to have phenotypical and functional features associated with tissue resident macrophages. These features present new opportunities to develop models of human disease and to understand the role of developmental or tissue context in innate immune cell function. iPSCs-derived macrophages have also been identified as a highly attractive source for cell and gene therapy in the treatment of diverse degenerative diseases based on their anti-inflammatory activity, their ability to clear scarred cells by phagocytosis, and providing extracellular matrices. We review and present a concise discussion on macrophage differentiation from stem cells highlighting their advantages over classical monocyte-derived macrophages in modelling organ specific macrophages. We summarize the various disease models utilizing iPSCs-derived macrophages including hereditary syndromes and host-pathogen interactions in tissue repair and the strategies used to mimic pathological phenotypes. Finally, we describe the pre-clinical studies that have addressed the application of iPSCs-derived macrophages as a therapeutic intervention.
Keywords
- iPSCs
- macrophages
- polarization
- inflammation
- regeneration
- cell therapy
1. Introduction
Macrophages were historically considered as specialized immune cells that are resident in every tissue. They are professional phagocytic cells and are considered to be one of the most evolutionary conserved components of the innate immune system [1]. However, studies of the past two decades identified several additional functions of macrophages particularly those involved in maintaining tissue homeostasis such as wound healing and regeneration [2]. Macrophage populations within the tissue were originally assumed to be continuously replaced by the differentiation of monocytes derived from peripheral blood [3, 4]. However recent studies using methods such as lineage tracing and single cell transcriptomics have established that several macrophage populations resident in organs including brain, lung, intestine and liver, originate from yolk-sac (YS) myeloid precursor cells that were seeded within the tissues during early embryonic hematopoiesis [5]. These myeloid precursors differentiate into macrophages (microglia in case of brain) within their resident tissue site, and are self-maintained throughout the life course of the organism [5]. Tissue resident macrophages (TRMs) are functionally distinct from macrophages derived from the more accessible circulating blood monocytes but their yolk sac origin makes them difficult to study [5]. In order to successfully study these cells
The ground-breaking discovery that human somatic cells could be reprogramed into induced pluripotent stem cells (iPSCs) that are capable of differentiating into any cell type has revolutionized many areas of medical research including macrophage biology [7]. Several studies have shown that human iPSCs can be differentiated into macrophage populations that are phenotypically and functionally comparable to human macrophages. The major advantage of the iPSCs-derived macrophages is that they share some phenotypic and functional profiles with both tissue resident macrophages and monocyte derived macrophages (MDMs) [8]. Here, we review the potential of iPSCs derived macrophages in both classical immune function as well as their tissue repair and regeneration properties. We summarize the various protocols that have been used for macrophage production from iPSCs, discuss their disease modeling potential including hereditary and pathogen associated diseases and describe some of the pre-clinical trials lay the foundations for their use in cell therapies.
2. Human induced pluripotent stem cells for macrophage production in vitro
The generation of iPSCs from adult somatic cells by the introduction of four genes encoding the “Yamanaka” transcription factors, octamer-binding transcription factor 3/4 (OCT3/4), sex determining region Y-box 2 (SOX2), Kruppel-like factor 4 (KLF4), and myelocytomatosis viral oncogene homolog (MYC), was first reported in 2006 [9]. Resultant iPSCs were shown to be comparable to embryonic stem cells (ESCs), thus providing an ethical strategy for creating clonal PSC lines that did not involve the destruction of human embryos [10]. Initially reprogramming of somatic cells to iPSCs was successfully performed using genome integrating retroviral or lentiviral vectors such as retroviruses and lentiviruses [7]. Though viral integration was efficient, it was associated with risks of random mutational insertions into the genome. Alternate non-viral integration methods such as plasmids, synthetic mRNA, minicircle DNA molecules and small chemical molecules to generate iPSCs reduced random mutations but the efficiency of generating iPSCs was low [7, 8]. Sendai virus is now commonly used for iPSC generation as its replication is limited within the cytoplasm and it does not integrate into the genome. Initially skin fibroblasts were used for reprogramming iPSCs, but other easily accessible cells such as peripheral blood monocytes and renal epithelial cells from urine have been successfully used as the starting cells [11, 12]. iPSCs are able to be differentiated into the three embryonic germ layers namely ectoderm, endoderm and mesoderm and can differentiate into somatic cells associated with all cell lineages [13].
Following the generation of stable iPSCs, their pluripotent properties are maintained using specific growth factors [9]. Removal of these maintenance factors results in spontaneous differentiation and, under appropriate conditions the formation of 3-dimensional (3D) embryoid bodies (EBs) that are considered to mimic early embryogenesis [14]. Differentiation factors such as cytokines and small molecules have been used to stimulate specific differentiation pathways resulting in the production of cells displaying phenotypes and gene expression patterns of almost any cell type including cells of the blood and immune system [15].
Doetschman and colleagues were the first to report that hematopoietic cell types could be produced from mouse embryonic stem cells (ESCs) by demonstrating structures resembling blood islands in cystic embryoid bodies (EBs) that were comparable to the primitive wave of hematopoiesis in the yolk sac [16]. Differentiation protocols were subsequently developed and refined to include the use of feeder cells, extracellular matrices and specific growth factors [17, 18, 19, 20]. Many of these protocols failed to generate the long-term reconstituting hematopoietic stem cells (HSCs) associated with the definitive wave of hematopoietic development and a significant amount of research has gone into addressing this problem in both the mouse and human systems [21, 22, 23, 24]. The production of macrophages was reported even in the first, rather crude differentiation protocols with the ability to harvest on regular basis for several weeks represented a significant advance in the field [18, 25]. The fact that these differentiation protocols most likely mimic the primitive wave of hematopoiesis it is not surprising that resultant cells have some features that are comparable to TRMs.
The production of macrophages from human iPSCs is now well established and they are considered to have features associated with both YS-derived TRMs as well as MDMs [5]. As iPSCs can been maintained indefinitely in culture and can be readily genetically manipulated, they can therefore provide an inexhaustible source of macrophages carrying any desired genetic alteration. The first protocols that were developed involved the co-culture of iPSCs with OP9 mouse stromal cell monolayers to induce hematopoietic differentiation, followed by expansion of myeloid progenitors and selective differentiation into macrophages by using growth factors to differentiate dendritic cells and macrophages [26, 27]. These protocols were further modified to establish embryoid body (EB)-based protocols for IPSC-derived macrophage differentiation. We have used a modified serum-free protocol in which EB-based hematopoiesis is used to generate monocyte-like cells in suspension that can then be differentiated into mature macrophages [28]. Briefly, differentiation from human iPSCs is initiated by the removal of pluripotency factors and the addition of stem cell factor (SCF), bone morphogenetic protein (BMP)-4 and vascular endothelial growth factor (VEGF) to induce EB formation. The addition of interleukin (IL)-3 and macrophage colony stimulating factor (M-CSF) to EBs that are then plated down onto the culture plates results in the production of monocyte precursors that are released into suspension. Monocyte-like cells are then plated down and differentiated into mature macrophages by the addition of M-CSF [29]. iPSCs derived macrophages express macrophage-specific markers including cluster of differentiation (CD)11b, CD163, and CD169 [30]. Macrophages generated from iPSCs that carried the Zeiss Green reporter gene integrated into the adeno-associated virus integration site 1 (
2.1 iPSCs-derived macrophages share features of MDM
Yeung and colleagues demonstrated that the iPSCs-derived macrophages exhibited gene expression profiles and responsiveness to external stimuli that were comparable to MDMs. Their data demonstrated that untreated iPSCs-derived macrophages and MDMs expressed 12,599 human genes overlapping and a further 93% of these genes were expressed to a similar level [31]. This pattern of gene expression iPSCs-derived macrophages and MDMs remained consistent even after stimulation, as upon
iPSCs-derived macrophages secrete comparable levels of cytokines as MDMs upon stimulation with pattern recognition receptors such as toll-like receptor (TLR) agonist [32]. They are able to phagocytose live
2.2 iPSCs derived macrophages model tissue resident macrophages
Although iPSCs-derived macrophages have demonstrated similar phenotypic, functional, and transcriptomic characteristics to MDMs as discussed above, they are also reported to have comparable characteristics to tissue resident macrophages (TRMs). This TRM like phenotype gives iPSCs-derived macrophages an advantage over other models such as MDMs or monocytic cell lines such as THP-1. It has been recognized that the standard hematopoietic differentiation protocols of iPSCs resemble the primitive rather than definitive wave of hematopoiesis
2.3 iPSCs derived macrophages in disease modeling
Another important feature of human iPSCs derived macrophages is that iPSCs are amenable to genetic engineering and thus can be manipulated to be model genetic disease. Disease modeling can be achieved either through the production of iPSCs from patients carrying disease-causing mutations and/or specific genome-wide associations or by targeted gene edited using the (CRISPR)/Cas9 system. iPSCs-derived macrophages are increasingly being used to study genetic disease, including validation of known causative genes or identifying novel mutations associated with single nucleotide polymorphisms (SNPs) [40]. iPSCs-derived macrophages helped overcome the limitations of the poor availability of disease-specific primary macrophages in studying these rare genetic diseases. The ability to derive macrophages from iPSCs provided new opportunities to develop models relevant to human genetics, resulting in a progressive accumulation of studies describing macrophage functions in both tissue homeostasis and disease. For example, patient iPSCs-derived macrophages have been utilized to investigate several genetically inherited diseases including Blau Syndrome, Tangier disease and Gaucher disease. In additions there are studies where a diseased condition such as Dyskeratosis Congenita has been generated in iPSCs using genetic engineering technology and macrophages or myeloid cells derived from these used to understand the disease mechanism [41, 42, 43, 44]. Table 1 lists some of the genetic studies performed using patient iPSCs-derived macrophages, that would not have been possible using primary cells.
Studies utilizing iPSCs derived macrophages for disease modeling | ||
---|---|---|
Disease | Research findings | References |
Tangier Disease (TD) | iPSC-derived macrophages from TD patients recapitulate the clinical defect of failed cholesterol efflux resulting in reverse cholesterol transport. | [45] |
TD effect of reverse cholesterol transport in macrophages derived from CRISPR/Cas9 induced adenosine triphosphate binding cassette subfamily A member 1 gene ( | [43] | |
Gaucher disease (GD) | iPSCs-derived macrophages from GD patients exhibited delayed clearance of phagocytosed RBC which was reversed when treated with recombinant glucocerebrosidase enzyme. | [42] |
Reversal of GD phenotype in iPSCs-derived macrophages using small-molecule chaperone drug. | [46] | |
Chronic granulomatous disease | iPSCs-derived macrophages from dihydronicotinamide-adenine dinucleotide phosphate (NADPH) oxidase defective patient showed normal phagocytic properties unlike patient MDMs, however showed a lack in reactive oxygen species production, correlating with clinical diagnosis. | [47] |
Blau syndrome | iPSCs-derived macrophages from nucleotide-binding oligomerization domain-containing protein 2 ( | [41] |
Type 1 diabetes | iPSCs-derived macrophages from Diabetic patient showed potential for antigen presentation to proinsulin-specific T cell receptors from donor-matched islet-infiltrating T cells. | [48] |
Familial Mediterranean fever | Patient iPSCs-derived macrophages exhibited the disease characteristics including enhanced IL-1β secretion and hyperactivation of the pyrin inflammasome. | [49] |
Mendelian Susceptibility to Mycobacterial Disease (MSMD) | iPSCs-derived macrophages from MSMD patients with autosomal recessive complete- and partial IFN-γR2 deficiency, partial IFN-γR1 deficiency and complete STAT1 deficiency demonstrated varying phenotypes including cytokine secretion for the partial and complete deficiencies. | [50] |
Idiopathic Parkinson’s disease | Patient iPSCs-derived microglia to confirmed findings in patients brain tissue of having elevated | [51] |
Chronic infantile neurologic cutaneous and articular syndrome | iPSCs-derived macrophages from NLRP3 mutated patient showed the disease relevant phenotype of abnormal IL-1β secretion which were inhibited by anti-inflammatory compounds. | [52] |
Immortalized iPSCs-derived myeloid cells from patient recapitulated the disease phenotypes | [53] |
As well as generating valuable disease models these studies also described the novel approach of immortalizing iPSCs-derived myeloid cells using transducing lentiviral vectors that encoded genes
2.4 iPSCs-derived macrophages in host-pathogen interactions
iPSCs-derived macrophages have also been used widely in studies relating to their classical role in infection biology. iPSCs-derived macrophages can polarized to a pro-inflammatory or anti-inflammatory phenotype by treating with lipopolysaccharide (LPS)/IFN-γ or IL-4/IL-10, respectively [30]. These features make them a powerful
One such example was the study of the interaction between
Viruses require a specific cellular host for replication and so the readily available supply of infectable cells is crucial in viral research. iPSCs are ideal for this purpose because they can be differentiated into the specific cell type associated with an infectious agent, including endothelial cells for cytomegalovirus, neurons for herpes simplex virus, hepatocytes for hepatitis viruses, CD4 T-cells for human immunodeficiency virus (HIV) [56]. The field of HIV research has used iPSCs-derived macrophages widely. Using various genetic editing techniques, Kambal
2.5 iPSCs-derived macrophages in cell and regenerative therapy
Several studies performed in the last decade identified macrophages to have a prominent role in tissue repair and regeneration by their injury response features including clearing cell debris by phagocytosis, activating and resolving inflammation and promoting fibrosis by providing growth factors [61, 62]. For example the transplantation of mouse bone marrow-derived macrophages into a CCL4 mediated advanced liver injury mice model resulted in the reduction of fibrosis by increased recruitment of host effector cells such as neutrophils and secretion of regenerative factors such as matrix metallopeptidase 9 (MMP9), insulin-like growth factor 1 (IGF-1), M-CSF, vascular endothelial growth factor (VEGF) and IL10 [63]. Similarly exogenous macrophage treatments were shown to promote injury resolution in a several murine models of inflammatory and degenerative diseases including pulmonary fibrosis and osteochondral defect [64, 65]. This successful demonstration of macrophage therapy in pre-clinical models led to the use of autologous macrophages in therapeutic interventions in clinical studies against chronic liver injury and neurodegenerative diseases [66, 67]. It is thought that these repair functions are performed by the anti-inflammatory or resolving M2-polarized macrophages and several studies identified iPSCs-derived macrophages to be able to be polarized into an M2-phenotype similar to MDMs [30]. The polarization potential together with their ability for unrestricted production makes the iPSCs-derived macrophages ideal candidates for future cell therapies. Some of the studies highlighting these
Studies demonstrating iPSCs derived macrophages as therapeutic interventions | ||
---|---|---|
Disease | Research findings | References |
Liver Fibrosis | Mouse ESC-derived macrophages showed repair capacity in CCL4 murine model. | [68] |
M1 and M2 polarized human iPSCs-derived macrophages ameliorated fibrosis in an immunodeficient CCL4 murine model. | [69] | |
Pulmonary Alveolar Proteinosis (PAP) | Mouse iPSCs-derived macrophages gained an alveolar phenotype lung of PAP model and improved alveolar protein deposition. | [34] |
Human iPSCs–derived macrophages transplanted into the lung of humanized PAP mice showed | [70] | |
Human iPSCs-derived macrophages engrafted into the lung of the PAP mouse model differentiated into alveolar macrophages and eliminated disease associated surfactant proteins. | [5] | |
Macrophages derived from gene corrected PAP patient-derived iPSCs showed restoration of normal phenotype. | [71] | |
Inflammatory Bowel Diseases (IBDs) | Very early onset of IBDs in patients leads to decreased bacterial killing ability in macrophages, which was reverted by the pharmacological inhibition of PGE2 synthesis and PGE2 receptor blockade. | [72] |
Genetic correction of patient iPSCs-derived macrophages | [73] | |
Solid Tumors | Engineered iPSCs-derived CAR-macrophages with antigen-dependent anti-cancer functions demonstrated pro-inflammatory/anti-tumor state, enhanced clearance of tumor cells by phagocytosis. | [74] |
Designer iPSCs-derived macrophage cell line to secrete IFN-β (opinion) | [75] |
Studies from our lab demonstrated that
The therapeutic potential of iPSCs-derived macrophages has also been assessed in lung fibrosis particularly in the case of pulmonary alveolar proteinosis (PAP). Hereditary PAP is a disorder known to be originated by a defect in the
Inflammatory bowel disease (IBD) is another group of inflammatory syndromes where the potential role of iPSCs-derived macrophage mediated therapy has been evaluated. Studies have demonstrated that these macrophages can be used in disease modeling and to reduce the disease pathology
The iPSCs-derived macrophage strategy has also been applied to the exciting field of cancer immunotherapy. Chimera antigen receptor (CAR)-T cells and NK cells re shown to have potent cytotoxicity against tumor cells with CAR-T cell therapy having gained great success in the clinic [77]. Recently CAR-macrophages have been developed by engineering an adenoviral vector to express a CAR targeted against human epidermal growth factor receptor 2, (a biomarker in many solid tumors) and imparted a sustained pro-inflammatory (M1) phenotype [78]. The model showed great success by demonstrating antigen-specific phagocytosis and tumor clearance
3. Conclusion
In summary, the phenotypic, functional, and transcriptomic characteristics of iPSCs-derived macrophages share many similarities with both tissue resident macrophages and MDMs. The unlimited replication potential of iPSCs and the ease of genetic manipulation thus provides a valuable platform for disease modeling, drug screening, and studying the mechanisms of infection biology in various genetic backgrounds. Their autologous nature and polarization potential could also make them ideal tools for cell and regeneration therapy. iPSCs-derived macrophages have enormous potential in advancing our understanding of diseases that involve human macrophages and to date have demonstrated proof of principle utility in the development of disease models and in novel cell therapies. The use of iPSCs-derived macrophages does not eliminate the need for other models such as MDMs or BM-derived macrophages, but rather provides a complementary or alternative approach to further ensure validity and reproducibility. Together with genetic manipulations techniques such as CRISPR/Cas9 they can facilitate clinical and therapeutic translation for diseases such as liver fibrosis or inflammatory lung diseases where macrophages play an important clinical modulatory role. This is well highlighted by a research article under peer-review where M2 polarized iPSCs-derived macrophages are studied in context with COVID19 therapy [80]. The clinical potential of the macrophage cell therapy is highlighted by several clinical trials approved for autologous macrophages as intervention in various diseases including chronic liver injury, spinal cord injury, non-acute stroke, chronic anal fissure and as an anti-fibrotic treatment following COVID-19 infection [clinicaltrials.gov]. The future of iPSCs-derived macrophage therapy could be focused toward increasing their universality or increasing their better storage and differentiation as demonstrated by the studies of developing iPSCs-derived myeloid lines, continuous differentiation or cryopreservation [53, 81, 82]. An overview of the iPSCs-derived macrophages features and applications covered in this review is summarized as Figure 1.
Acknowledgments
This work was funded by the UK Medical Research Council (MR/T013923/1) and Figure 1 was created using BioRender.com
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