Rationale An efficient and reproducible source of genotype-specific human macrophages is essential for study of human macrophage biology and related diseases. Objective To perform integrated functional and transcriptome analyses of human induced pluripotent stem cell-derived macrophages (IPSDM) and their isogenic PBMC-derived macrophages (HMDM) counterparts and assess the application of IPSDM in modeling macrophage polarization and Mendelian disease. Methods and Results We developed an efficient protocol for differentiation of IPSDM, which expressed macrophage-specific markers and took up modified lipoproteins in a similar manner to HMDM. Like HMDM, IPSDM revealed reduction in phagocytosis, increase in cholesterol efflux capacity and characteristic secretion of inflammatory cytokines in response to M1 (LPS+IFN-γ) activation. RNA-Seq revealed that non-polarized (M0) as well as M1 or M2 (IL-4) polarized IPSDM shared transcriptomic profiles with their isogenic HMDM counterparts while also revealing novel markers of macrophage polarization. Relative to IPSDM and HMDM of control individuals, patterns of defective cholesterol efflux to apoA-I and HDL3 were qualitatively and quantitatively similar in IPSDM and HMDM of patients with Tangier disease (TD), an autosomal recessive disorder due to mutations in ATP-binding cassette transporter A1. TD-IPSDM also revealed novel defects of enhanced pro-inflammatory response to LPS stimulus. Conclusions Our protocol-derived IPSDM are comparable to HMDM at phenotypic, functional and transcriptomic levels. TD-IPSDM recapitulated hallmark features observed in HMDM and reveal novel inflammatory phenotypes. IPSDM provide a powerful tool for study of macrophage-specific function in human genetic disorders as well as molecular studies of human macrophage activation and polarization.
Patients with Down syndrome (trisomy 21, T21) have hematologic abnormalities throughout life. Newborns frequently exhibit abnormal blood counts and a clonal preleukemia. Human T21 fetal livers contain expanded erythro-megakaryocytic precursors with enhanced proliferative capacity. The impact of T21 on the earliest stages of embryonic hematopoiesis is unknown and nearly impossible to examine in human subjects. We modeled T21 yolk sac hematopoiesis using human induced pluripotent stem cells (iPSCs). Blood progenitor populations generated from T21 iPSCs were present at normal frequency and proliferated normally. However, their developmental potential was altered with enhanced erythropoiesis and reduced myelopoiesis, but normal megakaryocyte production. These abnormalities overlap with those of T21 fetal livers, but also reflect important differences. Our studies show that T21 confers distinct developmental stage-and species-specific hematopoietic defects. More generally, we illustrate how iPSCs can provide insight into early stages of normal and pathological human development.
There has been much interest in magnetite (Fe 3 O 4 ) due to its utility in adsorbing high concentrations of arsenic in contaminated water. The magnetic properties of the material allow for simple dispersion and removal from an aqueous system. An inquiry-based laboratory has been developed that illustrates these unique properties of magnetite nanoparticles while developing cross-disciplinary and critical-thinking skills. The versatility of the pedagogic approach makes it suitable for both high school (grades 11 and 12) and college-level STEM (science, technology, engineering, and math) courses. The lab experience exposes students to experimental design, environmental remediation, adsorption, and the surface area-to-volume ratio concept of nanotechnology.
Tangier disease (TD) is an autosomal recessive disorder caused by mutations in the ATP-binding cassette transporter A1 (ABCA1). These result in a greatly reduced ability to transport cholesterol out of cells, leading to the accumulation of cholesterol in macrophages and many other body tissues expressing ABCA1. We recruited two TD individuals, TD1, compound heterozygote at S2046R/K531N, and TD2, homozygous for the E1005X/E1005X truncation mutation, as well as heterozygote/non-affected family members, and healthy controls. We reprogrammed PBMC of TD patients and controls to pluripotency by Sendai viral transduction with Oct3/4, Sox2, Klf4 and c-Myc. TD-iPSC expressed pluripotency markers including SSEA-3, SSEA-4, TRA-1.60, and TRA-1.81, and maintained a normal karyotype. TD iPSC differentiated efficiently to macrophages. iPSC-derived macrophages (IPSDM) characteristics paralleled those of primary PBMC-derived macrophages (HMDM) at morphological, phenotypic and transcriptomic levels. TD-IPSDM and HMDM showed no cholesterol efflux to apolipoprotein A-I (apoA-I) and impaired efflux to HDL 3 . Treatment of TD cells with LXR agonists, which upregulate ABCA1 expression, failed to enhance cholesterol efflux to apoA-I in TD-IPSDM and HMDM consistent with the absence of functional ABCA1. In both IPSDM and HMDM, the heterozygote ABCA1 mutation carrier had an intermediate defect in cholesterol efflux and a partial response to the LXR agonist consistent with the presence of one functional allele. Relative to control-IPSDM, TD-IPSDM also showed a higher cholesterol ester/total cholesterol (CE/TC) ratio upon acetylated-LDL loading. Compared with control-IPSDM, TD-IPSDM showed enhanced phagocytosis of zymosan particles and greater inflammatory response to ATP treatment in lipopolysaccharide (LPS)-primed IPSDM, evidenced by markedly elevated gene expression of IL-1beta, IL-6, IL-8 and CCL5, but not TNF-alpha. These observations provide further support for the utility of IPSDM in defining more subtle macrophage phenotypes that are less obvious manifestations of Mendelian disorders. We conclude that macrophages derived from TD IPS can effectively recapitulate pathologic hallmarks of the disease.
Genome‐wide association studies have suggested that, compared to non‐O blood types, the ABO O blood type phenotype and related genotypes are associated with protection from CVD. To test the hypothesis that ABO glycosyltransferase modulates endothelial functional pathways and glycan profile, we differentiated adipose stem cell (ASC)‐derived induced pluripotent stem cell (iPSC) from individuals of different ABO blood types to endothelial cells (ECs). iPSC‐ECs exhibited cobble‐stone morphology, demonstrated capillary tube formation on matrigel, and incorporated acetylated low‐density lipoprotein. FACS analysis indicates expression of both CD31 and vascular endothelial growth factor receptor‐2 (VEGFR2). Immunocytochemical analysis of iPSC‐ECs revealed positive staining for EC markers e.g., CD31, vascular endothelial cadherin (CD144), and von Willebrand factor (vWF). The phenotypic characteristics of iPSC‐ECs were similar to that of human coronary arterial endothelial cells (hCAECs). The adhesion pattern of iPSC‐EC to a variety of extracellular matrix (ECM) was similar across O and non‐O iPSC‐EC, but ABO blood group O showed lesser adhesion to vitronectin and laminin (mean±SD; 2.37±0.19 folds in non‐O vs. O for vitronectin [p=0.02] and 2.30±0.05 folds in non‐O vs. O for laminin [p=0.004]). Further, gap closure assay showed that O type iPSC‐EC had greater migration capacity. We examined glycan profiles of pooled iPSC‐ECs and pooled hCAECs from subjects with matched blood types. N‐linked glycan profiling showed that series of high‐mannose structures were observed as main components of iPSC‐EC and hCAECs. Complex structures were observed as minor components. iPSC‐ECs which gave similar profile to that of hCAECs. Main O‐glycans in iPSC‐ECs and hCAECs was a monosialylated core 1 structure. In summary, we established the feasibility of differentiating EC from subject‐specific ASC‐iPSC as tools to define disease‐relevant ABO modification of EC functions. Grant Funding Source: NIH U01, K24
Macrophages fulfill homeostatic functions beyond defense. Monocytes/macrophages derived from human induced pluripotent stem cells (hiPSCs) are useful tools to study vascular diseases and potential sources of cell-based therapies. The objective of this study is to generate PBMC-hiPSC derived monocytes, macrophages, as well as M1 and M2 type differentiated macrophages and to examine multiple macrophage characteristics of relevance to cardio-metabolic disease. We briefly cultured human adult PBMCs from healthy volunteers to enrich for the erythroid progenitors. These progenitors were used to generate hiPSCs using lentivirus-mediated transduction of Oct3/4, Sox2, Klf4 and c-Myc. A stepwise protocol was used for the differentiation of PBMC-hiPSCs to monocytes/macrophages. The protocol involves primitive streak and mesoderm induction (stage 1), hematopoietic specification (stage 2), hematopoietic cell maturation and myeloid expansion (stage 3), monocytes collection and differentiation to macrophages (stage 4). Differentiation cultures were set up in six-well plate format, from 20 embryoid bodies per well, and produced up to 30 million macrophages. hiPSC-derived macrophages (hiPSC-MΦ) exhibited spindle morphology and incorporated acetylated low-density lipoprotein. Immunocytochemical analysis of hiPSC-MΦ revealed positive staining for CD68, CD11b, and CCL2. FACS analysis indicates >90% expression of macrophage lineage markers, including CD45, CD14, CD16, CD115, CD68, CX3CR1, CD11b, and CD18. Migration of hiPSC-MΦ in response to M-CSF was confirmed by Boyden Chamber assay. hiPSC-MΦ were polarized to M1 (classical activation stimulated by TLR ligands LPS and IFN-gamma) and M2 (alternative activation stimulated by IL-4) types. M2 macrophages were characterized by more efficient phagocytic activity of zymosan particles and greater migration capacities compared with M1 macrophages. The results demonstrated the feasibility of differentiation/polarization of macrophages from PBMC-hiPSCs. This study advanced our understanding of the suitability of subject-specific hiPSC for deriving myeloid/monocyte/macrophages for study of monocyte-macrophage functions of direct relevance to cardio-metabolic disease.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.