Pulmonary Transplantation of Human Induced Pluripotent Stem Cell-derived Macrophages Ameliorates Pulmonary Alveolar Proteinosis

Abstract

Rationale: Although the transplantation of induced pluripotent stem cell (iPSC)-derived cells harbors enormous potential for the treatment of pulmonary diseases, in vivo data demonstrating clear therapeutic benefits of human iPSC-derived cells in lung disease models are missing.

Objectives: We have tested the therapeutic potential of iPSC-derived macrophages in a humanized disease model of hereditary pulmonary alveolar proteinosis (PAP). Hereditary PAP is caused by a genetic defect of the GM-CSF (granulocyte-macrophage colony-stimulating factor) receptor, which leads to disturbed macrophage differentiation and protein/surfactant degradation in the lungs, subsequently resulting in severe respiratory insufficiency.

Methods: Macrophages derived from human iPSCs underwent intrapulmonary transplantation into humanized PAP mice, and engraftment, in vivo differentiation, and therapeutic efficacy of the transplanted cells were analyzed.

Measurements and main results: On intratracheal application, iPSC-derived macrophages engrafted in the lungs of humanized PAP mice. After 2 months, transplanted cells displayed the typical morphology, surface markers, functionality, and transcription profile of primary human alveolar macrophages. Alveolar proteinosis was significantly reduced as demonstrated by diminished protein content and surfactant protein D levels, decreased turbidity of the BAL fluid, and reduced surfactant deposition in the lungs of transplanted mice.

Conclusions: We here demonstrate for the first time that pulmonary transplantation of human iPSC-derived macrophages leads to pulmonary engraftment, their in situ differentiation to an alveolar macrophage phenotype, and a reduction of alveolar proteinosis in a humanized PAP model. To our knowledge, this finding presents the first proof-of-concept for the therapeutic potential of human iPSC-derived cells in a pulmonary disease and may have profound implications beyond the rare disease of PAP.

Keywords: cell therapy; induced pluripotent stem cell; macrophages; pulmonary alveolar proteinosis.

Figure 1.

Generation and characterization of induced pluripotent stem cell (iPSC)-derived macrophages. (A) Generation of human iPSCs: Hematopoietic differentiation of iPSC-derived macrophages (iPSC Mϕ) with human macrophage colony–stimulating factor and human IL-3 and transplantation into humanized pulmonary alveolar proteinosis (HuPAP) mice. (B) Continuous generation of human iPSC Mϕ from embryoid body–derived structures (bright-field image; scale bar, 500 μm). (C) Morphology of iPSC Mϕ after cytospin preparation and May-Giemsa staining (scale bar, 50 μm). (D) Flow cytometric analysis of surface marker expression in iPSC Mϕ (gray, respective isotype control; red, stained surface marker; n = 3 experiments). (E) Overall numbers of macrophages from human iPSCs, generated in a six-well plate over 6 weeks of differentiation (n = 6, mean ± SEM). (F) Methylcellulose-based clonogenic assays performed with either iPSC Mϕ with various cell numbers or CD34⁺ cells derived from umbilical cord blood (n = 3, mean + SEM). (G) Chromosomal analysis of iPSC-derived macrophages by spectral R-banding. (H and I) Oil red O staining of iPSC-derived macrophages after incubation with BAL fluid from HuPAP mice (0 h). Cells were then washed and further cultured to demonstrate clearance of surfactant material after 24 hours (scale bars, 50 μm). C-MYC = MYC proto-oncogene; CFU = colony-forming unit; hIL3 = human IL-3; hiPSC = human induced pluripotent stem cell; hM-CSF = human macrophage colony–stimulating factor; KLF4 = Kruppel-like factor 4; mar = marker; n.d. = not detected; OCT4 = octamer-binding transcription 4; SOX2 = sex-determining region Y-box 2.

Figure 2.

Engraftment and clinical benefit of human induced pluripotent stem cell (iPSC)–derived macrophages in humanized pulmonary alveolar proteinosis (huPAP) mice analyzed 2 months after the first transplantation. (A) Detection of human CD45⁺ cells within the lungs of recipient mice (light blue, autofluorescence; yellow, human CD45 [hCD45, huCD45]), with less consolidated alveoli in regions with cell engraftment (white dashed frame) compared with adjacent regions where no cells occurred (pink dashed frame) (scale bar, 250 μm). (B) Flow cytometry of BAL fluid (BALF) and lung cells reveals clear population of hCD45⁺ cells within the SSC^hi gate after pulmonary iPSC-based macrophage transplantation (PiMT) but not in untreated (PAP) or untransplanted control nonobese diabetic severe combined immunodeficient (NSG) mice. (C and D) BALF and lung chimerism as assessed by flow cytometry analyzing human CD45⁺ cells (n = 8 mice in PAP group, n = 10 in PiMT group, and n = 5 in NSG group; pooled data from three experiments). (E) Detection of human GAPDH in lungs of huPAP mice transplanted with iPSC-derived macrophages (PiMT, three mice). Primers detecting murine β-actin (mβactin) were used to detect murine DNA. Lungs of nontransplanted huPAP mice (huPAP) or NSG mice (NSG), a human cell line (hCtrl) or murine liver samples (mCtrl) were used as controls. (F) Reduced BALF protein levels (n = 10 mice in PAP group, n = 9 in PiMT group, and n = 9 in NSG group; pooled data from three experiments). (G) Reduced BALF turbidity after PiMT (photograph of representative BALF flushes). (H) Reduced BALF surfactant-D levels (n = 5 mice in PAP group, n = 6 in PiMT group; pooled data from two experiments). (I) Staining for periodic acid–Schiff reagent (PAS)-positive mucus in lung slices of treated and untreated mice (scale bars, 200 μm). (J) Reduced PAS-positive pixels after PiMT (n = 8 mice in PAP group, n = 10 in PiMT group, and n = 10 in NSG group; pooled data from three experiments). All graphs display mean + SEM; one-way ANOVA testing for three-group or one-tailed t test for two-group comparison: *P < 0.05, **P < 0.01, ***P < 0.001. BG = background; HE = hematoxylin and eosin; hGAPDH = human GAPDH; n.s. = not significant; SSC = side scatter.

Figure 3.

Phenotypic changes and protein and gene expression of induced pluripotent stem cell (iPSC)–derived macrophages 2 months after pulmonary transplantation therapy. (A) Cytospin appearance of iPSC-derived macrophages before and after transplantation (pulmonary iPSC-based macrophage transplantation [PiMT]) compared with primary human alveolar macrophages (huAMs; scale bar, 50 μm; representative experiment of three). (B) Surface protein expression and (C) principal component analysis (PCA) of 10 surface markers analyzed by chip cytometry of cells before and after PiMT, compared with the phenotype of human primary AMs within a lung specimen of a healthy human organ donor (representative experiment of two). Scale bar in B, 20 μm. (D) In situ staining of recipient lung tissue for human CD45 and oil red O staining of respective cells reveal intracellular lipoprotein uptake within recipient humanized pulmonary alveolar proteinosis (huPAP) lungs (scale bar, 50 μm; representative experiment of two). (E) Hierarchical clustering of 2,381 transcripts differentially expressed in the analyzed cell populations. One-way ANOVA testing was conducted to identify transcripts showing the most reliable mRNA expression differences in any of the possible pairwise comparisons between cells before (blue; pre-PiMT) and after transplantation (yellow; post-PiMT), primary huAMs (green) and alveolar macrophage–like cells reisolated from huPAP lungs 5 months after CD34 cell–based pulmonary macrophage transplantation (orange; CD34 PMT) (corrected P value < 0.01 according to Benjamini-Hochberg correction). Samples and transcripts were hierarchically clustered (distance metric, Euclidean; linkage rule, Ward’s). Red color indicates elevated relative mRNA expression levels, whereas blue color represents diminished mRNA levels. (F) PCA was conducted with unfiltered quantified RNA-Seq data. A two-dimensional representation focusing on main components 1 (26.52% of variance) and 2 (11.55% of variance) is depicted. (G and H) Hierarchical clustering of selected gene sets for (G) iPSC-determining and (H) AM-relevant genes, further illustrating the changes of iPSC-derived macrophages into AM-like cells after PiMT (at least three biological replicates from at least two independent experiments per cell type). ABCG1 = ATP binding cassette subfamily G member 1; BG = background; CSF2RB = granulocyte–macrophage colony–stimulating factor 2 receptor B; hCD45 = human CD45; ITGB5 = integrin β-5; MRC1 = mannose receptor 1; PC = principal component; PPRG = peroxisome proliferator–activated receptor-γ; SPI = spleen focus forming virus (SFFV) proviral integration oncogene (transcription factor PU.1); TLR4 = Toll-like receptor 4.